Table of contents

Miscellaneous targets of MAP kinase

Son of sevenless-1 and -2 (Sos-1 and -2) (Drosophila homolog SOS) are guanosine nucleotide exchange factors implicated in the activation of Ras (See Drosophila Ras) by both the insulin and epidermal growth factor signal transduction pathways. Ras initiates the activation of cellular protein kinases including mitogen-activated protein (MAP) kinases. Sos proteins contain numerous sequences in their carboxyl-terminal regions that correspond to consensus sites for MAP kinase phosphorylation. To examine whether these sites are substrates for MAP kinases, the cDNA encoding Drosophila Sos (dSos) was tagged with sequences encoding the major antigenic epitope of the influenza virus hemagglutinin (HA) to create a dSosHA fusion construct. dSosHA is transiently expressed in COS-1 cells and immunoprecipitated with anti-HA antibodies. When immune complexes are incubated with purified MAP kinase and radioactive ATP, a phosphorylated band of 180 kDa is observed. This band is not present in immunoprecipitations from cells transfected with vector alone. No phosphorylation of the 180 kDa band is seen when immunoprecipitates are incubated with ATP in the absence of MAP kinase. There are two major phosphorylated species that are also found in dSosHA isolated from COS-1 cells. This supports the hypothesis that a feedback loop exists wherein growth factor-activated MAP kinases phosphorylate and regulate Sos proteins (Cherniack, 1994).

Mitogen-activated protein (MAP) kinases bind tightly to many of their physiologically relevant substrates. A new subfamily of murine serine/threonine kinases has been identified, whose members, MAP kinase-interacting kinase 1 (Mnk1) and Mnk2, bind tightly to the growth factor-regulated MAP kinases: Erk1 and Erk2. MNK1, but not Mnk2, also binds strongly to the stress-activated kinase, p38. MNK1 complexes more strongly with inactive than active Erk, implying that Mnk and Erk may dissociate after mitogen stimulation. Erk and p38 phosphorylate MNK1 and Mnk2, which stimulates Mnk in vitro kinase activity toward a substrate, eukaryotic initiation factor-4E (eIF-4E). Initiation factor eIF-4E is a regulatory phosphoprotein whose phosphorylation is increased by insulin in an Erk-dependent manner. In vitro, MNK1 rapidly phosphorylates eIF-4E at the physiologically relevant site, Ser209. In cells, Mnk1 is post-translationally modified and enzymatically activated in response to treatment with either peptide growth factors, phorbol esters, anisomycin or UV. Mitogen- and stress-mediated MNK1 activation is blocked by inhibitors of MAP kinase kinase 1 (Mkk1) and p38, demonstrating that Mnk1 is downstream of multiple MAP kinases. MNK1 may define a convergence point between the growth factor-activated and one of the stress-activated protein kinase cascades and is a candidate to phosphorylate eIF-4E in cells (Waskiewicz, 1997).

A novel expression screening method has been developed for identifying protein kinase substrates. In this method, a lambda phage cDNA expression library is screened by in situ, solid-phase phosphorylation using purified protein kinase and [gamma-32P]ATP. Screening a HeLa cDNA library with ERK1 MAP kinase yields cDNAs of previously characterized ERK substrates, c-Myc and p90RSK (see Drosophila S6kII), demonstrating the utility of this method for identifying physiological protein kinase substrates. A novel clone isolated in this screen, designated MNK1, encodes a protein-serine/threonine kinase, which is most similar to MAP kinase-activated protein kinase 2 (MAPKAP-K2), 3pK/MAPKAP-K3 and p90RSK. Bacterially expressed MNK1 is phosphorylated and activated in vitro by ERK1 and p38 MAP kinases but not by JNK/SAPK. Further, MNK1 is activated upon stimulation of HeLa cells with 12-O-tetradecanoylphorbol-13-acetate, fetal calf serum, anisomycin, UV irradiation, tumor necrosis factor-alpha, interleukin-1beta, or osmotic shock, and the activation by these stimuli is differentially inhibited by the MEK inhibitor PD098059 or the p38 MAP kinase inhibitor SB202190. Together, these results indicate that MNK1 is a novel class of protein kinase that is activated through both the ERK and p38 MAP kinase signaling pathways (Fukunaga, 1997).

Xenopus laevis oogenesis is characterized by an active transcription that ceases abruptly upon maturation. To survey changes in the characteristics of the transcriptional machinery that might contribute to this transcriptional arrest, the phosphorylation status of the RNA polymerase II largest subunit (RPB1 subunit) was analyzed during oocyte maturation. The RPB1 subunit accumulates in large quantities from previtellogenic early diplotene oocytes up to fully grown oocytes. The C-terminal domain (CTD) of the RPB1 subunit is essentially hypophosphorylated in growing oocytes from Dumont stage IV to stage VI. Upon maturation, the proportion of hyperphosphorylated RPB1 subunits increases dramatically and abruptly. The hyperphosphorylated RPB1 subunits are dephosphorylated within 1 h after fertilization or heat shock of the matured oocytes. Extracts from metaphase II-arrested oocytes show a much stronger CTD kinase activity than extracts from prophase stage VI oocytes. Most of this kinase activity can be attributed to the activated Xp42 mitogen-activated protein (MAP) kinase, a MAP kinase of the ERK type. Making use of artificial maturation of the stage VI oocyte through microinjection of a recombinant stable cyclin B1, a parallel activation of Xp42 MAP kinase and phosphorylation of RPB1 is observed. Both events required protein synthesis, which demonstrated that activation of p34cdc2 kinase is insufficient to phosphorylate RPB1 ex vivo and is consistent with a contribution of the Xp42 MAP kinase to RPB1 subunit phosphorylation. These results further support the possibility that the largest RNA polymerase II subunit is a substrate of the ERK-type MAP kinases during oocyte maturation (Bellier, 1997).

