MAPK6 and MAPK4 (also known as ERK3 and ERK4) are vertebrate-specific atypical MAP kinases. Atypical MAPK are less well characterized than their conventional counterparts, and are generally classified as such based on their lack of activation by MAPKK family members. Unlike the conventional MAPK proteins, which contain a Thr-X-Tyr motif in the activation loop, MAPK6 and 4 have a single Ser-Glu-Gly phospho-acceptor motif (reviewed in Coulombe and Meloche, 2007; Cargnello et al, 2011). MAPK6 is also distinct in being an unstable kinase, whose turnover is mediated by ubiquitin-dependent degradation (Coulombe et al, 2003; Coulombe et al, 2004). The biological functions and pathways governing MAPK6 and 4 are not well established. MAPK6 and 4 are phosphorylated downstream of class I p21 activated kinases (PAKs) in a RAC- or CDC42-dependent manner (Deleris et al, 2008; Perander et al, 2008; Deleris et al, 2011; De La Mota-Peynado et al, 2011). One of the only well established substrates of MAPK6 and 4 is MAPKAPK5, which contributes to cell motility by promoting the HSBP1-dependent rearrangement of F-actin (Gerits et al, 2007; Kostenko et al, 2009a; reviewed in Kostenko et al, 2011b). The atypical MAPKs also contribute to cell motility and invasiveness through the NCOA3:ETV4-dependent regulation of MMP gene expression (Long et al, 2012; Yan et al, 2008; Qin et al, 2008). Both of these pathways may be misregulated in human cancers (reviewed in Myant and Sansom, 2011; Kostenko et al, 2012)
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Signaling by AKT is one of the key outcomes of receptor tyrosine kinase (RTK) activation. AKT is activated by the cellular second messenger PIP3, a phospholipid that is generated by PI3K. In ustimulated cells, PI3K class IA enzymes reside in the cytosol as inactive heterodimers composed of p85 regulatory subunit and p110 catalytic subunit. In this complex, p85 stabilizes p110 while inhibiting its catalytic activity. Upon binding of extracellular ligands to RTKs, receptors dimerize and undergo autophosphorylation. The regulatory subunit of PI3K, p85, is recruited to phosphorylated cytosolic RTK domains either directly or indirectly, through adaptor proteins, leading to a conformational change in the PI3K IA heterodimer that relieves inhibition of the p110 catalytic subunit. Activated PI3K IA phosphorylates PIP2, converting it to PIP3; this reaction is negatively regulated by PTEN phosphatase. PIP3 recruits AKT to the plasma membrane, allowing TORC2 to phosphorylate a conserved serine residue of AKT. Phosphorylation of this serine induces a conformation change in AKT, exposing a conserved threonine residue that is then phosphorylated by PDPK1 (PDK1). Phosphorylation of both the threonine and the serine residue is required to fully activate AKT. The active AKT then dissociates from PIP3 and phosphorylates a number of cytosolic and nuclear proteins that play important roles in cell survival and metabolism. For a recent review of AKT signaling, please refer to Manning and Cantley, 2007.
The PAKs (p21-activated kinases) are a family of serine/threonine kinases mainly implicated in cytoskeletal rearrangements. All PAKs share a conserved catalytic domain located at the carboxyl terminus and a highly conserved motif in the amino terminus known as p21-binding domain (PBD) or Cdc42/Rac interactive binding (CRIB) domain. There are six mammalian PAKs that can be divided into two classes: class I (or conventional) PAKs (PAK1-3) and class II PAKs (PAK4-6). Conventional PAKs are important regulators of cytoskeletal dynamics and cell motility and are additionally implicated in transcription through MAPK (mitogen-activated protein kinase) cascades, death and survival signaling and cell cycle progression (Chan and Manser 2012).
PAK1, PAK2 and PAK3 are direct effectors of RAC1 and CDC42 GTPases. RAC1 and CDC42 bind to the CRIB domain. This binding induces a conformational change that disrupts inactive PAK homodimers and relieves autoinhibition of the catalytic carboxyl terminal domain (Manser et al. 1994, Manser et al. 1995, Zhang et al. 1998, Lei et al. 2000, Parrini et al. 2002; reviewed by Daniels and Bokoch 1999, Szczepanowska 2009). Autophosphorylation of a conserved threonine residue in the catalytic domain of PAKs (T423 in PAK1, T402 in PAK2 and T436 in PAK3) is necessary for the kinase activity of PAK1, PAK2 and PAK3. Autophosphorylation of PAK1 serine residue S144, PAK2 serine residue S141, and PAK3 serine residue S154 disrupts association of PAKs with RAC1 or CDC42 and enhances kinase activity (Lei et al. 2000, Chong et al. 2001, Parrini et al. 2002, Jung and Traugh 2005, Wang et al. 2011). LIMK1 is one of the downstream targets of PAK1 and is activated through PAK1-mediated phosphorylation of the threonine residue T508 within its activation loop (Edwards et al. 1999). Further targets are the myosin regulatory light chain (MRLC), myosin light chain kinase (MLCK), filamin, cortactin, p41Arc (a subunit of the Arp2/3 complex), caldesmon, paxillin and RhoGDI, to mention a few (Szczepanowska 2009).
