Ribosomal protein S6 kinase II: Biological Overview | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References
Gene name - Ribosomal protein S6 kinase II
Synonyms - dRSK
Cytological map position- 20C1-20C1
Function - signaling
Keywords - MAP kinase pathway, eye, wing, learning
Symbol - S6kII
FlyBase ID: FBgn0262866
Genetic map position - X
Classification - protein serine/threonine kinase activity
Cellular location - cytoplasmic
Although p90 ribosomal S6 kinase (RSK) is known as an important downstream effector of the ribosomal protein S6 kinase/extracellular signal-regulated kinase (Ras/ERK) pathway, its endogenous role, and precise molecular function remain unclear. Using gain-of-function and null mutants of RSK, its physiological role was successfully characterized in Drosophila. Surprisingly, RSK-null mutants are viable, but exhibit developmental abnormalities related to an enhanced ERK-dependent cellular differentiation such as ectopic photoreceptor- and vein-cell formation. Conversely, overexpression of RSK dramatically suppresses the ERK-dependent differentiation, which is further augmented by mutations in the Ras/ERK pathway. Consistent with these physiological phenotypes, RSK negatively regulates ERK-mediated developmental processes and gene expressions by blocking the nuclear localization of ERK in a kinase activity-independent manner. In addition, RSK-dependent inhibition of ERK nuclear migration is mediated by the physical association between ERK and RSK. Collectively, these studies reveal a novel regulatory mechanism of the Ras/ERK pathway by RSK, which negatively regulates ERK activity by acting as a cytoplasmic anchor in Drosophila (Kim, 2006).
The p90 ribosomal protein S6 kinase (RSK) is well conserved among the metazoan systems (Blenis, 1993; Nebreda, 1999). RSK was originally identified as a direct target of the ribosomal protein S6 kinase/extracellular signal-regulated kinase (Ras/ERK)-MAP kinase signaling pathway, which regulates the most critical cellular responses including cell proliferation, differentiation, and metabolism (Blenis, 1993; Pearson, 2001). RSK specifically binds to ERK (Scimeca, 1992) and is activated through phosphorylation by activated ERK (Sturgill, 1988; Chung, 1991). Since the activity of RSK tightly correlates with that of ERK, RSK has been thoroughly studied as one of the critical downstream effectors of ERK. Indeed, various physiologically important molecules such as lamin-C, glycogen synthase kinase 3, cAMP-responsive binding-element protein (CREB), histone 3B, anaphase-promoting complex (APC), C/EBP beta, Bub1, c-Fos, filamin A, and tuberous sclerosis complex (TSC) were suggested as putative targets mediating the molecular function of RSK (Schwab, 2001; Roux, 2004; Woo, 2004; reviewed in Frodin, 1999). Although these targets seem to appropriately explain the roles of RSK as a downstream effector of the Ras/ERK signaling pathway, there has been insufficient evidence to consider them as the actual downstream targets of RSK in vivo. Furthermore, some recent studies (Myers, 2004) suggest that RSK has an inhibitory role in the Ras/ERK pathway while neither the physiological relevance nor the molecular mechanism has yet been sufficiently addressed (Kim, 2006).
The existence of many RSK isoforms (RSK 1-4) in the mammalian genome has hampered extensive genetic researches on the physiological functions of RSK (Alcorta, 1989; Moller, 1994; Yntema, 1999). In this study, advantage was taken of the Drosophila system, which has only a single RSK gene in the genome (Wassarman, 1994). Null flies for Drosophila RSK were generated and its in vivo function was characterized. Surprisingly, RSK was found to be devoted to negatively regulate nuclear ERK function, restraining ERK in the cytoplasm during Drosophila eye and wing development, by physical association with ERK (Kim, 2006).
This study has found that overexpression of RSK strongly suppresses retinal (both in neuronal and non-neuronal cells) and wing vein cell differentiation, which is controlled by the Ras/ERK pathway. Conversely, RSK-null flies display ectopic differentiation of retinal and wing vein cells and deletion of abdominal denticle belts in embryos, that are reminiscent of the phenotypes caused by gain-of-function mutations of the Ras/ERK pathway components. Furthermore, various genetic interaction assays consistently indicate that the RSK-null phenotypes are caused by hyperactivation of the Ras/ERK pathway, and that the RSK overexpression phenotypes are caused by decreased activities of the Ras/ERK pathway. These genetic experiments strongly support RSK as a negative regulator of ERK-dependent cellular differentiation in vivo (Kim, 2006).
Many negative regulators of the Ras/ERK pathway including various dual-specificity phosphatases are transcriptionally induced by activation of the Ras/ERK pathway to form a negative feedback loop. Since RSK acts as a negative regulator of the Ras/ERK pathway, it is possible to hypothesize that expression of RSK may be induced by Ras/ERK signaling activity. However, the results clearly showed that RSK is ubiquitously expressed in all developmental stages, while Ras/ERK signaling is activated in a specific region and at specific times. Furthermore, although the expression of pnt-P1 was highly induced by hyperactive Ras (RasV12) and silenced by dominant-negative Ras (RasN17), RSK gene expression was not altered by Ras at all, suggesting the Ras/ERK signaling pathway does not transcriptionally induce RSK (Kim, 2006).
Genetic and biochemical analyses using kinase-dead mutants of RSK suggested that the kinase activity of RSK is dispensable for its role during Drosophila eye and wing development. This is in stark contrast to previous assertions on RSK as an important kinase that controls many crucial downstream targets of the Ras/ERK pathway through phosphorylation in mammals (Frodin, 1999). Supporting the results, there were no differences in the phosphorylation level of histone H3, a well-known target of RSK, between wild-type and RSK-null eye and wing discs. Therefore, it is believed that the substrate phosphorylation by RSK is largely unnecessary for its function in Drosophila. However, since some phenotypes including life span reduction, fertility reduction and growth retardation shown in RSK-null flies are not significantly rescued by expressing kinase-dead mutants of RSK by the da- or hs-Gal4 driver, the possibility that the kinase function plays a role in developmental processes other than eye and wing development cannot be entirely excluded. In addition, since only one RSK isoform exists in Drosophila, the physiological function of RSK shown in this study may not satisfactorily represent more specialized physiological roles of all the RSK isoforms (RSK1-4) in mammals (Kim, 2006).
Through a biochemical study using rat PC12 cell line, it has been claimed that RSK negatively regulates the Ras/ERK pathway by phosphorylating the Son of sevenless (Sos; see Drosophila Son of sevenless) Ras-GEF protein, an upstream activator of Ras (Douville, 1997). However, genetic analyses using Drosophila did not coincide with this result. Expression of RSK strongly suppressed the phenotypes of the constitutively active forms of Ras and Raf which are downstream signaling molecules of Sos, suggesting that RSK-mediated inhibition of the Ras/ERK pathway does not occur through Sos in Drosophila. Moreover, kinase-dead RSK also completely inhibited Ras/ERK-dependent signaling in a similar manner to wild-type RSK, which further undermined the possibility of phosphorylation-dependent inhibition of Sos by RSK in Drosophila eye development (Kim, 2006).
Since the phosphorylation of ERK was thought as a prerequisite for its nuclear entry, it was also determined whether RSK negatively regulates ERK phosphorylation by inducing gene expression of MAP kinase-specific phosphatases (MKP). Interestingly, although RSK dramatically inhibited the nuclear migration of ERK, it did not affect the status of ERK phosphorylation. Rather, direct protein-protein association between RSK and ERK is the essential mechanism to inhibit ERK signaling by RSK, since the mutant forms of RSK defective in binding ERK completely failed to rescue the phenotypes of RSK-null flies and since wild-type RSK failed to suppress the phenotypes of ERKSem (Kim, 2006).
Interestingly, recent reports have demonstrated that ERK enters the nucleus by diffusion in a temperature-dependent manner, which may explain the temperature-sensitive phenotypes of RSKD1 flies. This suggests that the binding partner of ERK is necessary for the tight regulation of the ERK nuclear localization. Since RSK is constitutively cytoplasmic even in the presence of upstream activators such as RasV12, it is very likely that RSK appropriately maintains ERK activity by restraining ERK in a cytoplasmic compartment, which would prevent ERK from activating its nuclear targets. Consistent with this argument, the nuclear entry of activated ERK is dramatically increased by the loss of RSK. Collectively, these studies demonstrate that RSK is a critical negative regulator of ERK in Drosophila by acting as a cytoplasmic anchor (Kim, 2006).
To understand the role of RSK at the organism level, a null mutant was generated for Drosophila RSK, a single orthologue of the mammalian RSK gene family (Wassarman, 1994). Surprisingly, the RSK null flies (RSKD1) that completely lack RSK transcripts and proteins survived to the adult stage. However, RSKD1 mutants displayed several developmental abnormalities including some developmental delay, reduced fertility, and shortened longevity, as well as learning defects (Putz, 2004). Since RSK is ubiquitously expressed throughout all developmental stages and tissues, it is inferred that RSK is involved in general developmental processes that are not essential for the survival of the organism (Kim, 2006).
To find out more about endogenous RSK function, focus was placed on the eye differentiation phenotypes of RSKD1 mutants since these phenotypes are highly correlated with the Ras/ERK pathway. As a result, it was unexpectedly found RSKD1 adult flies have disarrayed eye structure and some ommatidia with increased number of photoreceptor rhabdomeres (R cells). When RSKD1 mutants were grown at a higher temperature, the number of R cells was further increased and other eye defects were also exacerbated. To further examine these phenotypes in earlier developmental stages, the pupal retinal cells were stained with anti-Armadillo (Arm, Drosophila β-catenin) antibodies to mark adherens junctions. A characteristic staining pattern of seven 'dots' forming a circle at the center of each ommatidium was observed in the wild-type eye. However, in RSKD1 mutants, some pupal ommatidia were found aberrantly containing extra anti-Arm antibody-stained dots, consistent with the extra R-cell phenotype of the adult ommatidia in the mutant. These results strongly suggested that RSK plays a negative role in photoreceptor cell differentiation during eye development (Kim, 2006).
Additionally, the RSK-null mutants displayed severe irregularities in ommatidial spacing, presumably reflecting defects in the differentiation of non-neuronal retinal cells such as cone and pigment cells. To analyze the non-neuronal retinal cells, anti-Discs large (Dlg) antibodies were used to stain the membrane structure of cone and pigment cells. In the wild-type pupal retina, the regular arrangement of four cone cells and 11 pigment cells was discernable in each ommatidium. However, RSK-null flies displayed an increased number of cone and pigment cells with highly disrupted structures under the same developmental conditions (34 h after puparium formation). These results strongly supported that RSK regulates not only the neuronal photoreceptor cell differentiation but also the non-neuronal retinal cell development during Drosophila eye morphogenesis (Kim, 2006).
To provide further evidence for this hypothesis, the transallelic mutants of RSKD1 with Df(1)R8A, an RSK deficiency allele, also displayed identical phenotypes to RSKD1 mutants. Furthermore, weak ubiquitous expression of the wild-type RSK (RSKWT) transgene by the da-Gal4 driver fully rescued the eye phenotypes of RSKD1 mutants, demonstrating that the defects in Drosophila retinal cell differentiation indeed resulted from the absence of RSK (Kim, 2006).
Since null mutation of RSK ectopically induced differentiation of both neuronal and non-neuronal retinal cells, RSK was hypothesized to be a general negative regulator of retinal cell fate determination. To further confirm this possibility, RSKWT was expressed by using eye-specific Gal4 drivers. As expected, expression of RSKWT in Drosophila eye by the gmr-Gal4 induced a roughened eye phenotype with dramatically reduced number of photoreceptor cells. Moreover, RSKWT-overexpressing pupal eyes further revealed inhibited differentiation of both neuronal and non-neuronal retinal cells (Kim, 2006).
Collectively, these RSK null and overexpression experiments consistently demonstrated that RSK is a negative regulator of retinal cell differentiation in Drosophila (Kim, 2006).
To address the possibility of the involvement of RSK in differentiation processes other than retinal cell development, other adult structures such as the thorax, abdomen, legs, and wings were carefully observed. Notably, some RSKD1 mutants (~16%) had ectopic veins in various regions of the wing, suggesting that differentiation of wing vein cells is also promoted by RSK null mutation. The number of ectopic veins was significantly increased when the flies were grown at 29°C. Moreover, RSKD1 allele over Df(1)R8A also led to identical wing vein phenotypes to those of RSK-null flies, and weak ubiquitous expression of RSKWT transgene by the da-Gal4 driver completely rescued the ectopic wing vein phenotypes of RSKD1 flies, verifying that the ectopic differentiation of wing vein cells resulted from the absence of RSK (Kim, 2006).
Consistently, overexpression of RSKWT by the e16E-Gal4 (induces gene expression in the posterior part of wing) or the MS1096-Gal4 (induces gene expression in the whole wing) dramatically induced compartment-specific vein-loss phenotypes. In sum, the data demonstrated that RSK negatively regulates vein cell differentiation during Drosophila wing development (Kim, 2006).