While most untransformed cells require substrate attachment for growth (anchorage dependence), the oncogenically transformed cells lack this requirement (anchorage independence) and are often tumorigenic. However, the mechanism of loss of anchorage dependence is not fully understood. When normal rat fibroblasts are cultured in suspension without substrate attachment, the cell cycle arrests in G1 phase and the cyclin-dependent kinase inhibitor p27Kip1 protein and its mRNA accumulate. Conditional expression of oncogenic Ras induces the G1-S transition of the cell cycle and significantly shortens the half-life of p27Kip1 protein without altering its mRNA level. Inhibition of the activation of mitogen-activated protein (MAP) kinase by cyclic AMP-elevating agents and a MEK inhibitor prevents the oncogenic Ras-induced degradation of p27Kip1 (See Drosophila Dacapo). These results suggest that the loss of substrate attachment induces cell cycle arrest through the up-regulation of p27Kip1 mRNA, but the oncogenic Ras confers anchorage independence by accelerating p27Kip1 degradation through the activation of the MAP kinase signaling pathway. It appears that p27Kip1 is phosphorylated by MAP kinase in vitro and the phosphorylated p27Kip1 cannot bind to and inhibit cdk2 (Kawada, 1997).

During mitosis, chromatin is condensed into mitotic chromosomes and transcription is inhibited. These are two processes that might be thwarted by the chromatin remodeling activity of the SWI/SNF complexes. Brg1 and hBrm (see Drosophila Brahma), two components of human SWI/SNF (hSWI/SNF) complexes, have recently been shown to be phosphorylated during mitosis. This suggests that phosphorylation might be used as a switch to modulate SWI/SNF activity. Using an epitope-tag strategy, hSWI/SNF complexes were purified at different stages of the cell cycle: hSWI/SNF was found to be inactive in cells blocked in G2-M. Mitotic hSWI/SNF contains Brg1 but not hBrm, and is phosphorylated on at least two subunits, hSWI3 (Drosophila homolog: Moira) and Brg1 (Sif, 1998).

To test whether phosphorylation is the only modification required to inactivate the hSWI/SNF complex, a kinase was sought, capable of phosphorylating hSWI/SNF and altering its activity. Brg1 is known to be phosphorylated in unfertilized Xenopus eggs, which are naturally blocked in metaphase. Fractionation of Xenopus egg extracts revealed that a single peak of activity, which can perfectly phosphorylate Brg1, cofractionates with ERK1. To determine whether ERK1 can phosphorylate human SWI/SNF subunits, MEK1-activated ERK1 was tested for its ability to phosphorylate the hSWI/SNF complex. Wild-type GST-ERK1 can phosphorylate SWI/SNF subunits, whereas a kinase-deficient form of GST-ERK1 cannot. Other well-characterized mitotic kinases such as cdc2-cyclinA, cdc2-cyclinB and polo-like kinase 1, are unable to phosphorylate SWI/SNF subunits. When the hSWI/SNF complex that is phosphorylated in vitro by activated GST-ERK1 was tested for its ability to remodel chromatin templates, it was found to be inactive; however, when mutant GST-ERK1 (K63M) lacking kinase activity was used, hSWI/SNF was still able to remodel assembled templates in an ATP-dependent manner. Inhibition of hSWI/SNF activity depends on the presence of wild-type GST-ERK1, which is activated by GST-MEK1. hSWI/SNF which was isolated as cells traversed mitosis, regains activity when its subunits are dephosphorylated either in vitro or in vivo. It is proposed that this transitional inactivation and reactivation of hSWI/SNF is required for formation of a repressed chromatin structure during mitosis and reformation of an active chromatin structure as cells leave mitosis (Sif, 1998).

Active ERK1 can phosphorylate (and thus inactivate) hSWI/SNF complexes. The involvement of the mitogen-activated protein kinase (MAPK) signal transduction pathway in mitosis is not well understood; however, recently MEK1 has been shown to be involved in the golgi fragmentation that occurs during mitosis by activating a novel ERK, one associated with the golgi membrane. ERK1 can inhibit hSWI/SNF activity by phosphorylating Brg1, hBrm, and hSWI3 in vitro. These same SWI/SNF subunits are also phosphorylated in vivo. There are thirteen potential MAPK sites in Brg1, and seven in hSWI3, so it is not clear at this time what portion of these large proteins might be phosphorylated. It is possible that ERK1, or another MAPK with similar substrate specificity, might be involved in regulating hSWI/SNF activity during mitosis; however, it is also possible that other kinases are involved. Further experiments are needed to clarify the role of the MAPK family of proteins in regulating the activity of chromatin remodeling complexes in vivo. (Sif, 1998).