Class II PAKs also have a CRIB domain, but lack a defined autoinhibitory domain and proline-rich regions. They do not require GTPases for their kinase activity, but their interaction with RAC or CDC42 affects their subcellular localization. Only conventional PAKs will be annotated here.
Inactive p21-associated kinases (PAKs), PAK1, PAK2 and PAK3, form homodimers that are autoinhibited through in trans interaction between the inhibitory N-terminus of one PAK molecule and the catalytic domain of the other PAK molecule. All PAK isoforms are direct effectors of RAC1 and CDC42 GTPases. RAC1 and CDC42 bind to a highly conserved motif in the amino terminus of PAK known as p21-binding domain (PBD) or Cdc42/Rac interactive binding (CRIB) domain. This binding induces a conformational change that disrupts PAK homodimers and relieves autoinhibition of the catalytic carboxyl terminal domain, thereby inducing autophosphorylation at several sites and enabling the phosphorylation of exogenous substrates (Manser et al. 1994, Manser et al. 1995, Zhang et al. 1998, Lei et al. 2000, Parrini et al. 2002; reviewed by Daniels and Bokoch 1999, Szczepanowska 2009).
Binding of PAK1, PAK2 or PAK3 to GTP-bound RAC1 or CDC42 disrupts PAK homodimers and allows PAK autophosphorylation. Autophosphorylation of a conserved threonine residue in the catalytic domain of PAKs (T423 in PAK1, T402 in PAK2 and T436 in PAK3) is necessary for the kinase activity of PAK1, PAK2 and PAK3. Autophosphorylation of PAK1 serine residue S144, PAK2 serine residue S141, and PAK3 serine residue S154 disrupts association of PAKs with RAC1 or CDC42 GTPases and enhances kinase activity (Lei et al. 2000, Chong et al. 2001, Parrini et al. 2002, Jung and Traugh 2005, Wang et al. 2011).
IGF2BP1 is a cytosolic RNA-binding protein that recruits target transcripts to RNP particles for storage or transport. These RNP particles also restrict access of the translational machinery and micro-RNAs to the transcript and in this way affect rates of protein translation (reviewed in Bell et al, 2013). IGFBP1 binds to the 3' UTR of MAPK4 mRNA and inhibits its translation. This antagonizes MAPKAPK5 activation and HSBP1 phosphorylation and in this manner affects F-actin rearrangements and cell motility (Stohr et al, 2012; Kostenko et al, 2009a; reviewed in Kostenko et al, 2012).
MAPK6 is an unstable protein that is constitutively degraded by the ubiquitin-proteasome system. Degradation is promoted by two destabilization regions in the N-terminal region of MAPK6 which are required for the conjugation of ubiquitin to the free amino-terminal by an unknown ligase (Coulombe et al, 2003; Coulombe et al, 2004). Although in this reaction ubiquitination is depicted as occuring in the cytosol, it may also occur in the nucleus.
MYC binds to consensus sites in the MAPKAPK5 gene promoter as assessed by ChIP (Kress et al, 2011). Although not depicted here and not experimentally validated in this context, MYC likely binds the promoter in the context of a MYC:MAX heterodimer.
The atypical MAPKs MAPK6 (also known as ERK3) and MAPK4 (also known as ERK4) lack the conserved activation loop T-X-Y motif of the conventional MAPKs, and are thus not substrates for the dual-specificity MAPK kinases (reviewed in Coulombe and Meloche, 2007; Cargnello and Roux, 2011). The corresponding loop of MAPK6 and 4 instead contain a S-E-G motif that is phosphorylated at serine 189 and serine 186, respectively, by class I p21 activated kinases (PAKs) in a RAC- or CDC42-dependent manner (Deleris et al, 2008; Perander et al, 2008; Deleris et al, 2011; De La Mota-Peynado et al, 2011). Phosphorylation of the atypical MAPKs is not responsive to any identified extracellular stimulus, but rather occurs constitutively (Deleris et al, 2008).