Since the Ras/ERK pathway positively regulates the developmental processes of retinal and wing vein cell formation, and because all of the genetic studies consistently suggested RSK acts as a negative regulator of retinal and wing vein cell differentiation, the reliability of the established hypothesis on the downstream role of RSK in the Ras/ERK pathway in Drosophila has become unclear. Moreover, RSK-null embryos frequently displayed partial deletion of the abdominal denticle belts, which is highly similar to those caused by gain-of-function mutation of ERK and Torso. Therefore, it was thought that RSK may have an antagonizing function against the Ras/ERK pathway in Drosophila (Kim, 2006).
To substantiate whether RSK indeed suppresses the Ras/ERK signaling activity in Drosophila, various genetic interaction assays between RSK and the components of the Ras/ERK pathway were performed. First, whether downregulation of Ras/ERK signaling enhances the RSK-overexpression phenotypes was tested. Although the eyes with heterozygotic mutation of Ras, Raf, MEK, or ERK itself showed no change in the number of R cells, overexpression of RSKWT with heterozygotic mutation of Ras, Raf, MEK, or ERK resulted in more severe roughened-eye phenotypes with further decreased number of R cells, when compared to the eyes overexpressing RSKWT in a wild-type genetic background. Next, whether RSK-null mutation enhances gain-of-function phenotypes of the Ras/ERK pathway components was tested. Ectopic expression of constitutively active Ras (RasV12) or Raf (RafF179) cause roughened eye phenotypes with extra R cells in some ommatidia. Interestingly, these phenotypes are further enhanced in heterozygotic or hemyzygotic backgrounds of RSKD1. These two series of experiments consistently suggested that RSK negatively regulates the Ras/ERK pathway (Kim, 2006).
It was next asked whether downregulation of Ras/ERK signaling suppresses the RSK-null phenotypes. Interestingly, RSKD1 phenotypes are significantly suppressed by heterozygotic mutation of Ras or ERK, demonstrating that RSK-null phenotypes result from upregulation of the Ras/ERK pathway. Conversely, it was hypothesized that if RSK-null mutation indeed upregulates the Ras/ERK pathway, it should relieve the phenotypes caused by a low activity in the Ras/ERK signaling pathway. As expected, RSKD1 mutation strongly suppresses the eye roughness and R-cell number decrease phenotypes in a hypomorphic Raf mutant (RafHM7), and even rescues the temperature-sensitive lethality (although RafHM7 hemizygote males are viable at 18°C, they are lethal at 25°C) of RafHM7 mutants at 25°C. Collectively, these results coherently showed that RSK is a negative regulator of the Ras/ERK pathway in vivo (Kim, 2006).
Finally, whether RSK overexpression suppresses the phenotypes caused by the upregulation of the Ras/ERK pathway was tested. Strikingly, the gain-of-function phenotypes induced by a receptor tyrosine kinase (sevs11), Ras (sev>RasV12), and Raf (sev>RafF179) were almost completely suppressed by expression of RSKWT, supporting that RSK inhibits the Ras/ERK pathway at the downstream of these molecules. However, interestingly, RSKWT entirely failed to suppress the gain-of-function phenotypes of ERK (sev>ERKSem), showing the epistatic relationship between RSK and ERK (Kim, 2006).
It was asked how RSK inhibits the Ras/ERK pathway. From genetic results, it was found that RSK is epistatic to hyperactive RTK, Ras, and Raf, but not ERK. Hence, the direct regulation of ERK activity by RSK is presumed, without involving upstream components. Interestingly, mammalian ERK is constitutively associated with RSK in quiescent cells and the key residues of the docking domains of the two proteins play a crucial role in the interaction (mutation in the conserved Asp319 to Asn in the common docking (CD) domain of ERK (Dimitri, 2005) corresponding to ERKSem mutation in Drosophila) or a mutation in the conserved Arg742 (Arg902 in Drosophila RSK) of RSK to Ala in the ERK-docking (D) domain (Roux, 2003) nullified their interaction in mammals). It is predicted that the physical association between ERK and RSK also occurs in Drosophila since their interaction domains are highly conserved and both ERK and RSK are colocalized in the cytoplasm of larval eye discs. Indeed, this hypothesis was confirmed by the fact that RSK physically associates with ERK under overexpressed or endogenous conditions. Moreover, ERKSem (ERKD334N) and RSKR902A mutants failed to bind RSK and ERK, respectively. These results demonstrated that RSK-ERK interaction in Drosophila occurs via key residues in the D domain of RSK and the CD domain of ERK, as in mammals (Kim, 2006).
Since various ERK-binding molecules modulate ERK activity by altering its subcellular localization and because retinal cell fate determination is fully dependent on the nuclear localization of activated ERK in Drosophila, it was asked whether RSK affects the intracellular localization pattern of ERK during Drosophila eye development. Thus, a hemagglutinin (HA)-tagged form of Drosophila ERK was expressed in the eye disc to detect ERK localization using anti-HA antibody. HA-ERK was predominantly distributed in the cytoplasm, but hyperactive Ras strongly induced migration of HA-ERK into the nucleus. Surprisingly, co-expression of RSKWT dramatically suppressed the Ras-induced nuclear localization of HA-ERK, while co-expression of RSKWT/RA completely failed to generate the same result as RSKWT. Consistently, even though RSKWT and RSKWT/RA were expressed at similar levels in the eye and wing, expression of RSKWT/RA could not inhibit retinal and wing vein cell differentiation, showing that RSK-ERK association is essential for the physiological functions of RSK (Kim, 2006).
Next, to determine whether endogenous RSK controls ERK localization, the subcellular localization of HA-ERK was examined in RSK-null mutants. Surprisingly, in RSKD1 mutant eye discs, ERK migrated into the nucleus in a substantial portion of the Gal4-expressing retinal cells unlike those in the control eye discs, showing that endogenous RSK plays a critical role in preventing ERK nuclear localization. To further examine whether endogenous ERK is indeed regulated by RSK, the localization of endogenous ERK was monitored using anti-phospho-ERK (anti-dpERK) antibodies. Although the endogenous ERK activated by the hyperactive Ras migrated into the nucleus, RSKWT overexpression dramatically inhibited this nuclear translocation by retaining dpERK in the cytoplasm without significantly diminishing its concentration, suggesting that RSK-dependent control of ERK localization does not affect the phosphorylation of ERK. Furthermore, it was observed that endogenous dpERK is predominantly cytoplasmic in the morphogenetic furrow of the wild-type eye discs, but RSK-null mutation strongly induced its nuclear translocation. Taken together, it was clearly confirmed that RSK suppresses the nuclear translocation of activated ERK molecules in vivo (Kim, 2006).
With the nuclear migration of ERK being prerequisite for phosphorylation of its nuclear targets such as transcription factors, it was speculated that RSK may downregulate the activities of nuclear ERK targets by preventing ERK nuclear localization. To test this, the expression levels of ERK-dependent genes, rhomboid (rho) and pointed-P1 (pnt-P1), were examined. The transcription of rho can be visualized by anti-β-galactosidase staining using the rho-lacZ reporter. As expected, rho-lacZ expression was dramatically decreased by co-expression of RSKWT in eye discs but not by RSKWT/RA, compared to the control. Consistently, RSK-null flies showed an increased expression of endogenous pnt-P1 transcripts, comparable to the flies overexpressing RasV12 (hs>RasV12). Therefore, it is concluded that RSK reduces the transcription of ERK downstream targets by retaining the ERK protein in the cytosol (Kim, 2006).
Previous mammalian studies indicated that the kinase activity of RSK is critical for the molecule's functions in vivo. However, biochemical and genetic data suggested that RSK does not act as a signal transducer of ERK, but as a spatial regulator of ERK by blocking the nuclear localization of activated ERK in Drosophila. Hence, it was unclear whether the kinase activity of RSK is crucial for its physiological function. To address this issue, transgenic fly lines expressing a kinase-dead mutant of RSK (RSKKR, K231R) were generated as well as a kinase-dead and binding-defective mutant of RSK (RSKKR/RA). The K231R mutation was expected to nullify the kinase activity of RSK by disrupting the conserved lysine residue in the ATP-binding site on the N-terminal kinase (NTK) domain, which is mainly responsible for the phosphorylation of its downstream substrates (Roux, 2003; Woo, 2004). Through immunohistochemical and immunoblot analyses, it was confirmed that RSKKR and RSKKR/RA transgenes are expressed efficiently to a similar level as that of RSKWT and RSKWT/RA. As expected, RSKKR was completely incapable of phosphorylating its substrate (transphosphorylation) as well as the kinase itself (autophosphorylation), while RSKWT was capable of performing both phosphorylating activities, showing that RSKKR is indeed catalytically inactive. However, surprisingly, RSKKR prevented the differentiation of retinal and vein cells as did RSKWT, while RSKKR/RA did not. Moreover, weak transgenic expression of RSKKR using the da-Gal4 driver completely suppressed the ectopic retinal and vein cell differentiation phenotypes of RSKD1 homozygous flies, while RSKKR/RA did not. Furthermore, RSKK231R/K597R (RSKKR/KR) and RSKK231M/K597M (RSKKM/KM) mutants, whose N-terminal kinase and C-terminal kinase activities are completely eliminated, also inhibited retinal and vein cell differentiation, and rescued the RSK-null eye and wing phenotypes. Thus, it was deduced that the ERK-binding activity of RSK, but not the phosphotransferase activity, is essential for the in vivo function of RSK in Drosophila eye and wing development (Kim, 2006).
To further examine whether RSKKR displays an identical molecular function as RSKWT, the same genetic and histological analyses were conducted as performed for RSKWT. Expectedly, RSKKR genetically interacted with various loss-of-function mutants of the Ras/ERK pathway to induce severer roughened-eye phenotypes with dramatically reduced R cells, compared to the eyes overexpressing RSKKR in a wild-type genetic background. Moreover, RSKKR suppressed the gain-of-function phenotypes induced by a receptor tyrosine kinase (sevs11), Ras (sev>RasV12), and Raf (sev>RafF179), but not by ERK (sev>ERKSem). Furthermore, while RSKKR was able to prevent the nuclear localization of HA-ERK or endogenous dpERK and to suppress the transcriptional induction of rho, RSKKR/RA was neither able to suppress the nuclear localization of HA-ERK or endogenous dpERK nor able to reduce the expression of rho. These results consistently demonstrated that kinase-dead RSK functions through a similar molecular mechanism with wild-type RSK during eye development. Collectively, it is concluded that RSK negatively regulates Ras/ERK-dependent eye differentiation via direct physical association with ERK in a kinase-independent manner (Kim, 2006).
The insulin/insulin-like growth factor-1 signaling pathway promotes growth in invertebrates and vertebrates by increasing the levels of phosphatidylinositol 3,4,5-triphosphate through the activation of p110 phosphatidylinositol 3-kinase. Two key effectors of this pathway are the phosphoinositide-dependent protein kinase 1 (PDK1) and Akt/PKB. Although genetic analysis in C. elegans has implicated Akt as the only relevant PDK1 substrate, cell culture studies have suggested that PDK1 has additional targets. In Drosophila, dPDK1 controls cellular and organism growth by activating Akt1 and S6 kinase, dS6K. Furthermore, dPDK1 genetically interacts with dRSK but not with dPKN (FlyBase name: Protein kinase related to protein kinase N), encoding two substrates of PDK1 in vitro. Thus, the results suggest that dPDK1 is required for dRSK but not dPKN activation and that it regulates insulin-mediated growth through two main effector branches, dAkt and dS6K (Rintelen, 2001).
The pronounced effect of loss of dPDK1 function on head size suggests that it is a dominant constituent in the dInr pathway. To test this possibility, the ability of complete and partial loss-of-function alleles of dPDK1 to reverse phenotypes caused by either overexpression of dInr or by mutations in dPTEN, the 3-phosphatidylinositide phosphatase, was evaluated. Overexpression of a wild-type dInr cDNA under the control of GMR-Gal4 leads to a marked increase in eye size and a slightly rough eye surface, an effect dominantly suppressed by removing one copy of dPDK1. Further reduction of dPDK1 function by the dPDK11/4 heteroallelic combination reduces the eye to almost wild-type size, suggesting that the amount of dPDK1 protein is rate-limiting for the dInr overgrowth phenotype. Null mutations in dPTEN cause lethality, and removal of dPTEN function in clones stimulates cell autonomous growth, suggesting that increased levels of PIP3 promote growth and are the likely cause of lethality. Thus, if dPDK1 is an essential target of PIP3, mutations in dPDK1 may suppress the dPTEN phenotype. Surprisingly, some dPTEN/dPDK1 double mutant flies survive to adulthood, indicating that the presumed PIP3-induced lethality is primarily caused by the hyperactivation of dPDK1 or of one of its targets (Rintelen, 2001).