The nucleosomal response refers to the rapid phosphorylation of histone H3 on serine 10 and HMG-14 on serine 6 that occurs concomitantly with immediate-early (IE) gene induction in response to a wide variety of stimuli. Using antibodies against the phosphorylated residues, it has been shown that H3 and HMG-14 phosphorylation is mediated via different MAP kinase (MAPK) cascades, depending on the stimulus. The nucleosomal response elicited by TPA is ERK-dependent, whereas that elicited by anisomycin is p38 MAPK-dependent. In intact cells, the nucleosomal response can be selectively inhibited using the protein kinase inhibitor H89. MAPK activation and phosphorylation of transcription factors are largely unaffected by H89, whereas induction of IE genes is inhibited and its characteristics markedly altered. MSK1 (Drosophila homolog JIL-1) is considered the most likely kinase to mediate this response because (1) it is activated by both ERK and p38 MAPKs; (2) it is an extremely efficient kinase for HMG-14 and H3, utilizing the physiologically relevant sites; and (3) its activity towards H3/HMG-14 is uniquely sensitive to H89 inhibition. Thus, the nucleosomal response is an invariable consequence of ERK and p38 but not JNK/SAPK activation, and MSK1 potentially provides a link to complete the circuit between cell surface and nucleosome (Thomson, 1999).

The baculovirus/Sf9 cell system can be used to dissect signaling pathways involved in transmitting activating signals from the cell surface to the nucleus. Different combinations of the critical signaling proteins pp60v-src, p21v-ras, Raf-1 and ERK-1 were coexpressed the effects of resulting signaling cascades on the modifications of coexpressed transcription factors c-jun or c-fos were assayed. Activation of ERK-1 via Raf-1 and p21ras dependent signals can result in the hyperphosphorylation of c-jun. In contrast, c-fos appears to be the target of two Raf-1 activated modifying signals: one independent of ERK-1 and the other dependent on ERK-1 stimulation. Thus, coexpression of c-fos with pp60v-src, p21v-ras or constitutively active forms of Raf-1 results in a dramatic reduction of its electrophoretic mobility in the absence of coexpressed ERK-1. Activation of this ERK-1-independent pathway together with the ERK-1 dependent pathway that modifies c-jun results in additional modification of c-fos. The observation of a Raf-1 activated, ERK-independent signaling pathway is consistent with previous reports that constitutively active Raf-1 can, in some cell types, result in transformation or differentiation without activation of ERKs. These data indicate the presence of multiple Raf-1 activated pathways that lead to modification of transcription factors (Agarwal, 1995).

Recent studies have indicated that serine phosphorylation regulates the activities of STAT1 and STAT3 (see Drosophila Marelle). However, the kinase(s) responsible and the role of serine phosphorylation in STAT function remain unresolved. In the present studies, the growth factor-dependent serine phosphorylation of STAT1 and STAT3 were examined. The ERK family of mitogen-activated protein (MAP) kinases, but not JNK or p38, specifically phosphorylates STAT3 at serine 727 in response to growth factors. Evidence for additional mitogen-regulated serine phosphorylation is also provided. STAT1 is a relatively poor substrate for all MAP kinases tested both in vitro and in vivo. STAT3 serine phosphorylation, not its tyrosine phosphorylation, results in retarded mobility of the STAT3 protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Importantly, serine 727 phosphorylation negatively modulates STAT3 tyrosine phosphorylation, which is required for dimer formation, nuclear translocation, and the DNA binding activity of this transcriptional regulator. Interestingly, the cytokine interleukin-6 also stimulates STAT3 serine phosphorylation, but in contrast to growth factors, this occurs by an ERK-independent process (Chung, 1997).

The growth factor TGF-beta, bone morphogenetic proteins (BMPs) and related factors regulate cell proliferation, differentiation and apoptosis, controlling the development and maintenance of most tissues. Their signals are transmitted through the phosphorylation of the tumour-suppressor SMAD (See Drosophila MAD) proteins by receptor protein serine/threonine kinases (RS/TKs), leading to the nuclear accumulation and transcriptional activity of SMAD proteins. Smad1, which mediates BMP signals, is also a target of mitogenic growth-factor signaling through epidermal growth factor and hepatocyte growth factor receptor protein tyrosine kinases (RTKs). Phosphorylation occurs at specific serines within the region linking the inhibitory and effector domains of Smad1, and is catalyzed by the Erk family of mitogen-activated protein kinases. In contrast to the BMP-stimulated phosphorylation of Smad1, which affects carboxy-terminal serines and induces nuclear accumulation of Smad1, Erk-mediated phosphorylation specifically inhibits the nuclear accumulation of Smad1. Thus, Smadl receives opposing regulatory inputs through RTKs and RS/TKs; it is this balance that determines the level of Smad1 activity in the nucleus, and so possibly the role of Smad1 in the control of cell fate (Kretzschmar, 1997).

