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 (FlyBase name: Protein kinase 61C) controls cellular and organism growth by activating Akt1 and S6 kinase, dS6K (FlyBase name: RPS6-p70-protein kinase). 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).
Reference names in red indicate recommended papers.
Search PubMed for articles about Drosophila Rsk
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Nebreda, A. R. and Gavin, A. C. (1999). Perspectives: signal transduction. Cell survival demands some Rsk. Science 286: 1309-1310. 10610536
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date revised: 31 January 2007
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