These results show that dPDK1 is an essential component in the insulin signaling pathway in the control of cell growth and body size through its two substrates, dAkt and dS6K. These results are distinct from the genetic evidence in C. elegans where Akt is the primary target of PDK1 in dauer formation. Because mutations in the insulin signaling pathway do not show an autonomous alteration of cell size in C. elegans, the regulation of the rate of protein synthesis through S6K does not seem to be a primary target of this pathway. However, the fact that dPDK1 may yet have additional substrates is suggested by the genetic interaction with dRSK gain-of-function mutations and because viable dPDK1 males are almost completely sterile. Although mutations in components of the insulin signaling pathway such as dInr, chico, Dp110/PI(3)K, and dAkt cause female sterility, male sterility is not observed. Further genetic dissection of dPDK1 function is required to determine the role of dPDK1 in male fertility. These findings in Drosophila are consistent with the absence of insulin growth factor-1-induced activation of S6K, Akt, and RSK in mammalian PDK1-/- embryonic stem cells, and therefore provide evidence for the functional conservation of branch points in kinase networks during evolution (Rintelen, 2001).
In an attempt to dissect classical and operant conditioning in Drosophila melanogaster, the gene for ribosomal S6 kinase II (S6KII) was isolated. This enzyme is part of a family of serine-threonine kinases that in mammals have been implicated in the MAPK (mitogen-activated protein kinase) signaling cascade controlling (among other processes) synaptic plasticity (long-term potentiation/long-term depression) and memory formation. The human homolog rsk2 has been linked to mental retardation (Coffin-Lowry syndrome). Mutant analysis in Drosophila shows that S6KII serves different functions in operant place learning and classical (pavlovian) olfactory conditioning. Whereas in the null mutant only pavlovian olfactory learning is affected, a P-element insertion mutant reducing the amount of S6KII only affects operant place learning. A mutant lacking part of the N-terminal kinase domain and performing poorly in both learning tasks is dominant in the operant paradigm and recessive in the pavlovian paradigm. The behavioral defects in the pavlovian task can be rescued by the genomic S6KII transgene. Overexpression of S6KII in wild type has a dominant-negative effect on the operant task that is rescued by the null mutant, whereas in the pavlovian task overexpression may even enhance learning performance (Putz, 2004; full text of article).
Operant place learning in the heat box was used to screen 1221 homozygous viable, X-chromosomal P-element insertion (P[lacW]) lines. Flies were trained to avoid part of an elongated, narrow chamber that was quickly heated from 20 to 40°C if the fly entered this part. In one line called ignorantP1 (ignP1), males showed reduced performance during the training (tr) and in the subsequent memory test (te). No significant defect was observed in females on any genetic background (against w1118 on a wild-type Berlin background. After recombining the chromosomal region carrying the P-element onto a WT-CS X chromosome carrying the w+ gene and extensive additional backcrossing of the autosomes to WT-CS, a robust male-specific defect during the training phase remained, whereas performance in the memory test was close to normal in both genders. Thermo-sensitivity was not impaired in ignP1 mutant males. Because the memory phenotype of ignP1 seemed to be under polygenic control and was not apparent in the WT-CS genetic background, it was decided to refer only to the performance deficit during training (Putz, 2004).
Sequencing of DNA flanking the P-element in the ignP1 line showed that it was inserted in the 5' untranslated region of the first exon of the gene coding for S6KII (CG17596). The P-element insertion is located 355 bp in front of the predicted translational start site. Sequencing of expressed-sequence tag clones SD05277, GH08264, and GH21818 confirmed Flybase information about the structure of S6KII. The P-element insertion is located at position 27/28 of clone SD05277. To test whether the P-element insertion in ignP1 flies is responsible for the behavioral defect, and second, to obtain additional S6KII mutants, P-element excision lines of ignP1 flies were generated. The P-element was remobilized crossing ignP1 to a source of transposase. Based on the loss of the mini-w+ marker of P[lacW], 128 jump-out lines were obtained from F1 females and 227 from F1 males. In a first screen of jump-outs in females, 13 lines were found by Southern blot to be candidate precise jump-out lines. Two lines, ignDelta1P1 and ignDelta2P1, restored wild-type sequence at the P-element insertion site. Line ignDelta1P1 does not show any nucleotide changes close to this site, whereas line ignDelta2P1 has several nucleotide changes surrounding the P-element insertion site in the untranslated region. The remaining jump-out lines were screened by PCR, and 13 of them contained deletions larger than 1 kb. Six of them were characterized in more detail. Sequencing of the complete region revealed that they had lost part or all of the S6KII gene. Deletion line Df(1)ignDelta24-3 had a loss of 1322 bp of genomic sequence in the 5' region of the S6KII gene, removing part of the first exon. In a similar mutant, Df(1)ignDelta30-2, 2197 bp were deleted. In excision lines Df(1)ignDelta67-1 and Df(1)ignDelta58-1, the coding region of S6KII was completely removed. Lines Df(1)ignDelta24-3, Df(1)ignDelta30-2, and Df(1)ignDelta58-1 still had retained part of the P-element, ranging from 16 bp to 5.3 kb. The largest of the deletions, Df(1)ignDelta67-1, removed the entire S6KII gene, the P-element, and a neighboring gene encoding a serine-threonine phosphatase (CG17598). All deletion lines were homozygous viable. The name ignorant (ign) was retained (as a synonym of S6KII) for all mutants derived from ignP1 (Putz, 2004).
The Drosophila S6KII is, perhaps surprisingly, encoded by a non-essential gene. Null mutants are viable and normal by morphological criteria and casual behavioral observation. Also, brain structure seems not to be severely affected. As the only apparent defect, Df(1)ignDelta58-1 shows a 40% reduced memory score in pavlovian olfactory learning. Regarding this memory component, the properties of the S6KII mutants are in line with what one might expect from S6KII in vertebrates (Bjorbaek, 1995; Nebreda, 1999). If it is the kinase domain in the N-terminal part of the enzyme that is phosphorylating target proteins, deletion of that domain should abolish phosphorylation. Indeed, in the mutant Df(1)ignDelta24-3, lacking part of the N-terminal kinase domain olfactory memory is reduced as much as in the null mutant [Df(1)ignDelta58-1]. The small and the large deletions are recessive, indicating that 50% S6KII enzymatic activity in the heterozygote is sufficient to provide the full function. In the mutant ignP1, again the reduced overall amount of protein is enough to allow for near to normal learning/memory. Also, the transgene in the genetic background of the null mutant or the partial deletion Df(1)ignDelta24-3 provides enough enzyme to fully support the behavioral task. Finally, overexpression of S6KII as a transgene has no deleterious effect. If at all, it enhances memory (Putz, 2004).
The involvement of the S6KII gene in operant place learning could hardly be more different. In wild-type Drosophila, S6KII is not essential for place learning, because removing the whole gene has no apparent phenotype. An influence of the S6KII gene on heat-box learning is seen only in mutants that leave the gene or part of it intact. The P insertion, the N-terminally truncated gene, and the S6KII transgenes all reduce learning performance. In the P-insertion mutant, the learning defect seems not to be related to the overall amount of S6KII because females learn well and show the same reduced amount of S6KII-like immunoreactivity as males. The heat-box phenotypes need to be studied in detail to find out whether the three kinds of mutations (P insertion, partial deletions, overexpression) interfere with heat-box learning in the same manner. Whether S6KII exerts its various effects in adulthood or during development can now be addressed using conditional expression systems (Putz, 2004).
The S6KII gene is nested in a large intron of a putative gene (CG17602) of unknown function. It should be noted that the mutant phenotypes cannot be assigned to this nearby gene because the genomic transgenes do not contain CG17602 and are inserted far away from the original genomic location. Their phenotypes correspond to and even interact with those of the deficiency mutants (Putz, 2004).
Assuming that it is at the protein level in which S6KII exerts its deleterious effect on heat-box learning in the mutants, a smaller protein must be synthesized from the C-terminal part of the S6KII gene in the small-deletion mutants. If this is true for the small deletions, it may well be true also for the wild type. This hypothetical small isoform might be mostly suppressed by the large isoform in wild type. Kinases with a large and a small isoform are not uncommon, e.g., in the protein kinase C family. It is too early to speculate about mechanisms before a small isoform is found (Putz, 2004).
Both olfactory learning and place learning require cAMP signaling. Because it is now well established that the memory trace for olfactory learning is localized in sets of intrinsic neurons (Kenyon cells) of the mushroom bodies, it will be of interest to determine, by the same reconstitution approach, whether the molecular functions of S6KII documented here colocalize in the same cells. For operant place learning, the localization of the memory trace is not as clear cut as in olfactory learning because several groups of neurons are still candidate locations for the memory trace. However, using the reconstitution strategy, it can be determined whether the same GAL4 lines that rescue in the case of rutabaga also do so with S6KII-cDNA in Df(1)ignDelta58-1 (Putz, 2004).
Current schemes for vertebrates place cAMP upstream of S6KII in the same signaling pathway (Impey, 1999). This may hold true in Drosophila for pavlovian olfactory conditioning, whereas for operant place learning, S6KII may not lie in the direct pathway (because it is dispensable) but rather in a side branch. With the phenotypes of the mutants and overexpression lines, one is inclined to assume that a close interaction partner or a protein similar to S6KII should be directly involved in place learning. For instance, the signaling pathways for the two learning tasks might diverge at the level of the MAPK that could be blocked by an excess of S6KII and, in particular, by the hypothetical small C-terminal isoform of S6KII. Recent studies in Xenopus oocytes already showed that an N-terminally truncated RSK mutant can constitutively interact with ERK (Gavin, 1999; Biondi, 2003) (Putz, 2004).
This assessment of S6KII function in Drosophila has focused on learning and memory. This does not exclude that the gene might be involved in other important processes as well. Also, future work will have to show whether the differential effects of the mutants on operant and classical conditioning can be generalized to other operant and classical learning tasks. The study, therefore, cannot claim to have shown that operant and classical associative processes require different biochemical mechanisms. Yet, the strikingly different mutant phenotypes of Drosophila S6KII in operant place learning and pavlovian olfactory conditioning may help to address this question (Putz, 2004).
Ribosomal S6 kinases (RSKs) are growth factor-regulated serine-threonine kinases participating in the RAS-ERK signaling pathway. RSKs have been implicated in memory formation in mammals and flies. To characterize the function of RSK at the synapse level, the effect was investigated of mutations in the rsk gene on the neuromuscular junction (NMJ) in Drosophila larvae. Immunostaining revealed transgenic expressed RSK in presynaptic regions. In mutants with a full deletion or an N-terminal partial deletion of rsk, an increased bouton number was found. Restoring the wild-type rsk function in the null mutant with a genomic rescue construct reverted the synaptic phenotype, and overexpression of the rsk-cDNA in motoneurons reduced bouton numbers. Based on previous observations that RSK interacts with the Drosophila ERK homologue Rolled, genetic epistasis experiments were performed with loss- and gain-of-function mutations in Rolled. These experiments provided evidence that RSK mediates its negative effect on bouton formation at the Drosophila NMJ by inhibition of ERK signaling (Fischer, 2009).
This study investigated the effect of rsk loss of function mutations in Drosophila, and found higher numbers of synaptic boutons in these mutants. The effect could be rescued by transgenic rsk expression. Vice versa overexpression of RSK reduced bouton numbers. Furthermore, removal of one allele of the Drosophila erk/rl gene normalized the effect of rsk loss of function on bouton formation, indicating that RSK mediates its effect through ERK/RL. Indeed, RSK and ERK/RL proteins interact directly with each other, and this interaction is abolished in the rlSem mutant. Furthermore, rlSem mutants show enhanced bouton numbers, similarly as rsk mutants, indicating that RSK negatively regulates ERK/RL activity at the NMJ and thus modulates bouton formation (Fischer, 2009).
A role of vertebrate RSK2 in inhibition of the RAS/ERK pathway has been proposed in several studies, but different underlying mechanisms have been suggested. In isolated mouse motoneurons, RSK2 is a negative regulator of axon growth by inhibiting ERK phosphorylation. In skeletal muscles of RSK2 knock-out mice, increased ERK activation has been observed. This could be explained by lack of inhibition of the ERK pathway via RAS guanine exchange factor SOS. In Drosophila, this inhibition seems not to occur through SOS. Knockdown of RSK2 leads to increased ERK phosphorylation in PC12 cells and cortical neurons. Moreover basal and 5HT2A receptor-mediated ERK 1/2 phosphorylation is increased in RSK2 knock-out fibroblasts These data are consistent with the current results showing that RSK interacts with ERK/RL and that this interaction leads to inhibition of ERK/RL activity in bouton formation at the NMJ (Fischer, 2009).
Previous studies on RSK and RL in the developing eye and wing imaginal disc provided evidence that RSK inhibits translocation of ERK/RL from the cytoplasm to the nucleus and thereby controls RL dependent gene transcription. However, the NMJ constitutes a separate part of the cell, and it is also conceivable that the effects of RSK and RL are mediated locally and do not involve nuclear translocation of these proteins. RSK seems to be present in the presynapse, but its distribution is diffuse and not restricted to active zones. This corresponds to the known distribution of RL in axon terminals. Thus, it is possible that RSK determines the localization of RL within synaptic boutons. Interestingly, an antibody that only recognizes active, phosphorylated RL showed a restricted localization to spots most likely corresponding to active zones. Thus one could speculate that RSK binds ERK/RL in axon terminals, thus inhibiting its activation, and only ERK/RL that is unbound can be activated by phosphorylation and move to active zones (Fischer, 2009).