The tumor suppressor PTEN (see Drosophila Pten) dephosphorylates focal adhesion kinase (FAK) and inhibits integrin-mediated cell spreading and cell migration. Expression of PTEN selectively inhibits activation of the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway. PTEN expression in glioblastoma cells lacking the protein results in inhibition of integrin-mediated MAP kinase activation. Epidermal growth factor (EGF) and platelet-derived growth factor (PDGF)- induced MAPK activation are also blocked. To determine the specific point of inhibition in the Ras/Raf/ MEK/ERK pathway, these components were examined after stimulation by fibronectin or growth factors. Shc phosphorylation and Ras activity are inhibited by expression of PTEN, whereas EGF receptor autophosphorylation is unaffected. The ability of cells to spread at normal rates is partially rescued by coexpression of constitutively activated MEK1, a downstream component of the pathway. In addition, focal contact formation is enhanced as indicated by paxillin staining. The phosphatase domain of PTEN is essential for all of these functions, because PTEN with an inactive phosphatase domain does not suppress MAP kinase or Ras activity. In contrast to its effects on ERK, PTEN expression does not affect c-Jun NH2-terminal kinase (JNK) or PDGF-stimulated Akt. These data suggest that a general function of PTEN is to down-regulate FAK and Shc phosphorylation, Ras activity, downstream MAP kinase activation, and associated focal contact formation and cell spreading (Gu, 1998).

Heat shock transcription factor 1 (HSF-1) activates the transcription of heat shock genes in eukaryotes. Under normal physiological growth conditions, HSF-1 is a monomer. Its transcriptional activity is repressed by constitutive phosphorylation. Upon activation, HSF-1 forms trimers, acquires DNA binding activity, increases transcriptional activity, and appears as punctate granules in the nucleus. In this study, using bromouridine incorporation and confocal laser microscopy, it has been demonstrated that newly synthesized pre-mRNAs colocalize to the HSF-1 punctate granules after heat shock, suggesting that these granules are sites of transcription. Evidence that glycogen synthase kinase 3beta (GSK-3beta) and extracellular signal-regulated kinase mitogen-activated protein kinase (ERK MAPK) participate in the down regulation of HSF-1 transcriptional activity. Transient increases in the expression of GSK-3beta facilitate the disappearance of HSF-1 punctate granules and reduce hsp-70 transcription after heat shock. ERK is shown to be the priming kinase for GSK-3beta. Taken together, these results indicate that GSK-3beta and ERK MAPK facilitate the inactivation of activated HSF-1 after heat shock by dispersing HSF-1 from the sites of transcription (He, 1998).

GSK-3 consists of two isoforms: GSK-3alpha (51 kDa) and GSK-3beta (46 kDa). It was first identified as an enzymatic activity that phosphorylates and inactivates glycogen synthase. A second role of GSK-3 was found when studies showed that inhibition of phosphatase type I activity is relieved when GSK-3 phosphorylates phosphatase inhibitor 2. At least 15 other substrates have been reported to be phosphorylated by GSK-3, including the transcription factors c-Jun, JunD, c-myb, c-myc, L-myc, CREB, and NF-AT, most of which become inactivated when phosphorylated by GSK-3. GSK-3 tends to phosphorylate serine/threonine residues located next to a proline which, in turn, is near another serine residue that has been prephosphorylated by some other protein kinase (referred to as priming kinase). GSK-3 is constitutively active and, as a result, suppresses many of its substrates under normal physiological growth conditions. It appears that the activity of HSF-1 can be down regulated by protein kinases that are activated by diverse signal transduction pathways. The ERK MAPK pathway is activated during cell growth and development by multiple signaling pathways, which in turn are activated by growth factor receptors, G protein-coupled receptors, ceramide production, and a protein kinase C-dependent pathway. Recent evidence has suggested that ERK MAPK activation by heat shock may be through ceramide activation of protein kinase Raf-1. The pathways leading to GSK-3beta regulation are complex. GSK-3beta activity is down regulated by PKB/Akt, p70S6K, or p90rsk as a result of phosphorylation on serine residues. Activation of PKB/Akt leads to increased cell survival, as is the case with activation of ERK. The ability of ERK to mediate cell survival is dependent on the activation of transcription factors such as Elk1 and repair of damaged proteins. The ability of PKB/Akt to mediate cell survival is likely to be dependent on downstream effectors such as p70S6K and protein translation, activation of FRAP/TOR, and inhibition of GSK-3beta. Interestingly, heat shock stimulates the activity of GSK-3beta and ERK MAPK. The increase in GSK-3beta activity may occur through its phosphorylation on a tyrosine residue by an unknown tyrosine kinase. Thus, it appears that when activated, HSF-1 reduces the expression of most other genes and must be inactivated in a timely manner for cell proliferation to continue. The cell has developed an elegant mechanism for doing this, since some of the enzymes that control cell proliferation are capable of inactivating HSF-1 (He, 1998 and references).

Genetic and biochemical studies have identified kinase suppressor of Ras (KSR) to be a conserved component of Ras-dependent signaling pathways. To better understand the role of KSR in signal transduction, studies investigating the effect of phosphorylation and protein interactions on KSR function have been initiated. Five in vivo phosphorylation sites of KSR have been identified. In serum-starved cells, KSR contains two constitutive sites of phosphorylation (Ser297 and Ser392), which mediate the binding of KSR to the 14-3-3 family of proteins. In the presence of activated Ras, KSR contains three additional sites of phosphorylation (Thr260, Thr274, and Ser443), all of which match the consensus motif (Px[S/T]P) for phosphorylation by mitogen-activated protein kinase (MAPK). Treatment of cells with the MEK inhibitor PD98059 blocks phosphorylation of the Ras-inducible sites and activated MAPK associates with KSR in a Ras-dependent manner. Together, these findings indicate that KSR is an in vivo substrate of MAPK. Mutation of the identified phosphorylation sites does not alter the ability of KSR to facilitate Ras signaling in Xenopus oocytes, suggesting that phosphorylation at these sites may serve other functional roles, such as regulating catalytic activity. Interestingly, during the course of this study, it was found that the biological effect of KSR varies dramatically with the level of KSR protein expressed. In Xenopus oocytes, KSR functions as a positive regulator of Ras signaling when expressed at low levels, whereas at high levels of expression, KSR blocks Ras-dependent signal transduction. Likewise, overexpression of Drosophila KSR blocks R7 photoreceptor formation in the Drosophila eye. Therefore, the biological function of KSR as a positive effector of Ras-dependent signaling appears to be dependent on maintaining KSR protein expression at low or near-physiological levels (Cacace, 1999).

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).

A variety of transmembrane proteins, such as transforming growth factor-alpha (TGF-alpha), tumor necrosis factor-alpha (TNF-alpha) and L-selectin, undergo shedding, i.e. cleavage of the ectodomain, resulting in release of a soluble protein. Although the physiological relevance of ectodomain shedding is well recognized, little is known about the signaling mechanisms activating this process. Growth factor activation of cell surface tyrosine kinase receptors induces ectodomain cleavage of transmembrane TGF-alpha through activation of the Erk MAP kinase signaling cascade, without the need for new protein synthesis. In addition, expression of constitutively activated MEK1 or its downstream target Erk2 MAP kinase is sufficient to stimulate TGF-alpha shedding. The basal cleavage level in the absence of exogenous growth factor stimulation is due to p38 MAP kinase signaling. Accordingly, a constitutively activated MKK6, a p38 activator, activates TGF-alpha shedding in the absence of exogenous stimuli. In addition to TGF-alpha shedding, these mechanisms also mediate L-selectin and TNF-alpha cleavage. Thus, L-selectin shedding by neutrophils, induced by N-formylmethionyl-leucyl-phenylalanine, is strongly inhibited by inhibitors of Erk MAP kinase or p38 MAP kinase signaling. These results indicate that activation of Erk and p38 signaling pathways may represent a general physiological mechanism to induce shedding of a variety of transmembrane proteins. Considering the nature of the p38 and Erk MAP kinase pathways, it is tempting to speculate that a proximal phosphorylation event will play a key role in the activation of the protease(s) in response to the very diverse extracellular signals (Fan, 1999).

Histone acetylation and phosphorylation have separately been suggested to affect chromatin structure and gene expression. These two modifications are synergistic. Stimulation of mammalian cells by epidermal growth factor (EGF) results in rapid and sequential phosphorylation and acetylation of H3, and these modified H3 molecules are preferentially associated with the EGF-activated c-fos promoter in a MAP kinase-dependent manner. In addition, the prototypical histone acetyltransferase Gcn5 displays an up to 10-fold preference for phosphorylated (Ser-10) H3 over nonphosphorylated H3 as substrate in vitro, suggesting that H3 phosphorylation can affect the efficiency of subsequent acetylation reactions. Together, these results illustrate how the addition of multiple histone modifications may be coupled during the process of gene expression (Cheung, 2000).

Differential gene expression through alternative pre-mRNA splicing is crucial to various physiological and pathological conditions. Upon activation of B and T lymphocytes during an immune response, variant isoforms of the cell surface molecule CD44 are generated by alternative pre-mRNA splicing. In primary mouse T cells and in the murine LB-17 T-cell line upregulation of variant CD44 mRNA species upon T-cell activation requires activation of the MEK-ERK pathway. By employing mutant signaling molecules and a novel luciferase-based splice reporter system it has been demonstrated that the Ras-Raf-MEK-ERK signaling cascade, but not the p38 MAP-kinase pathway, activates a mechanism that retains variant CD44 exon v5 sequence in mature mRNA. In an inducible T-cell line phorbol-ester, constitutively active mutants of Ras, Rac, Raf, MEK and ERK induce the inclusion of CD44 v5 sequences into mature mRNA depending on signal-responsive exonic silencer sequences. Activation of the MEK-ERK pathway or of the JNK pathway suffices to inactivate splice silencing. The findings demonstrate that a highly conserved pleiotropic signaling pathway links extracellular cues to splice regulation, providing an avenue for tissue-specific, developmental or pathology-associated splicing decisions (Weg-Remers, 2001).