In conclusion, these data indicate that RSK negatively regulates bouton formation at the NMJ, and that negative regulation of RL signaling is involved in this effect. Thus, Drosophila RSK seems to have a similar function as the RSK2 isoform in vertebrates. Therefore, the memory defects observed in flies, mice, and human CLS patients with mutations in rsk could be caused by dysregulated synapse architecture, as observed in the Drosophila model (Fischer, 2009).
A detailed structure/function analysis of Drosophila p90 ribosomal S6 kinase (S6KII) or its mammalian homolog RSK has not been performed in the context of neuronal plasticity or behavior. A previous study has reported that S6KII is required for normal circadian periodicity. This study reports a site-directed mutagenesis of S6KII and analysis of mutants, in vivo, that identifies functional domains and phosphorylation sites critical for the regulation of circadian period. A role is demonstrated for the S6KII C-terminal kinase that is independent of its known role in activation of the N-terminal kinase. Both S6KII C-terminal kinase activity and its ERK-binding domain are required for wild-type circadian period and normal phosphorylation status of the protein. In contrast, the N-terminal kinase of S6KII is dispensable for modulation of circadian period and normal phosphorylation of the protein. Particular sites of S6KII phosphorylation, Ser-515 and Thr-732, are essential for normal circadian behavior. Surprisingly, the phosphorylation of S6KII residues, in vivo, does not follow a strict sequential pattern, as implied by certain cell-based studies of mammalian RSK protein (Tangredi, 2012).
p90 ribosomal S6 kinase (RSK) is a well-conserved serine/threonine kinase and a target of the Ras-mitogen-activated protein kinase (MAPK)/extracellular-signal-regulated kinase (ERK) signaling cascade (Frodin, 1999). RSK was first identified as a kinase that phosphorylates the 40 S ribosomal subunit protein S6 in Xenopus larval extracts, although it has since been determined that S6 protein may not be a relevant substrate in vivo. As with other elements of the Ras-ERK pathway, RSK kinase activity has been linked to a wide range of critical cellular processes including transcriptional and translational regulation, cell proliferation, survival, growth, and cell motility (Tangredi, 2012).
RSK is unique among serine/threonine kinases in that it contains two distinct and functional kinase domains joined by a linker region. The kinases are thought to be sequentially activated by a series of phosphorylation events, beginning with phosphorylation by activated ERK. However, recent studies indicate that kinase activation may occur in the absence of this full sequential series of modifications (Cohen, 2007; Richards, 2001). As the majority of these studies were conducted in vitro, there is insufficient evidence as to whether sequential phosphorylation/kinase activation is required in vivo. The sole known purpose of the RSK carboxyl (C)-terminal kinase is to activate the amino (N)-terminal kinase through autophosphorylation. In contrast, the N-terminal kinase is responsible for phosphorylation of other protein substrates. N-terminal kinase activity was thought to be essential for RSK function until a recent study demonstrated an alternative role for RSK in fly eye development as a non-catalytic, scaffolding protein (Kim, 2006; Tangredi, 2012 and references therein).
The mammalian genome encodes four isoforms of RSK (RSK 1-4) while only a single isoform has been described for Drosophila melanogaster (dRSK or S6KII) that has ∼60% amino acid identity with RSK1 (Wassarman, 1994). Whereas there have been extensive studies of RSK structure and function using mammalian cell-based assays, detailed studies of fly S6KII functional domains have not been reported even though the kinase is known to be important for memory functions and circadian behavior (Akten, 2009; Neuser, 2008; Putz, 2004). The use of Drosophila for such an analysis will permit critical domains and phosphorylation sites of S6KII to be delineated, in vivo, in the context of behavior and plasticity, with obvious relevance for mammalian RSK function (Tangredi, 2012).
Even though S6KII is ubiquitously expressed throughout Drosophila development and in all embryonic tissues (Kim, 2006), S6KII null mutant flies are viable (Kim, 2006; Putz, 2004). Interestingly, S6KII null (S6KIIignorant or S6KIIign) flies exhibit circadian molecular and behavioral phenotypes (Akten, 2009). Specifically, the mutant exhibits a short-period phenotype that appears to result from alteration of the Period (Per)-based circadian oscillator. Based on those and other studies, a role has been proposed for S6KII in modulating the Drosophila circadian molecular oscillator that involves interaction and cooperation with the known clock kinase casein kinase 2 (CK2) (Akten, 2009). Given that S6KII also interacts with numerous other partners in a variety of ERK pathway roles, it is possible that S6KII modulation of oscillator function is controlled by ERK signaling. In addition, it is not known whether S6KII serves as a kinase or alternatively as a scaffolding protein in the circadian system. Finally, it would be of interest to discover whether the sequence of RSK phosphorylation and kinase activation observed in mammals is relevant, in vivo, for S6KII modulation of fly circadian behavior (Tangredi, 2012).
To further understand the role of S6KII in the circadian system, this study set out to evaluate the importance of conserved protein domains and phosphorylation sites for kinase regulation of locomotor activity rhythms. It is reported that S6KII's circadian function does not require its N-terminal kinase activity, similar to findings reported for Drosophila eye development (Kim, 2006). In contrast, C-terminal kinase activity, previously thought to be responsible only for N-terminal kinase activation, is required for normal circadian behavior. These studies also suggest that ERK binding to and phosphorylation of S6KII threonine 732 (T732) within clock neurons is essential for normal rhythmicity. Whereas S6KII was shown to negatively regulate ERK in the fly eye (Kim, 2006) and at the neuromuscular junction (Fischer, 2008), this work indicates that activation of S6KII by ERK is required for modulation of the circadian clock. Further, it is show that both ERK binding and C-terminal kinase activity are important for autophosphorylation of S6KII serine 515 (S515) and T732 phosphorylation, whereas phosphorylation at S357, which activates the N-terminal kinase, is not dependent on these activities. Phosphorylation of S6KII S515 or T732 is not required for normal phosphorylation of the protein, but it is required for wild-type circadian behavior. These studies provide novel insights about the function of S6KII, in vivo, and support a model in which ERK regulates S6KII in clock cells, thereby, modulating circadian behavior (Tangredi, 2012).
This study utilized wild-type and mutant forms of S6KII in genetic rescue experiments to identify domains that are critical for the protein's function in circadian behavior. This is the first study to identify domains of S6KII (RSK) that are required, in vivo, for a behavioral function. Although in many cases S6KII isoforms were expressed at higher than normal levels in transgenic flies, it is not thought that results can be attributed to overexpression of the protein. Expression of wild-type S6KII at high levels has no discernible effects on circadian behavior or the phosphorylation pattern of S6KII. For example high level expression of a C-terminal kinase-dead mutant (S6KIIKm) does not rescue behavior nor phosphorylation defects observed at several sites including S357, a postulated PDK1 docking site within the N kinase domain. However there may be effects of S6KII overexpression that are not discernible in these molecular and behavioral assays (Tangredi, 2012).
In agreement with a previous study of fly development, this study shows that the S6KII N-terminal kinase is dispensable for its circadian function. This result contrasts with previous studies showing that RSK functions in the Ras/MAPK pathway as a kinase; it suggests that phosphorylation of downstream targets by the N-terminal kinase is not essential for modulation of the circadian clock. In support of a non-critical role for the N-terminal kinase, certain mutants that fail to rescue behavior nonetheless exhibit phosphorylation of S357, an event thought to activate RSK kinase activity. In addition, an S6KII mutant (S6KIIignΔ24−3) missing a large portion of the N-terminal region, including the N-terminal kinase, has been shown to partially rescue an S6KII-null mutant (Tangredi, 2012).
In contrast, the current studies emphasize the importance of S6KII C-terminal kinase activity for modulation of the Drosophila circadian clock. This is the first evidence, in either vertebrate or invertebrate systems, of a function for the S6KII C-terminal kinase that is independent of activation of the N-terminal kinase. It is also the first direct link between the C-terminal kinase and behavior. Heretofore, the only known function of the RSK C-terminal kinase was autophosphorylation, which leads to activation of the N-terminal kinase. The results suggest that either autophosphorylation serves an independent purpose (such as altering protein-protein interactions) or that the C-terminal kinase phosphorylates other proteins (Tangredi, 2012).
The C-terminal kinase was shown to promote phosphorylation of S515, a presumed autophosphorylation site and a residue within the hydrophobic motif site of AGC-type kinases. This region is important for stabilization of the catalytic domain of such kinases, including RSK, cAMP-dependent kinase and protein kinase C. It is noted that there is residual pS515 signal in a S6KII C-terminal kinase-dead mutant (S6KIIK597M) which may indicate that other kinases phosphorylate the site or that the mutant retains an undetectable amount of activity (Tangredi, 2012).
The work also suggests that S6KII S357 and T732 phosphorylation events are modulated by the C-terminal kinase. The N-terminal kinase is dispensable for circadian regulation, but nevertheless the data suggests that it is activated by the C-terminal kinase via phosphorylation of S357; in agreement with cell-based studies of RSK. The C-terminal kinase may promote S357 phosphorylation through recruitment of PDK1 or another factor. While the mechanism for C-terminal kinase modulation of T732 phosphorylation is unknown, it is possible that kinase activity that is stimulated by ERK binding feeds back to activate ERK phosphorylation of T732. This and other alterations may also involve the actions of phosphatases as there is undoubtedly a dynamic interplay between the two types of modifying enzymes. Of interest, pS515 levels are not altered in the T732A/T732E mutants, indicating that T732 phosphorylation is not a prerequisite for S515 phosphorylation (Tangredi, 2012).
There is a positive correlation between S6KII variants that rescue S6KIIign mutant behavior and robust phosphorylation of S515; this suggests that phosphorylation of this residue is essential for normal circadian behavior. It is noted, however, that while S515 phosphorylation is correlated with rhythmicity, it is not sufficient for normal circadian behavior. Therefore, pS515 may simply be indicative of a functional C-terminal kinase whose kinase activity is necessary for modulating circadian behavior via phosphorylation of other unknown targets. In addition, phosphorylation of this residue does not affect the phosphorylation of other S6KII domains; instead C-terminal kinase activity and modification of S515 may serve to alter S6KII protein conformation and relevant protein-protein interactions. While this study shows that C-terminal kinase activity is important for rhythmicity, the experiments do not exclude the idea that S6KII functions as a scaffold in the circadian system, analogous to its role in Drosophila eye and wing development (Tangredi, 2012).
ERK binding to S6KII is required for transgenic rescue of circadian behavior, as it is for rescue of Drosophila eye development phenotypes. Consistent with a role for ERK in this pathway, it was shown that phosphorylation of S6KII at T732 (a known ERK phosphorylation site on RSK) is required for rescue of behavioral rhythms. ERK phosphorylation of T732, previously demonstrated for RSK in mammalian cell-based assays, was verified in the fly by the observation that pT732 is reduced in ERK binding-deficient mutants. ERK binding may promote S6KII function by facilitating activation of the C-terminal kinase, as evidenced by the decreased autophosphorylation of ERK-binding mutants. Alternatively, ERK binding may alter S6KII localization and/or binding to clock-related proteins such as CK2, similar to S6KII's regulation of ERK in fly eye development. Whatever the precise mechanism, phosphorylation of T732 and S515 are likely to be important for ERK's interaction with S6KII and the regulation of circadian period (Tangredi, 2012).
It was demonstrated in fly photoreceptor cells that S6KII negatively regulates ERK by retaining it in the cytoplasm. Using immunostaining procedures, however, this study has shown that the localization pattern of ERK and diphosphorylated (activated) ERK are the same within PDF clock neurons (primarily cytoplasmic) in wild-type flies (w1118), S6KIIign-null mutants, and ERK-binding mutants (S6KIIign;timUG4>S6KIIR902A). Hence, ERK may bind to and activate S6KII in clock cells, but there is no evidence that S6KII regulates ERK localization in this cell type (Tangredi, 2012).
RSK protein is thought to be activated by a sequence of protein binding and phosphorylation events, based on cell-based investigations of the protein. More recent cell-based assays question the validity of this mode and give added relevance to the current studies as the first to examine this model in vivo (Tangredi, 2012).
This study provides the first evidence that phosphorylation/activation of Drosophila S6KII can occur in the absence of a strict sequence of binding and phosphorylation events, but it is noted that there is some dependence of certain events on others. Although there is extremely low pS515 immunoreactivity in C-terminal kinase and ERK-binding mutants, indicating that S515 phosphorylation is a downstream event, there is residual phospho-signal on this residue in such flies. Thus, ERK binding and C-terminal kinase activation may not be the only events contributing to S515 phosphorylation. Consistent with this idea, in vitro analysis of mammalian RSK has demonstrated that S380 phosphorylation (S515 in S6KII) and C-terminal kinase activation can occur in the absence of RSK-ERK interactions. Similarly, ERK binding and C-terminal kinase activity are not the only contributors to S6KII T732 phosphorylation because residual pT732 signal exists in mutants lacking these functions. The current results also indicate that neither ERK binding nor phosphorylation at S515 or T732 is essential for phosphorylation S357 although C-terminal kinase activity influences this event. This result is in agreement with mammalian cell-based studies demonstrating that N-terminal kinase activation is not fully dependent upon C-terminal kinase activity. Altogether, ERK-binding and C-terminal catalytic activity appear to play an important role in regulating phosphorylation of the S6KII protein, but the phosphorylation of individual sites is not absolutely required for the downstream phosphorylation of others (Tangredi, 2012).