IEX-1 is an early response and NF-kappaB target gene implicated in the regulation of cellular viability. IEX-1 is a substrate for ERKs and IEX-1 and ERK regulate each other's activities. IEX-1 was isolated by phosphorylation screening with active ERK2 and is subsequently phosphorylated in vivo upon ERK activation. IEX-1 interacts with phosphorylated ERKs but not with c-jun N-terminal kinase (JNK) or p38. Upon phosphorylation by ERKs, IEX-1 acquires the ability to inhibit cell death induced by various stimuli. In turn, IEX-1 potentiates ERK activation in response to various growth factors. By using various IEX-1 mutants in which the ERK phosphoacceptor and/or ERK docking sites were mutated, it has been shown that the IEX-1 pro-survival effect is dependent on its phosphorylation state but not on its ability to potentiate ERK activation. Conversely, IEX-1-induced modulation of ERK activation requires ERK-IEX-1 association but is independent of IEX-1 phosphorylation. Thus, IEX-1 is a new type of ERK substrate that has a dual role in ERK signaling by acting both as an ERK downstream effector mediating survival and as a regulator of ERK activation (Garcia, 2002).

Evolution of human organismal complexity from a relatively small number of genes -- only approximately twice that of worm or fly -- is explained mainly by mechanisms generating multiple proteins from a single gene, the most prevalent of which is alternative pre-messenger-RNA splicing. Appropriate spatial and temporal generation of splice variants demands that alternative splicing be subject to extensive regulation, similar to transcriptional control. Activation by extracellular cues of several cellular signalling pathways can indeed regulate alternative splicing. This study addresses the link between signal transduction and splice regulation. The nuclear RNA-binding protein Sam68 is a new extracellular signal-regulated kinase (ERK) target. It binds exonic splice-regulatory elements of an alternatively spliced exon that is physiologically regulated by the Ras signalling pathway, namely exon v5 of CD44. Forced expression of Sam68 enhances ERK-mediated inclusion of the v5-exon sequence in mRNA. This enhancement is impaired by mutation of ERK-phosphorylation sites in Sam68, whereas ERK phosphorylation of Sam68 stimulates splicing of the v5 exon in vitro. Finally, Ras-pathway-induced alternative splicing of the endogenous CD44-v5 exon is abolished by suppression of Sam68 expression. These data define Sam68 as a prototype regulator of alternative splicing whose function depends on protein modification in response to extracellular cues (Matter, 2002).

Cell migration on extracellular matrix requires the turnover of integrin-dependent adhesions. The nonreceptor tyrosine kinases Src and FAK regulate focal-adhesion turnover by poorly understood mechanisms. ERK/MAP kinase-mediated activation of the protease Calpain 2 also promotes focal-adhesion turnover; however, it is not known if this is linked to the activities of Src and FAK. Calpain 2 has previously been demonstrated to colocalize with focal-adhesion structures and can cleave several focal-adhesion complex components, including FAK. Studies utilizing Calpain inhibitors or Calpain-deficient cells confirm that Calpain's role in regulating focal-adhesion turnover is necessary for cell migration. A novel and kinase-independent function has been identified for FAK as an adaptor molecule that mediates the assembly of a complex consisting of at least Calpain 2 and p42ERK. Mutation of proline residues (Pro2) in the amino-terminal region of FAK blocks direct binding with Calpain 2 and also prevents formation of the Calpain 2/p42ERK complex in cells. Both complex formation and MEK/ERK activity are associated with Calpain-mediated proteolysis of FAK and focal adhesion turnover during transformation and migration. Furthermore, FAK is necessary for recruiting both Calpain 2 and p42ERK/MAPK to peripheral adhesion sites facilitating maximal Calpain activity (Carragher, 2003).

These results indicate that FAK combines to spatially couple Calpain 2 to its upstream regulator ERK/MAP kinase. In live cells fluorescence intensity relating to Calpain activity is visibly reduced in FAK-/- cells relative to wild-type MEFs. In addition, Calpain activity localizes to the cell membrane in wild-type MEFs but not in FAK-/- cells. Total Calpain activity in extracted cell lysates is reduced in FAK-/- cells relative to wild-type MEFs. These results are consistent with the conclusion that FAK is required for assembly of a complex containing both Calpain 2 and phospho-ERK, the upstream activating kinase Calpain, and for recruitment of this complex to the plasma membrane, which is known to promote full Calpain activation (Carragher, 2003). In summary, this study describes a novel function for FAK as an adaptor molecule that permits the assembly of a Calpain 2/FAK/p42ERK complex. FAK-dependent complex assembly and localization to the cell periphery facilitates ERK/MAPK-induced activation of Calpain. This subsequently permits Calpain-mediated cleavage of FAK (and most likely other adhesion components), focal adhesion turnover, and cell migration. These findings provide a mechanistic explanation for the similar adhesion turnover and migratory defects of FAK null and Calpain-deficient cells (Carragher, 2003).

Loss of extracellular matrix (ECM) attachment leads to metabolic impairments that limit cellular energy production. Characterization of the metabolic alterations induced by ECM detachment revealed a dramatic decrease in uptake of glucose, glutamine, and pyruvate, and a consequent decrease in flux through glycolysis, the pentose phosphate pathway, and the tricarboxylic acid (TCA) cycle. However, flux through pyruvate dehydrogenase (PDH) is disproportionally decreased, concomitant with increased expression of the PDH inhibitory kinase, PDH kinase 4 (PDK4), and increased carbon secretion. Overexpression of ErbB2 maintains PDH flux by suppressing PDK4 expression in an Erk-dependent manner, and Erk signaling also regulates PDH flux in ECM-attached cells. Additionally, epidermal growth factor (EGF), a potent inducer of Erk, positively regulates PDH flux through decreased PDK4 expression. Furthermore, overexpression of PDK4 in ECM-detached cells suppresses the ErbB2-mediated rescue of ATP levels, and in attached cells, PDK4 overexpression decreases PDH flux, de novo lipogenesis, and cell proliferation. Mining of microarray data from human tumor data sets revealed that PDK4 mRNA is commonly down-regulated in tumors compared with their tissues of origin. These results identify a novel mechanism by which ECM attachment, growth factors, and oncogenes modulate the metabolic fate of glucose by controlling PDK4 expression and PDH flux to influence proliferation (Grassian, 2011).

Convergence of BMP and MAPK signaling pathways: impact of differential Smad1 phosphorylation on development and homeostasis

Integration of diverse signaling pathways is essential in development and homeostasis for cells to interpret context-dependent cues. BMP and MAPK signaling converge on Smads, resulting in differential phosphorylation. To understand the physiological significance of this observation, Smad1 mutant mice were generated carrying mutations that prevent phosphorylation of either the C-terminal motif required for BMP downstream transcriptional activation (Smad1C mutation) or of the MAPK motifs in the linker region (Smad1L mutation). Smad1C/C mutants recapitulate many Smad1-/- phenotypes, including defective allantois formation and the lack of primordial germ cells (PGC), but also show phenotypes that are both more severe (head and branchial arches) and less severe (allantois growth) than the null. Smad1L/L mutants survive embryogenesis but exhibit defects in gastric epithelial homeostasis correlated with changes in cell contacts, actin cytoskeleton remodeling, and nuclear ß-catenin accumulation. In addition, formation of PGCs is impaired in Smad1L/L mutants, but restored by allelic complementation in Smad1C/L compound mutants. These results underscore the need to tightly balance BMP and MAPK signaling pathways through Smad1 (Aubin, 2004).

Histological analysis reveals that the cytology of the Smad1L/L gastric mucosa was perturbed. The stomach of rodents consists of a proximal keratinized epithelium and a distal glandular mucosa. The glandular stomach is subdivided in three zones: a proximal zymogenic, a middle mucoparietal, and a distal pure mucus zone. In the zymogenic zone, the four main cell types show a stereotyped distribution: mucus-producing and zymogenic cells are found in the upper third and at the base of the unit, respectively, whereas parietal and enteroendocrine cells are distributed along the entire length. A common stem cell progenitor located in the isthmus repopulates each unit. In the Smad1L/L stomach, all the expected cell types were represented albeit with variations in their relative proportion. The zymogenic cells recognizable by their strong affinity for hematoxylin were severely depleted in linker mutants compared with wild-type samples. In addition, the parietal cells were more numerous in mutant compared with wild-type stomachs, as revealed by immunostaining with the H+K+ATPase proton pump antibody specific for this cell type. Mucous-producing and enteroendocrine cells were not substantially affected. Stomach morphogenesis and primordial gastric unit formation was similar in wild-type and Smad1L/L mutants (tested at E13.5, E18.5, and postnatal day 0). The altered cellularity of Smad1L/L gastric epithelium was not associated with altered specification of the stomach epithelium into an intestinal identity, as revealed at P0 and in adults by the absence of alkaline phosphatase staining, a hallmark of intestinal transformation. Thus, preventing MAPK phosphorylation of Smad1 alters stomach homeostasis (Aubin, 2004).

The analysis of the Smad1 linker phenotype provides the first evidence for a role of integrating BMP and MAPK signaling in epithelial homeostasis. Rendering the Smad1 protein resistant to MAPK-mediated phosphorylation alters homeostasis of the gastric epithelium as reflected by the increased parietal cells and decreased zymogenic cell populations. This characterization provides some hints on how MAPK signals affect homeostasis via Smad1. Smad1 is expressed at high levels in parietal cells, a cell population that is expanded in Smad1L/L mutants. Parietal cell depletion experiments in transgenic mice have demonstrated that this cell type influences decision-making among gastric epithelial cell precursors and modulates the migration-associated terminal differentiation programs of the pit (mucous-producing) and zymogenic lineages. Changes in the parietal cell population in Smad1L/L mutants are therefore most likely to impede this migration-associated differentiation and underlie the perturbed homeostasis. However, a cell-autonomous role in the differentiation of zymogenic cells cannot be excluded because Smad1 is also expressed at low levels in these cells (Aubin, 2004).

The severity of the stomach alteration in some of the Smad1L/- mutants unveils the importance of fine-tuning BMP signaling during organogenesis. Stomach morphogenesis is known to rely on several signaling cascades, and requires integration of TGFß and FGF signals. The source of MAPK signaling that is involved in this process remains to be identified, but FGF10 and its cognate receptor FGFR2b constitute potential candidates. The glandular stomach is particularly sensitive to the action of BMP and FGF signaling; they are both essential for its formation . This potential scenario is reminiscent of the role of FGF/IGF signaling in antagonizing BMP action in neural development in Xenopus. The extent of the defect on both the epithelium and the muscular layer in Smad1L/- stomach, however, may indicate that cell autonomous as well as non-cell-autonomous factors are involved in this phenotype (Aubin, 2004).