Previous work indicated that S6KII modulates circadian function by negatively regulating the activity of the clock kinase CK2, via physical interaction with the CK2β subunit (Akten, 2009). Thus, it is possible that a prerequisite for the S6KII-CK2 interaction is activation of the S6KII C-terminal kinase or a conformational change in the protein resulting from ERK binding. CK2β may be a phosphorylation target of the S6KII C-terminal kinase (although there is no evidence of this), and this would provide a mechanism by which S6KII could regulate CK2 activity. Alternatively, a change in S6KII conformation might regulate interaction with CK2, thus modulating the previously documented effects of the kinases on the PER-based clock. Finally, the possibility exists that S6KII regulates circadian clock function through a CK2-independent pathway. Further analysis of the S6KII binding partners and substrates may yield insights about the precise role of the C-terminal kinase and ERK-binding domains in circadian regulation (Tangredi, 2012).
Orientation and navigation in a complex environment requires path planning and recall to exert goal-driven behavior. Walking Drosophila flies possess a visual orientation memory for attractive targets which is localized in the central complex of the adult brain. This study shows that this type of working memory requires the cGMP-dependent protein kinase encoded by the foraging gene in just one type of ellipsoid-body ring neurons. Moreover, genetic and epistatic interaction studies provide evidence that Foraging functions upstream of the Ignorant Ribosomal-S6 Kinase 2, thus revealing a novel neuronal signaling pathway necessary for this type of memory in Drosophila (Kuntz, 2012).
For signaling has previously been implicated in different types of memories; however, in contrast to the working memory in the detour paradigm, these memories require a longer time frame to be established. In mammals, nitric oxide, the initiating molecule of the cGMP/PKG-pathway, is thought to act as a retrograde messenger during the induction of long-term potentiation (LTP). A LTP enhancement has been reported after adding PKG activators and a long-term depression after the addition of PKG inhibitors. Mice carrying a knock-out for the Pkg gene show reduced ability of motor learning due to a loss of synaptic plasticity in the cerebellum. Furthermore, mice lacking Pkg in the amygdala exhibit an impairment in fear conditioning and cGMP/PKG signaling in the hippocampus is required for novel object recognition. In insects, For is involved in different types of food searching behavior and associative memories in which establishing the learning traces takes at least seconds. In contrast, the orientation memory observed in the detour paradigm presented in this study represents a form of working memory which has to be updated continuously in fractions of seconds. Whereas the phosphorylation and activation of For and Ignorant might be the mechanism by which these kinases affect longer-lasting memories, it is thought unlikely that this mechanism is involved in the constantly and rapidly changing orientation memory. Both kinases would have to be activated or inactivated in an online fashion during every turn of the fly. On the other hand, RSK2 has been implicated in multiple cellular processes and transcriptional control. It is therefore speculated that the biochemical pathway both kinases work in is necessary to endow the ring neurons with the capacity to efficiently change signaling rapidly to encode orientation. For instance, ring neurons might need a higher density of synaptic release sites and/or dendritic neurotransmitter receptors to exert their specific function (Kuntz, 2012).
Plastic changes in synaptic properties are considered as fundamental for adaptive behaviors. Extracellular-signal-regulated kinase (ERK)-mediated signaling has been implicated in regulation of synaptic plasticity. Ribosomal S6 kinase 2 (RSK2) acts as a regulator and downstream effector of ERK. In the brain, RSK2 is predominantly expressed in regions required for learning and memory. Loss-of-function mutations in human RSK2 cause Coffin-Lowry syndrome, which is characterized by severe mental retardation and low IQ scores in affected males. Knockout of RSK2 in mice or the RSK ortholog in Drosophila results in a variety of learning and memory defects. However, overall brain structure in these animals is not affected, leaving open the question of the pathophysiological consequences. Using the fly neuromuscular system as a model for excitatory glutamatergic synapses, this study shows that removal of RSK function causes distinct defects in motoneurons and at the neuromuscular junction. Based on histochemical and electrophysiological analyses, it was found that RSK is required for normal synaptic morphology and function. Furthermore, loss of RSK function interferes with ERK signaling at different levels. Elevated ERK activity is evident in the somata of motoneurons, whereas decreased ERK activity is observed in axons and the presynapse. In addition, a novel function of RSK in anterograde axonal transport was uncovered. These results emphasize the importance of fine-tuning ERK activity in neuronal processes underlying higher brain functions. In this context, RSK acts as a modulator of ERK signaling (Beck, 2015).
One emerging common picture from studies in animal models for CLS is that loss of RSK2 function in neurons is associated with deregulation of ERK signaling and synaptic properties. In general, ERK signaling is not only required for cell proliferation, differentiation and survival, but also for synaptic plasticity and memory. The molecular functions of RSK proteins as an interaction partner of ERK proteins are discussed ambivalently in the literature. On the one hand, RSK2 mediates ERK signaling by phosphorylation of numerous targets; on the other hand, it is described as a negative regulator of ERK. This ambivalent picture is also reflected by genetic interaction experiments between RSK and ERK mutants, which do not provide a conclusive answer about the relationship between RSK and ERK with respect to pre- and postsynaptic functions. Further complexity is added because subcellular localization of pERK is changed in RSKΔ58/1 motoneurons, with elevated pERK levels in the somata and strongly decreased levels at the NMJ. Thus, even in a single cell, opposing effects with respect to ERK targets in different subcellular compartments can be expected (Beck, 2015).
These results coincide at several points with findings in the vertebrate nervous system. Elevated pERK levels have been observed in the hippocampus of RSK2 knockout mice, resulting in deregulation of ERK-mediated gene transcription. For instance, transcription of the Gria2 gene encoding the GLUR2 subunit of the AMPA receptor is upregulated. Nevertheless, electrophysiological, biochemical and ultrastructural analyses carried out with isolated cortical neurons and in the hippocampus reveals impaired AMPA-receptor-mediated synaptic transmission. This can be explained, at least in part, by the requirement of RSK2 for phosphorylation of postsynaptic PDZ [post synaptic density protein-95 (PSD95), discs large 1 (DLG1), zonula occludens-1 (ZO1)] domain-containing proteins to regulate channel properties. Morphological and electrophysiological data at the NMJ are also consistent with a postsynaptic requirement of RSK for synaptic transmission. In addition, RSK mutants display a number of defects in the presynaptic motoneuron, including upregulation and relocalization of pERK and a reduction in active zone numbers. How do these phenotypes relate to known functions of ERK in Drosophila motoneurons? First, alterations in ERK activity at the NMJ are inversely correlated with levels of the neural cell adhesion molecule Fasciclin II. Given that Fasciclin II has been found to be excluded from pERK-positive spots at synapses, a direct regulatory mechanism at the protein level seems plausible. Thus, it is conceivable that synaptic RSK contributes to Fasciclin II-mediated cell adhesion either directly, by acting as an upstream kinase, or by feedback inhibition of ERK activity. Second, besides RSK, the serine-threonine kinase UNC-51 also acts as a negative regulator of ERK in motoneurons. It could therefore be expected that RSK and UNC-51 mutations display similar synaptic phenotypes. Indeed, NMJ size, number of active zones and eEPSC amplitudes are decreased in both mutants. Interestingly, transgenic rescue experiments for the electrophysiological defects fail to work in both mutants, emphasizing the importance of fine-tuning ERK activity for maintaining normal synaptic functions. However, there are also significant differences between the two mutants. In general, UNC-51 phenotypes are much more pronounced. In the UNC-51 mutant, many postsynaptic GluRs are unapposed to presynaptic BRP, a phenotype that was not observed in the case of loss of RSK. Both mutants show a decrease in eEPSC amplitude, but although this is attributable to defective transmitter release at UNC-51 mutant synapses, no such presynaptic defect is observed in RSK mutants. Instead, the reduced mini amplitude at RSK mutant synapses indicates impaired postsynaptic sensitivity, which in turn is unaltered in UNC-51 mutants. Thus, although RSK and UNC-51 act as negative regulators for ERK, their relative contribution to ERK signaling in different cell types appears to be different. At least in the case of the RSK mutant, hyperactivation of ERK is modest and has no effect on development or viability of the fly, which implies a subtle modulatory function of RSK (Beck, 2015).
Finally, analyses uncovers aberrant axonal BRP and CSP localization and anterograde transport defects of mitochondria. Transport of presynaptic components and their appropriate delivery at synaptic terminals require a complex interplay between motor proteins, the different transported components and local signaling events. In addition, mechanisms must exist to restrain localization of presynaptic components at the nerve terminals. Interfering with these processes in Drosophila motoneurons causes distinct phenotypes. For instance, loss of Liprin-α results in ectopic accumulation of synaptic vesicles and presynaptic cytomatrix proteins in distal axon regions close to the synaptic terminals without affecting mitochondria or motor protein localization. SR protein kinase 79D (SRPK79D) is required to prevent formation of large axonal agglomerates of BRP. Given that axonal transport processes and other synaptic proteins are not affected in SRPK79D mutants, a function of this kinase for site-specific active zone assembly at presynaptic membranes has been suggested. Large organelle-filled axonal swellings have been observed in mutants defective for motor protein components; however, these aggregates do not serve as physical barriers for mitochondrial transport. Local effects caused by changes in axonal transport are seen in dAcsl mutations. Here, mitochondrial transport is unaffected, but an increased velocity of anterograde transport and reduced velocity of retrograde transport of vesicles results in aggregates in distal axon regions. Mutation of the human ortholog ACSL4 (acyl-CoA synthase long chain family member 4) causes non-syndromic X-linked mental retardation (Beck, 2015).
The axonal phenotypes seen in RSK mutants differ in several respects from these phenotypes. Large axonal swellings are not evident, and the increase in the number of BRP and CSP particles is largely confined to the proximal portion of the nerve (close to the ventral nerve cord). Together with the finding of more stationary mitochondria and fewer mitochondria transported in the anterograde direction, one explanation could be a function of RSK at the level of motor-cargo interaction. Specificity of cargo transport in the anterograde direction is determined at the levels both of individual Kinesins and of cargo-specific adaptor proteins. For example, the catalytic subunit Kinesin-1 in Drosophila (kinesin heavy chain, KHC) recruits mitochondria via the adaptor protein Milton, whereas UNC-76 provides a link to the synaptic vesicle protein Synaptotagmin. Motor-cargo interactions are also regulated in a phosphorylation-dependent manner, as exemplified by the UNC-51 kinase-dependent interaction of UNC-76 with Synaptotagmin. Loss of either UNC-51 or UNC-76 results in accumulations of synaptic vesicles along motoneuron axons. Another example is glycogen synthase kinase 3 (GSK-3), which has been proposed to inhibit anterograde transport by phosphorylating Kinesin light chain and thereby causing dissociation of membrane-bound organelles from KHC. Based on genetic analyses in Drosophila, an alternative model proposes a function of GSK-3 in regulating motor protein activity rather than cargo binding. Interestingly, RSK2 has been reported to inhibit GSK-3 activity in different cellular contexts and is able to phosphorylate GSK-3, at least in vitro. Future studies are required to clarify a function of RSK in GSK-3-mediated control of anterograde transport processes. So far, there is no evidence for a direct or an indirect requirement of RSK for phosphorylation of motor protein components and, if so, whether this might have an impact on their in vivo function (Beck, 2015).
In summary, an emerging common picture from knockout studies in mice and flies as animal models for CLS is a postsynaptic requirement of RSK proteins for efficient synaptic transmission. In addition, this study uncovered changes in the presynaptic neuron; in particular, defects in anterograde axonal transport and changes in localization of activated ERK. Whether these phenotypes reflect independent functions of RSK or whether they are interdependent remains to be determined. Future studies will also have to aim at understanding the function of RSK at central brain synapses in learning and memory processes (Beck, 2015).
Activation of the various mitogen-activated protein (MAP) kinase pathways converts many different extracellular stimuli into specific cellular responses by inducing the phosphorylation of particular groups of substrates. One important determinant for substrate specificity is likely to be the amino-acid sequence surrounding the phosphorylation site; however, these sites overlap significantly between different MAP kinase family members. The idea is now emerging that specific docking sites for protein kinases are involved in the efficient binding and phosphorylation of some substrates. The MAP kinase-activated protein (MAPKAP) kinase p90(rsk) contains two kinase domains: the amino-terminal domain (D1) is required for the phosphorylation of exogenous substrates whereas the carboxy-terminal domain (D2) is involved in autophosphorylation. Association between the extracellular signal-regulated kinase (Erk) MAP kinases and p90(rsk) family members has been detected in various cell types including Xenopus oocytes, where inactive p90(rsk) is bound to the inactive form of the Erk2-like MAP kinase p42(mpk1). A new MAP kinase docking site has been identified that is located at the carboxyl terminus of p90(rsk). This docking site is required for the efficient phosphorylation and activation of p90(rsk) in vitro and in vivo and is also both necessary and sufficient for the stable and specific association with p42(mpk1). The sequence of the docking site is conserved in other MAPKAP kinases, suggesting that it might represent a new class of interaction motif that facilitates efficient and specific signal transduction by MAP kinases (Gavin, 1999).
Stimulation of the Ras/extracellular signal-regulated kinase (ERK) pathway can modulate cell growth, proliferation, survival, and motility. The p90 ribosomal S6 kinases (RSKs) comprise a family of serine/threonine kinases that lie at the terminus of the ERK pathway. Efficient RSK activation by ERK requires its interaction through a docking site located near the C terminus of RSK, but the regulation of this interaction remains unknown. This report shows that RSK1 and ERK1/2 form a complex in quiescent HEK293 cells that transiently dissociates upon mitogen stimulation. Complex dissociation requires phosphorylation of RSK1 serine 749, which is a mitogen-regulated phosphorylation site located near the ERK docking site. Using recombinant RSK1 proteins, it was found that serine 749 is phosphorylated by the N-terminal kinase domain of RSK1 in vitro, suggesting that ERK1/2 dissociation is mediated through RSK1 autophosphorylation of this residue. Consistent with this hypothesis, it was found that inactivating mutations in the RSK1 kinase domains disruptes the mitogen-regulated dissociation of ERK1/2 in vivo. Analysis of different RSK isoforms revealed that RSK1 and RSK2 readily dissociate from ERK1/2 following mitogen stimulation but that RSK3 remains associated with active ERK1/2. RSK activity assays revealed that RSK3 also remains active longer than RSK1 and RSK2, suggesting that prolonged ERK association increases the duration of RSK3 activation. These results provide new evidence for the regulated nature of ERK docking interactions and reveal important differences among the closely related RSK family members (Roux, 2003).
Inhibitors of the oncogenic Ras-MAPK pathway have been intensely pursued as therapeutics. Targeting this pathway, however, presents challenges due to the essential role of MAPK in homeostatic functions. The phosphorylation and activation of MAPK substrates is regulated by protein-protein interactions with MAPK docking sites. Active ERK1/2 (extracellular signal-regulated kinase 1/2)-MAPKs localize to effectors containing DEF (docking site for ERK, (F)/(Y) -X-(F)/(Y) -P)- or D-domain (docking domain) motifs. The in vivo activity was examined of ERK2 mutants with impaired ability to signal via either docking site. Mutations in the DEF-domain binding pocket prevent activation of DEF-domain-containing effectors but not RSK (90 kDa ribosomal S6 kinase), which contains a D domain. Conversely, mutation of the ERK2 CD domain, which interacts with D domains, prevents RSK activation but not DEF-domain signaling. Uncoupling docking interactions does not compromise ERK2 phosphotransferase activity. ERK2 DEF mutants undergo regulated nuclear translocation but are defective for Elk-1/TCF transactivation and target gene induction. Thus, downstream branches of ERK2 signaling can be selectively inhibited without blocking total pathway activity. Significantly, several protooncogenes contain DEF domains and are regulated by ERK1/2. Therefore, disrupting ERK-DEF domain interactions could be an alternative to inhibiting oncogenic Ras-MAPK signaling (Dimitri, 2005).
Glutathione S-transferase (GST)-fusion proteins containing the carboxyl-terminal tails of three p90 ribosomal S6 kinase (RSK) isozymes (RSK1, RSK2, and RSK3) interact with extracellular signal-regulated kinase (ERK) but not c-Jun-NH2-kinase (JNK) or p38 mitogen-activated protein kinase (MAPK). Within the carboxyl-terminal residues of the RSK isozymes is a region of high conservation corresponding to residues 722LAQRRVRKLPSTTL735 in RSK1. Truncation of the carboxyl-terminal 9 residues, 727VRKLPSTTL735, completely eliminates the interaction of the GST-RSK1 fusion protein with purified recombinant ERK2, whereas the truncation of residues 731PSTTL735 has no effect on the interaction with purified ERK2. ERK1 and ERK2 co-immunoprecipitate with hemagglutinin-tagged wild type RSK2 (HA-RSK2). However, ERK does not co-immunoprecipitate with HA-RSK2(1-729), a mutant missing the carboxyl-terminal 11 amino acids, similar to the minimal truncation that eliminates in vitro interaction of ERK with the GST-RSK1 fusion protein. Kinase activity of HA-RSK2 increases 6-fold in response to insulin. HA-RSK2(1-729) has a similar basal kinase activity to that of HA-RSK2 but is not affected by insulin treatment. Immunoprecipitated HA-RSK2 and HA-RSK2(1-729) can be activated to the same extent in vitro by active ERK2, demonstrating that HA-RSK2(1-729) is properly folded. These data suggest that the conserved region of the RSK isozymes (722LAQRRVRKL730 of RSK1) provides for a specific ERK docking site approximately 150 amino acids carboxyl-terminal to the nearest identified ERK phosphorylation site (Thr573). Complex formation between RSK and ERK is essential for the activation of RSK by ERK in vivo. Comparison of the docking site of RSK with the carboxyl-terminal tails of other MAPK-activated kinases reveals putative docking sites within each of these MAPK-targeted kinases. The number and placement of lysine and arginine residues within the conserved region correlate with specificity for activation by ERK and p38 MAPKs in vivo (Smith, 1999).
Protein kinase B (PKB), and the p70 and p90 ribosomal S6 kinases (p70 S6 kinase and p90 Rsk, respectively), are activated by phosphorylation of two residues, one in the 'T-loop' of the kinase domain and, the other, in the hydrophobic motif carboxy terminal to the kinase domain. The 3-phosphoinositide-dependent protein kinase 1 (PDK1), which binds with high affinity to the PI 3-kinase lipid product phosphatidylinositol-3,4,5-trisphosphate, activates many AGC kinases in vitro by phosphorylating the T-loop residue, but whether PDK1 also phosphorylates the hydrophobic motif and whether all other AGC kinases are substrates for PDK1 is unknown. Mouse embryonic stem (ES) cells in which both copies of the PDK1 gene were disrupted are viable. In PDK1-/- ES cells, PKB, p70 S6 kinase and p90 Rsk are not activated by stimuli that induced strong activation in PDK1+/+ cells. Other AGC kinases namely, protein kinase A (PKA), the mitogen- and stress-activated protein kinase 1 (MSK1) and the AMP-activated protein kinase (AMPK) have normal activity or are activated normally in PDK1-/- cells. The insulin-like growth factor 1 (IGF1) induces PKB phosphorylation at its hydrophobic motif, but not at its T-loop residue, in PDK1-/- cells. IGF1 does not induce phosphorylation of p70 S6 kinase at its hydrophobic motif in PDK1-/- cells. It is concluded PDK1 mediates activation of PKB, p70 S6 kinase and p90 Rsk in vivo, but is not rate-limiting for activation of PKA, MSK1 and AMPK. Another kinase phosphorylates PKB at its hydrophobic motif in PDK1-/- cells. PDK1 phosphorylates the hydrophobic motif of p70 S6 kinase either directly or by activation of another kinase (Williams, 2000).
G protein-coupled receptors (GPCRs) are essential for normal central CNS function and represent the proximal site(s) of action for most neurotransmitters and many therapeutic drugs, including typical and atypical antipsychotic drugs. Similarly, protein kinases mediate many of the downstream actions for both ionotropic and metabotropic receptors. Genetic deletion of p90 ribosomal S6 kinase 2 (RSK2) potentiates GPCR signaling. Initial studies of 5-hydroxytryptamine (5-HT)2A receptor signaling in fibroblasts obtained from RSK2 wild-type (+/+) and knockout (-/-) mice showed that 5-HT2A receptor-mediated phosphoinositide hydrolysis and both basal and 5-HT-stimulated extracellular signal-regulated kinase 1/2 phosphorylation are augmented in RSK2 knockout fibroblasts. Endogenous signaling by other GPCRs, including P2Y-purinergic, PAR-1-thrombinergic, beta1-adrenergic, and bradykinin-B receptors, was also potentiated in RSK2-deficient fibroblasts. Importantly, reintroduction of RSK2 into RSK2-/- fibroblasts normalized signaling, thus demonstrating that RSK2 apparently modulates GPCR signaling by exerting a 'tonic brake' on GPCR signal transduction. These results imply the existence of a novel pathway regulating GPCR signaling, modulated by downstream members of the extracellular signal-related kinase/mitogen-activated protein kinase cascade. The loss of RSK2 activity in humans leads to Coffin-Lowry syndrome, which is manifested by mental retardation, growth deficits, skeletal deformations, and psychosis. Because RSK2-inactivating mutations in humans lead to Coffin-Lowry syndrome, these results imply that alterations in GPCR signaling may account for some of its clinical manifestations (Sheffler, 2006).
A signaling pathway has been elucidated whereby growth factors activate the transcription factor cyclic adenosine monophosphate response element-binding protein (CREB), a critical regulator of immediate early gene transcription. Growth factor-stimulated CREB phosphorylation at serine-133 is mediated by the RAS-mitogen-activated protein kinase (MAPK) pathway. MAPK activates CREB kinase, which in turn phosphorylates and activates CREB. Purification, sequencing, and biochemical characterization of CREB kinase reveals that it is identical to a member of the pp90(RSK) family, RSK2. RSK2 mediates growth factor induction of CREB serine-133 phosphorylation both in vitro and in vivo. These findings identify a cellular function for RSK2 and define a mechanism whereby growth factor signals mediated by RAS and MAPK are transmitted to the nucleus to activate gene expression (Xing, 1996).
Although Ca2+-stimulated cAMP response element binding protein- (CREB-) dependent transcription has been implicated in growth, differentiation, and neuroplasticity, mechanisms for Ca2+-activated transcription have not been defined. Extracellular signal-related protein kinase (ERK) signaling is obligatory for Ca2+-stimulated transcription in PC12 cells and hippocampal neurons. The sequential activation of ERK and Rsk2 by Ca2+ leads to the phosphorylation and transactivation of CREB. The Ca2+-induced nuclear translocation of ERK and Rsk2 to the nucleus requires protein kinase A (PKA) activation. Interestingly, Ca2+-mediated CREB phosphorylation in wild-type PC12 cells is decreased by a selective PKA inhibitor. With a high efficiency transfection protocol, expression of dominant negative PKA also attenuates Ca2+-stimulated CREB phosphorylation. In addition, treatment with the PKA inhibitors also inhibits depolarization-mediated CREB phosphorylation in primary hippocampal neurons. These results suggest that in PC12 cells and hippocampal neurons, PKA activity is required for Ca2+-induced CREB phosphorylation (Impey, 1998).
Because the nuclear translocation of ERK may be necessary for ERK-activated transcription, and PKA is required for Ca2+ stimulation of CREB phosphorylation, nuclear translocation of ERK was monitored when PKA was inhibited. To efficiently induce the nuclear translocation of ERK by Ca2+, PC12 cells were treated with KCl and a direct activator of L-type Ca2+ channels. Depolarization induces the phosphorylation of ERK and its translocation to the nucleus in both PC12 cells and hippocampal neurons. The specific PKA inhibitors inhibited the nuclear translocation of Erk in PC12 cells and hippocampal neurons. Western blotting of cytosolic fractions shows that the inhibition of Erk translocation by treatment with PKA inhibitors is not the result of an effect on Erk activation. To verify that PKA is required for the nuclear translocation of ERK, the cytosolic-to-nuclear ratio of phospho-ERK in KCl-stimulated hippocampal neurons was also quantitated. A specific PKA inhibitor significantly inhibited the translocation of ERK to the nucleus. The importance of PKA activity for ERK nuclear translocation was confirmed by transiently transfecting PC12 cells with a dominant negative PKA fused to green fluorescent protein. Only cells that express dominant negative PKA-GFP show impaired nuclear translocation of phospho-ERK. These results suggest that PKA is required for the phosphorylation and transactivation of CREB by Ca2+, because PKA is required for the nuclear translocation of ERK. However, since Rsk2 [a member of the pp90(RSK) family] is a major Ca2+-activated CREB kinase in PC12 cells, inhibition of Erk translocation should also block the activation of nuclear but not cytosolic Rsk2. Accordingly, inhibition of PKA blocks the activation of Rsk2 in the nuclear fraction but not in the cytosolic fraction. Treatment with PKA inhibitors attenuates the nuclear translocation of Rsk2. This is not surprising, because it is known that both ERK and Rsk2 are tightly associated in vivo and that they cotranslocate to the nucleus. Collectively, these data indicate that PKA may be necessary for the phosphorylation and transactivation of CREB by Ca2+, because PKA is required for the nuclear translocation of ERK and subsequent nuclear activation of the CREB kinase Rsk2. Inhibition of PKA also significantly impairs the translocation of ERK to the nucleus in response to NGF. Interestingly, coexpression of dominant negative PKA attenuates NGF-stimulated Elk1 transcriptional activation. Evidently, the modulation of ERK translocation by PKA activity plays a general role in the activation of transcription by mitogens and neurotrophic factors. NGF does not detectably elevate intracellular cAMP, suggesting that basal PKA activity is sufficient for neurotrophic factors and other strong ERK activators to induce nuclear translocation of ERK. Nevertheless, in the case of depolarization, which activates ERK to a lesser degree, the concomitant depolarization-mediated increase in cAMP levels enhances ERK translocation. These results may explain why PKA activity is required for Ca2+-stimulated CREB-dependent transcription. Furthermore, the full expression of the late phase of long-term potentiation (L-LTP) and L-LTP-associated CRE-mediated transcription requires ERK activation, suggesting that the activation of CREB by ERK plays a critical role in the formation of long lasting neuronal plasticity (Impey, 1998).