Taken together, this study underscores the importance of fine-tuning the balance of BMP and MAPK signaling through Smad1 in a physiological context. The unforeseen germ-cell and gastric epithelial phenotype, as well as the cellular consequences of the linker mutation, raise interesting questions about the underlying mechanisms of BMP-MAPK cross-talk. They also support the notion that MAPK-dependent Smad1 phosphorylation may not only serve to inhibit BMP signaling but may serve other important cellular functions as well. An outstanding issue remains to identify the source of MAPK signaling in affected tissues. Another interesting aspect is how Smad1 linker phosphorylation impinges on Wnt signaling through ß-catenin. The Smad1C and Smad1L mutant lines provide useful tools to tackle these questions and to further dissect the functional significance of integrating diverse signaling pathways (Aubin, 2004).

Map kinase and cell cycle

Induction of G2/M phase transition in mitotic and meiotic cell cycles requires activation by phosphorylation of the protein phosphatase Cdc25. Although Cdc2/cyclin B and polo-like kinase (PLK) can phosphorylate and activate Cdc25 in vitro, phosphorylation by these two kinases is insufficient to account for Cdc25 activation during M phase induction. This study demonstrates that p42 MAP kinase (MAPK), the Xenopus ortholog of ERK2, is a major Cdc25 phosphorylating kinase in extracts of M phase-arrested Xenopus eggs. In Xenopus oocytes, p42 MAPK interacts with hypophosphorylated Cdc25 before meiotic induction. During meiotic induction, p42 MAPK phosphorylates Cdc25 at T48, T138, and S205, increasing Cdc25's phosphatase activity. In a mammalian cell line, ERK1/2 interacts with Cdc25C in interphase and phosphorylates Cdc25C at T48 in mitosis. Inhibition of ERK activation partially inhibits T48 phosphorylation, Cdc25C activation, and mitotic induction. These findings demonstrate that ERK-MAP kinases are directly involved in activating Cdc25 during the G2/M transition (Wang, 2007).

Having established the role of ERK-MAP kinases in the Cdc25C-activation system, the next question is whether MAPK is the only previously unrecognized kinase involved in Cdc25C activation during M phase induction. Although phosphorylation of Cdc25 by p42 MAPK accounts for Mos-induced Cdc2 activation in Xenopus oocyte extracts, this finding does not explain how the phosphatase inhibitor OA, which can bypass the requirement for progesterone to induce Xenopus oocyte maturation, induces Cdc2 activation without activation of p42 MAPK. In some of the previous studies, inhibition of MAPK activation delays, but does not block, progesterone-induced Xenopus oocyte maturation. In contrast to the natural stimulus progesterone, forced activation of MAPK does not always induce Cdc2 activation in Xenopus oocytes or cell-free systems. When it does, there is a significant delay between robust MAPK activation and Cdc2 dephosphorylation. These multiple unexplained observations indicate that progesterone stimulation of Xenopus oocytes activates at least one additional kinase that is involved in Cdc25 activation. While sufficient activation of p42 MAPK is probably able to initiate and amplify Cdc25 activation in cooperation with Cdc2 kinase and Plx1, composite roles of p42 MAPK and this additional kinase may cause quicker and more robust activation of Cdc25 and Cdc2/cyclin B during Xenopus oocyte maturation. In agreement with this hypothesis, gel filtration of MEE resulted in recovery of a much broader peak of Cdc25-phosphorylating activity than can be accounted for by p42 MAPK and/or Cdc2 activities. Depletion of MAPK from QE1, which contained most of the unaccounted-for Cdc25 phosphorylating activity in Xenopus egg extracts, removed only 50% of the Cdc25-phosphorylating activity. The remaining activity can be stabilized by OA and thiophosphorylation and is almost certainly due to a kinase complex of >200 kDa that does not contain Cdc2, Plx1, or p42 MAPK. Thus, it is hypothesized that this yet-to-be-identified Cdc25-phosphorylating activity represents the additional kinase or one of the additional kinases involved in Cdc25 activation during Xenopus oocyte maturation. The presence of alternative regulators in Cdc25 activation may explain why studies by different investigators have yielded differing results with respect to the requirement of MAPK for Cdc2 activation during Xenopus oocyte maturation (Wang, 2007).

Dual-mode regulation of the APC/C by CDK1 and MAPK controls meiosis I progression and fidelity

Female meiosis is driven by the activities of two major kinases, cyclin-dependent kinase 1 (Cdk1) and mitogen-activated protein kinase (MAPK). To date, the role of MAPK in control of meiosis is thought to be restricted to maintaining metaphase II arrest through stabilizing Cdk1 activity. This study finds that MAPK and Cdk1 play compensatory roles to suppress the anaphase-promoting complex/cyclosome (APC/C) activity early in prometaphase, thereby allowing accumulation of APC/C substrates essential for meiosis I. Furthermore, inhibition of MAPK around the onset of APC/C activity at the transition from meiosis I to meiosis II led to accelerated completion of meiosis I and an increase in aneuploidy at metaphase II. These effects appear to be mediated via a Cdk1/MAPK-dependent stabilization of the spindle assembly checkpoint, which when inhibited leads to increased APC/C activity. These findings demonstrate new roles for MAPK in the regulation of meiosis in mammalian oocytes (Nabti, 2014).

Table of contents

rolled/MAPK: Biological Overview | Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References

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