The keratinocyte growth factor receptor (KGFR) is a member of the fibroblast growth factor receptor (FGFR) superfamily. The proximal signaling molecules of FGFRs are much less characterized compared with other growth factor receptors. Using the yeast two-hybrid assay, ribosomal S6 kinase (RSK) was identified as a protein that associates with the cytoplasmic domain of the KGFR. The RSK family of kinases controls multiple cellular processes, and these studies show association between the KGFR and RSK. Using a lung-specific inducible transgenic system, protective effects of KGF on the lung epithelium has been demonstrated, as well as KGF-induced activation of the prosurvival Akt pathway. A kinase inactive RSK mutant blocks KGF-induced Akt activation and KGF-mediated inhibition of caspase 3 activation in epithelial cells subjected to oxidative stress. RSK2 recruits PDK1, the kinase responsible for both Akt and RSK activation. When viewed collectively, it appears that the association between the KGFR and RSK plays an important role in KGF-induced Akt activation and consequently in the protective effects of KGF on epithelial cells (Pan, 2004).
Receptor tyrosine kinase (RTK) signals regulate the specification of a varied array of tissue types by utilizing distinct modules of proteins to elicit diverse effects. The RSK proteins are part of the RTK signal transduction pathway and are thought to relay these signals by acting downstream of extracellular signal-regulated kinase (ERK). Ribosomal S6 kinase 4 (Rsk4) is an inhibitor of RTK signals. Among the RSK proteins, RTK inhibition is specific to RSK4 and, in accordance, is dependent upon a region of the RSK4 protein that is divergent from other RSK family members. Rsk4 inhibits the transcriptional activation of specific targets of RTK signaling as well as the activation of ERK. Developmentally, Rsk4 is expressed in extraembryonic tissue, where RTK signals are known to have critical roles. Further examination of Rsk4 expression in the extraembryonic tissues demonstrates that its expression is inversely correlated with the presence of activated ERK 1/2. These studies demonstrate a new and divergent function for RSK4 and support a role for RSK proteins in the specification of RTK signals during early mouse development (Myers, 2004)
Tuberous sclerosis complex (TSC) is a genetic disorder caused by mutations in either of the two tumor suppressor genes TSC1 or TSC2, which encode hamartin and tuberin, respectively. Tuberin and hamartin form a complex that inhibits signaling by the mammalian target of rapamycin (mTOR), a critical nutrient sensor and regulator of cell growth and proliferation. Phosphatidylinositol 3-kinase (PI3K) inactivates the tumor suppressor complex and enhances mTOR signaling by means of phosphorylation of tuberin by Akt. Importantly, cellular transformation mediated by phorbol esters and Ras isoforms that poorly activate PI3K promote tumorigenesis in the absence of Akt activation. This study shows that phorbol esters and activated Ras also induce the phosphorylation of tuberin and collaborates with the nutrient-sensing pathway to regulate mTOR effectors, such as p70 ribosomal S6 kinase 1 (S6K1). The mitogen-activated protein kinase (MAPK)-activated kinase, p90 ribosomal S6 kinase (RSK) 1, was found to interact with and phosphorylate tuberin at a regulatory site, Ser-1798, located at the evolutionarily conserved C terminus of tuberin. RSK1 phosphorylation of Ser-1798 inhibits the tumor suppressor function of the tuberin/hamartin complex, resulting in increased mTOR signaling to S6K1. Together, these data unveil a regulatory mechanism by which the Ras/MAPK and PI3K pathways converge on the tumor suppressor tuberin to inhibit its function (Roux, 2004).
The mechanism by which LKB1 is regulated in cells is not known. Stimulation of Rat-2 or embryonic stem cells with activators of ERK1/2 or of cAMP-dependent protein kinase induces phosphorylation of endogenously expressed LKB1 at Ser(431). Pharmacological and genetic evidence is presented that p90(RSK) mediates this phosphorylation in response to agonists that activate ERK1/2, and cAMP-dependent protein kinase mediates this phosphorylation in response to agonists that activate adenylate cyclase. Ser(431) of LKB1 lies adjacent to a putative prenylation motif, and full-length LKB1 expressed in 293 cells is prenylated by addition of a farnesyl group to Cys(433). These data suggest that phosphorylation of LKB1 at Ser(431) does not affect farnesylation and that farnesylation does not affect phosphorylation at Ser(431). Phosphorylation of LKB1 at Ser(431) does not alter the activity of LKB1 to phosphorylate itself or the tumor suppressor protein p53 or alter the amount of LKB1 associated with cell membranes. The reintroduction of wild-type LKB1 into a cancer cell line that lacks LKB1 suppressed growth, but mutants of LKB1 in which Ser(431) was mutated to Ala to prevent phosphorylation of LKB1 were ineffective in inhibiting growth. In contrast, a mutant of LKB1 that cannot be prenylated is still able to suppress the growth of cells (Sapkota, 2001).
The Ras-mitogen-activated protein (Ras-MAP) kinase pathway regulates various cellular processes, including gene expression, cell proliferation, and survival. Ribosomal S6 kinase (RSK), a key player in this pathway, modulates the activities of several cytoplasmic and nuclear proteins via phosphorylation. The cytoskeletal protein filamin A (FLNa) is a membrane-associated RSK target. The N-terminal kinase domain of RSK phosphorylates FLNa on Ser(2152) in response to mitogens. Inhibition of MAP kinase signaling with UO126 or mutation of Ser(2152) to Ala on FLNa prevents epidermal growth factor (EGF)-stimulated phosphorylation of FLNa in vivo. Furthermore, phosphorylation of FLNa on Ser(2152) is significantly enhanced by the expression of wild-type RSK and antagonized by kinase-inactive RSK or specific reduction of endogenous RSK. Strikingly, EGF-induced, FLNa-dependent migration of human melanoma cells is significantly reduced by UO126 treatment. Together, these data provide substantial evidence that RSK phosphorylates FLNa on Ser(2152) in vivo. Given that phosphorylation of FLNa on Ser(2152) is required for Pak1-mediated membrane ruffling, these results suggest a novel role for RSK in the regulation of the actin cytoskeleton (Woo, 2004).
Evidence is presented that RSK1 (ribosomal S6 kinase 1), a downstream target of MAPK (mitogen-activated protein kinase), directly phosphorylates nNOS (neuronal nitric oxide synthase) on Ser847 in response to mitogens. The phosphorylation thus increases greatly following EGF (epidermal growth factor) treatment of rat pituitary tumour GH3 cells and is reduced by exposure to the MEK (MAPK/extracellular-signal-regulated kinase kinase) inhibitor PD98059. Furthermore, it is significantly enhanced by expression of wild-type RSK1 and antagonized by kinase-inactive RSK1 or specific reduction of endogenous RSK1. EGF treatment of HEK-293 (human embryonic kidney) cells, expressing RSK1 and nNOS, led to inhibition of NOS enzyme activity, associated with an increase in phosphorylation of nNOS at Ser847, as is also the case in an in vitro assay. In addition, these phenomena were significantly blocked by treatment with the RSK inhibitor Ro31-8220. Cells expressing mutant nNOS (S847A) proved resistant to phosphorylation and decrease of NOS activity. Within minutes of adding EGF to transfected cells, RSK1 associated with nNOS and subsequently dissociated following more prolonged agonist stimulation. EGF-induced formation of the nNOS-RSK1 complex was significantly decreased by PD98059 treatment. Treatment with EGF further revealed phosphorylation of nNOS on Ser847 in rat hippocampal neurons and cerebellar granule cells. This EGF-induced phosphorylation was partially blocked by PD98059 and Ro31-8220. Together, these data provide substantial evidence that RSK1 associates with and phosphorylates nNOS on Ser847 following mitogen stimulation and suggest a novel role for RSK1 in the regulation of nitric oxide function in brain (Song, 2007).
The eukaryotic translation initiation factor 4B (eIF4B) plays a critical role in recruiting the 40S ribosomal subunit to the mRNA. In response to insulin, eIF4B is phosphorylated on Ser422 by S6K in a rapamycin-sensitive manner. This study demonstrates that the p90 ribosomal protein S6 kinase (RSK; see Drosophila) phosphorylates eIF4B on the same residue. The relative contribution of the RSK and S6K modules to the phosphorylation of eIF4B is growth factor-dependent, and the two phosphorylation events exhibit very different kinetics. The S6K and RSK proteins are members of the AGC protein kinase family, and require PDK1 phosphorylation for activation. Consistent with this requirement, phosphorylation of eIF4B Ser422 is abrogated in PDK1 null embryonic stem cells. Phosphorylation of eIF4B on Ser422 by RSK and S6K is physiologically significant, as it increases the interaction of eIF4B with the eukaryotic translation initiation factor 3 (Shahbazian, 2006).
The viability of vertebrate cells depends on a complex signaling interplay between survival factors and cell-death effectors. Subtle changes in the equilibrium between these regulators can result in abnormal cell proliferation or cell death, leading to various pathological manifestations. Death-associated protein kinase (DAPK) is a multidomain calcium/calmodulin (CaM)-dependent Ser/Thr protein kinase with an important role in apoptosis regulation and tumor suppression. The molecular signaling mechanisms regulating this kinase, however, remain unclear. This study shows that DAPK is phosphorylated upon activation of the Ras-extracellular signal-regulated kinase (ERK) pathway. This correlates with the suppression of the apoptotic activity of DAPK. DAPK is a novel target of p90 ribosomal S6 kinases (RSK) 1 and 2, downstream effectors of ERK1/2. Using mass spectrometry, Ser-289 was identified as a novel phosphorylation site in DAPK, which is regulated by RSK. Mutation of Ser-289 to alanine results in a DAPK mutant with enhanced apoptotic activity, whereas the phosphomimetic mutation (Ser289Glu) attenuates its apoptotic activity. These results suggest that RSK-mediated phosphorylation of DAPK is a unique mechanism for suppressing the proapoptotic function of this death kinase in healthy cells as well as Ras/Raf-transformed cells (Anjum, 2005).
Extracellular signal-regulated kinase (ERK) signaling is important for neuronal synaptic plasticity. The protein kinase ribosomal S6 kinase (RSK)2, a downstream target of ERK, uses a C-terminal motif to bind several PDZ domain proteins in heterologous systems and in vivo. Different RSK isoforms display distinct specificities in their interactions with PDZ domain proteins. Mutation of the RSK2 PDZ ligand does not inhibit RSK2 activation in intact cells or phosphorylation of peptide substrates by RSK2 in vitro but greatly reduces RSK2 phosphorylation of PDZ domain proteins of the Shank family in heterologous cells. In primary neurons, NMDA receptor (NMDA-R) activation leads to ERK and RSK2 activation and RSK-dependent phosphorylation of transfected Shank3. RSK2-PDZ domain interactions are functionally important for synaptic transmission because neurons expressing kinase-dead RSK2 display a dramatic reduction in frequency of AMPA-type glutamate receptor-mediated miniature excitatory postsynaptic currents, an effect dependent on the PDZ ligand. These results suggest that binding of RSK2 to PDZ domain proteins and phosphorylation of these proteins or their binding partners regulates excitatory synaptic transmission (Thomas, 2005).
M-phase entry in eukaryotic cells is driven by activation of MPF, a regulatory factor composed of cyclin B and the protein kinase p34(cdc2). In G2-arrested Xenopus oocytes, there is a stock of p34(cdc2)/cyclin B complexes (pre-MPF), which is maintained in an inactive state by p34(cdc2) phosphorylation on Thr14 and Tyr15. This suggests an important role for the p34(cdc2) inhibitory kinase(s) such as Wee1 and Myt1 in regulating the G2-->M transition during oocyte maturation. MAP kinase (MAPK) activation is required for M-phase entry in Xenopus oocytes, but its precise contribution to the activation of pre-MPF is unknown. The C-terminal regulatory domain of Myt1 specifically binds to p90(rsk), a protein kinase that can be phosphorylated and activated by MAPK. In turn, p90(rsk) phosphorylates the C-terminus of Myt1 and down-regulates its inhibitory activity on p34(cdc2)/cyclin B in vitro. Consistent with these results, Myt1 becomes phosphorylated during oocyte maturation, and activation of the MAPK-p90(rsk) cascade can trigger some Myt1 phosphorylation prior to pre-MPF activation. Myt1 preferentially associates with hyperphosphorylated p90(rsk), and complexes can be detected in immunoprecipitates from mature oocytes. These results suggest that during oocyte maturation MAPK activates p90(rsk) and that p90(rsk) in turn down-regulates Myt1, leading to the activation of p34(cdc2)/cyclin B (Palmer, 1998).
During oocyte maturation in Xenopus, progesterone induces entry into meiosis I, and the M phases of meiosis I and II occur consecutively without an intervening S phase. The mitogen-activated protein (MAP) kinase is activated during meiotic entry, and it has been suggested that the linkage of M phases reflects activation of the MAP kinase pathway and the failure to fully degrade cyclin B during anaphase I. To analyze the function of the MAP kinase pathway in oocyte maturation, U0126, a potent inhibitor of MAP kinase kinase, and a constitutively active mutant of the protein kinase p90Rsk, a MAP kinase target, were used. Even with complete inhibition of the MAP kinase pathway by U0126, up to 90% of oocytes were able to enter meiosis I after progesterone treatment, most likely through activation of the phosphatase Cdc25C by the polo-like kinase Plx1. Subsequently, however, U0126-treated oocytes fail to form metaphase I spindles, fail to reaccumulate cyclin B to a high level and fail to hyperphosphorylate Cdc27, a component of the anaphase-promoting complex (APC) that controls cyclin B degradation. Such oocytes enter S phase rather than meiosis II. U0126-treated oocytes expressing a constitutively active form of p90Rsk are able to reaccumulate cyclin B, hyperphosphorylate Cdc27 and form metaphase spindles in the absence of detectable MAP kinase activity. It is concluded that the MAP kinase pathway is not essential for entry into meiosis I in Xenopus but is required during the onset of meiosis II to suppress entry into S phase, to regulate the APC so as to support cyclin B accumulation, and to support spindle formation. Moreover, one substrate of MAP kinase, p90Rsk, is sufficient to mediate these effects during oocyte maturation (Gross, 2000).
The kinetochore attachment (spindle assembly) checkpoint arrests cells in metaphase to prevent exit from mitosis until all the chromosomes are aligned properly at the metaphase plate. The checkpoint operates by preventing activation of the anaphase-promoting complex (APC), which triggers anaphase by degrading mitotic cyclins and other proteins. This checkpoint is active during normal mitosis and upon experimental disruption of the mitotic spindle. In yeast, the serine/threonine protein kinase Bub1 and the WD-repeat protein Bub3 are elements of a signal transduction cascade that regulates the kinetochore attachment checkpoint. In mammalian cells, activated MAPK is present on kinetochores during mitosis and activity is upregulated by the spindle assembly checkpoint. In vertebrate unfertilized eggs, a special form of meiotic metaphase arrest by cytostatic factor (CSF) is mediated by MAPK activation of the protein kinase p90(Rsk), which leads to inhibition of the APC. However, it is not known whether CSF-dependent metaphase arrest caused by p90(Rsk) involves components of the spindle assembly checkpoint. This study shows that xBub1 is present in resting oocytes and its protein level increases slightly during oocyte maturation and early embryogenesis. In Xenopus oocytes, Bub1 is localized to kinetochores during both meiosis I and meiosis II, and the electrophoretic mobility of Bub1 upon SDS-PAGE decreases during meiosis I, reflecting phosphorylation and activation of the enzyme. The activation of Bub1 can be induced in interphase egg extracts by selective stimulation of the MAPK pathway by c-Mos, a MAPKKK. In oocytes treated with the MEK1 inhibitor U0126, the MAPK pathway does not become activated, and Bub1 remains in its low-activity, unshifted form. Injection of a constitutively active target of MAPK, the protein kinase p90(Rsk), restores the activation of Bub1 in the presence of U0126. Moreover, purified p90(Rsk) phosphorylates Bub1 in vitro and increases its protein kinase activity. It is concluded that Bub1, an upstream component of the kinetochore attachment checkpoint, is activated during meiosis in Xenopus in a MAPK-dependent manner. Moreover, a single substrate of MAPK, p90(Rsk), is sufficient to activate Bub1 in vitro and in vivo. These results indicate that in vertebrate eggs, kinetochore attachment/spindle assembly checkpoint proteins, including Bub1, are downstream of p90(Rsk) and may be effectors of APC inhibition and CSF-dependent metaphase arrest by p90(Rsk) (Schwab, 2001).
The cell cycle in oocytes generally arrests at a particular meiotic stage to await fertilization. This arrest occurs at metaphase of meiosis II (meta-II) in frog and mouse, and at G1 phase after completion of meiosis II in starfish. Despite this difference in the arrest phase, both arrests depend on the same Mos-MAPK (mitogen-activated protein kinase) pathway, indicating that the difference relies on particular downstream effectors. Immediately downstream of MAPK, Rsk [p90 ribosomal S6 kinase, p90(Rsk)] is required for the frog meta-II arrest. However, the mouse meta-II arrest challenges this requirement, and no downstream effector has been identified in the starfish G1 arrest. To investigate the downstream effector of MAPK in the starfish G1 arrest, a neutralizing antibody was used against Rsk and a constitutively active form of Rsk. Rsk was activated downstream of the Mos-MAPK pathway during meiosis. In G1 eggs, inhibition of Rsk activity released the arrest and initiated DNA replication without fertilization. Conversely, maintenance of Rsk activity prevented DNA replication following fertilization. In early embryos, injection of Mos activated the MAPK-Rsk pathway, resulting in G1 arrest. Moreover, inhibition of Rsk activity during meiosis I led to parthenogenetic activation without meiosis II. It is concluded that immediately downstream of MAPK, Rsk is necessary and sufficient for the starfish G1 arrest. Although CSF (cytostatic factor) was originally defined for meta-II arrest in frog eggs, distinguishing 'G1-CSF' for starfish from 'meta-II-CSF' for frog and mouse is proposed. The present study thus reveals a novel role of Rsk for G1-CSF (Mori, 2006).
A mechanism by which the Ras-mitogen-activated protein kinase (MAPK) signaling pathway mediates growth factor-dependent cell survival has been characterized. The neurotrophin BDNF (brain-derived neurotrophic factor) and its receptor TrkB regulate the survival of newly generated granule neurons within the developing cerebellum. BDNF promotes the survival of cultured rat cerebellar granule neurons; upon BDNF withdrawal, these neurons die by apoptosis. BDNF induces phosphorylation of MAPK. Inhibition of MAPK activity by PD098059, a pharmacological agent that blocks MEK activity, diminishes the effect of BDNF on the survival of cerebellar granule cells. Likewise, the introduction of a dominant interfering form of MEK (MEK-KA97) blocks BDNF-enhancement of neuronal survival. These results indicate that activation of MAPK is required for BDNF-induced survival of cerebellar granule neurons (Bonni, 1999).
Like BDNF, insulin-like growth factor 1 (IGF-1) (or a high concentration of insulin that stimulates the IGF-1 receptor) promotes the survival of cerebellar granule neurons. Both BDNF and IGF-1 activate phosphatidylinositol 3-kinase (PI-3K) and the protein kinase Akt (PKB: Drosophila homolog Akt1) cascade in cerebellar granule neurons. Although the PI-3K-Akt signaling pathway mediates the survival-promoting effects of BDNF and IGF-1, inhibition of MAPK in cerebellar neurons has no effect on IGF-1 receptor-mediated cell survival. These results suggest that BDNF and IGF-1 promote cell survival at least in part by distinct mechanisms (Bonni, 1999).
The MAPK-activated kinases, the Rsks, catalyze the phosphorylation of the pro-apoptotic protein BAD at serine 112 both in vitro and in vivo. The Rsk-induced phosphorylation of BAD at serine 112 suppresses BAD-mediated apoptosis in neurons. Rsks also are known to phosphorylate the transcription factor CREB (cAMP response element-binding protein) at serine 133. Activated CREB promotes cell survival, and inhibition of CREB phosphorylation at serine 133 triggers apoptosis. These findings suggest that the MAPK signaling pathway promotes cell survival by a dual mechanism comprising the posttranslational modification and inactivation of a component of the cell death machinery and the increased transcription of pro-survival genes (Bonni, 1999).
To determine whether CREB contributes to BDNF's ability to enhance cerebellar granule cell survival, the effects of two distinct dominant interfering forms of CREB on the BDNF survival response were tested. K-CREB, in which Arg287 is converted to Leu, forms dimers with endogenous CREB proteins via its leucine zipper domain. K-CREB inhibits the binding of endogenous CREB to the promoters of CREB-responsive genes. M1-CREB, in which Ser133 is converted to Ala, competes with endogenous CREB proteins for binding to the promoters of CREB-responsive genes. However, once bound to DNA, M1-CREB does not activate transcription. When transfected into cerebellar granule neurons, either K-CREB or M1-CREB inhibits the effect of BDNF on cell survival. However, the dominant interfering forms of CREB do not inhibit IGF-1-mediated cerebellar granule cell survival; this finding suggests that these proteins act specifically to block the BDNF response. In addition, M1-CREB does not lead to inhibition of Rsk function because its expression in 293T cells does not inhibit the MEK-induced phosphorylation of BAD at Ser112 (Bonni, 1999).
CREB has been implicated in mediating adaptive responses of neurons to trans-synaptic stimuli. These findings indicate that CREB may also have a function in the regulation of neuronal survival in the developing central nervous system. Mice in which the CREB gene has been disrupted die perinatally before the majority of cerebellar granule neurons are generated However, analysis of the CREB-/- mouse embryos has revealed a number of abnormalities in brain development that may reflect the contribution of CREB to the regulation of the survival of neurons. These findings suggest that the MAPK signaling pathway promotes cell survival by a dual mechanism that modulates the cell death machinery directly by phosphorylating and thereby inhibiting the pro-apoptotic protein BAD, and by inducing the expression of pro-survival genes in a CREB-dependent manner. Suppression of BAD-mediated cell death by Rsk occurs relatively early after the removal of extracellular survival factors, whereas the contribution of CREB-mediated cell survival is detected significantly later. Therefore, the two arms of the MAPK-Rsk-regulated mechanism might act with different kinetics or at different times in developing neurons (Bonni, 1999).
Growth factors activate an array of cell survival signaling pathways. Mitogen-activated protein (MAP) kinases transduce signals emanating from their upstream activators: MAP kinase kinases (MEKs). The MEK-MAP kinase signaling cassette is a key regulatory pathway promoting cell survival. The downstream effectors of the mammalian MEK-MAP kinase cell survival signal have not been previously described. Identified here is a pro-survival role for the serine/threonine kinase S6 kinase p90 ribosomal S6 kinase Rsk1, a downstream target of the MEK-MAP kinase signaling pathway. In cells that are dependent on interleukin-3 (IL-3) for survival, pharmacological inhibition of MEKs antagonize the IL-3 survival signal. In the absence of IL-3, a kinase-dead Rsk1 mutant eliminates the survival effect afforded by activated MEK. Conversely, a novel constitutively active Rsk1 allele restores the MEK-MAP kinase survival signal. Experiments in vitro and in vivo have demonstrated that Rsk1 directly phosphorylates the pro-apoptotic protein Bad at the serine residues that, when phosphorylated, abrogate Bad's pro-apoptotic function. Constitutively active Rsk1 causes constitutive Bad phosphorylation and protection from Bad-modulated cell death. Kinase-inactive Rsk1 mutants antagonize Bad phosphorylation. Bad mutations that prevent phosphorylation by Rsk1 also inhibit Rsk1-mediated cell survival. These data support a model in which Rsk1 transduces the mammalian MEK-MAP kinase signal in part by phosphorylating Bad (Shimamura, 2000).
Search PubMed for articles about Drosophila Rsk
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Bonni, A., et al. (1999). Cell survival promoted by the Ras-MAPK signaling pathway by transcription-dependent and -independent mechanisms. Science 286: 1358-1362.
Chung, J., Pelech, S. L. and Blenis, J. (1991). Mitogen-activated Swiss mouse 3T3 RSK kinases I and II are related to pp44mpk from sea star oocytes and participate in the regulation of pp90rsk activity. Proc. Natl. Acad. Sci. 88: 4981-4985.
Cohen, M. S., Hadjivassiliou, H. and Taunton, J. (2007). A clickable inhibitor reveals context-dependent autoactivation of p90 RSK. Nat Chem Biol 3: 156-160. PubMed ID: 17259979
Dimitri, C. A., et al. (2005). Spatially separate docking sites on ERK2 regulate distinct signaling events in vivo. Curr Biol 15: 1319-1324. 16051177
Douville, E. and Downward, J. (1997). EGF induced SOS phosphorylation in PC12 cells involves P90 RSK-2. Oncogene 15: 373-383. 9242373
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Kuntz, S., Poeck, B., Sokolowski, M. B. and Strauss, R. (2012). The visual orientation memory of Drosophila requires Foraging (PKG) upstream of Ignorant (RSK2) in ring neurons of the central complex. Learn Mem 19: 337-340. PubMed ID: 22815538
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Pan, Z. Z., Devaux, Y. and Ray, P. (2004). Ribosomal S6 kinase as a mediator of keratinocyte growth factor-induced activation of Akt in epithelial cells. Mol. Biol. Cell 15(7): 3106-13. 15107468
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Richards, S. A., Dreisbach, V. C., Murphy, L. O. and Blenis, J. (2001). Characterization of regulatory events associated with membrane targeting of p90 ribosomal S6 kinase 1. Mol Cell Biol 21: 7470-7480. PubMed ID: 11585927
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date revised: 2 January 2016
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