MAPKAPK5 is phosphorylated at serine 115 by the catalytic subunit of PKA, which translocates into the nucleus in response to elevated cellular cAMP levels. Phosphorylation at serine 115 promotes the cytoplasmic relocalization of MAPKAPK5 and is required for HSBP1-dependent rearrangements of F-actin in response to PKA (Gerits et al, 2007; Kostenko et al, 2011a; Kostenko et al, 2009; reviewed in Kostenko et al, 2011b)
MAPK6 is proposed to phosphorylate NCOA3 at serine 857. This phosphorylation is required for NCOA3 to interact with the transcription factor ETV4 (also known as PEA3). Together, ETV4 and NCOA3 bind to the promoters and regulate the expression of metalloprotease genes such as MMP2 and MMP10 and in this way contribute to cell motility and invasiveness in lung cancer (Long et al, 2012; Qin et al, 2008; Yan et al, 2008; Li et al, 2008; reviewed in Kostenko et al, 2012).
In proliferating cells, p-S189 MAPK6 and pS-186 MAPK4 bind to the MAPK effector kinase MAPKAPK5 (also known as MK5) through an FRIEDE motif in the C-terminal region (Perander et al, 2008; Deleris et al, 2008; Aberg et al, 2009). This motif, which is unique to MAPK6 and MAPK4 binds to the C-terminal 50 residues of MAPKAKP5 and is required for both the cytoplasmic accumulation and the activation of MAPKAPK5 (Aberg et al, 2009; Aberg et al, 2006; Seternes et al, 2004; Deleris et al, 2008). Cytoplasmic redistribution of MAPKAPK5 depends on the protein-protein interaction with MAPK6 or 4 and not the activity of any of the kinases, as cytoplasmic localization is abrogated by disruption of the interaction interface but not by kinase-dead versions of MAPK6, 4 or MAPKAPK5 itself (Aberg et al, 2006; Seternes et al, 2004).
Activated MAPK6 and MAPK4 promote the phosphorylation of MAPKAPK5 on threonine 182, activating it (Deleris et al, 2008; Aberg et al, 2006; Aberg et al, 2009; Seternes et al, 2004; Perander et al, 2008). Thr182 phosphorylation may result in part from autophosphorylation stimulated by MAPK6 binding, rather than direct phosphorylation by MAPK6, as an ATP-binding pocket mutant of MAPKAKP5 is not phosphorylated in response to MAPK6 (Seternes et al, 2004; Schumacher et al, 2004). There is conflicting evidence as to whether a catalytically inactive MAPK6 mutant can promote MAPKAPK5 phosphorylation (Schumacher et al, 2004; Seternes et al, 2004; Deleris et al, 2008). These conflicting results can be reconciled by the suggestion that inactive MAPK6 promotes MAPKAPK5 phosphorylation through heterodimerization with active MAPK4 (Kant et al, 2006). Phosphorylation of MAPKAPK5 in response to MAPK4/6 signaling promotes its cytoplasmic relocalization (Shumacher et al, 2004; Aberg et al, 2006; Deleris et al, 2008; Seternes et al, 2004).
Phosphorylated FOXO3 binds to consensus sites in the promoter of MIR34B and C genes as assessed by ChIP, promoting expression of the microRNAs (Kress et al, 2011).
NCOA3 interacts with ETV4 (also known as PEA3) in a manner that depends on S857 phosphorylation (Long et al, 2012). ETV4 and NCOA3 coactivate expression of a number of MMP genes, which play roles in cell motility and invasiveness in a subset of lung carcinomas (Long et al, 2012; Qin et al, 2008; Yan et al, 2008).
MAPK6-dependent phosphorylation of NCOA3 S857 promotes its interaction with the transcription factor ETV4 and increases the occupancy at promoters of the MMP2 and 10 genes in vivo as assessed by ChIP (Long et al, 2012; Qin et al, 2008; Yan et al, 2008). MMP gene expression is associated with invasiveness in lung and breast cancer, and MAPK6 is highly expressed in a subset of human lung carcinomas (Long et al, 2012; Qin et al, 2008; Yan et al, 2008; Li et al, 2008; reviewed in Kostenko et al, 2012).
Activated MAPKAPK5 phosphorylates FOXO3 at serine 215, promoting its activation and translocation to the nucleus. In the nucleus, FOXO3 promotes the expression of miR-34B and C, which in turn represses translation of MYC RNA (Kress et al, 2011; reviewed in Myant and Sansom, 2011; Kostenko et al, 2012).
Binding of IGF2BP1 to the 3' UTR of MAPK4 mRNA inhibits its translation and in this way antagonizes the MAPKAPK5-dependent phosphorylation of HSBP1 (Stohr et al, 2012; Kostenko et al, 2009a; reviewed in Kostenko et al, 2012).
Despite differences in their overall cellular distribution (MAPK6 is found in both the nucleus and the cytosol, while MAPK4 is predominantly found in the cytosol), both MAPK4 and 6 shuttle between the cytosol and the nucleus. Nuclear import of both proteins occurs through an active temperature sensitive pathway, while nuclear export depends on XPO1 (Aberg et al, 2006; Julien et al, 2003).
Despite differences in their overall cellular distribution (MAPK6 is found in both the nucleus and the cytosol, while MAPK4 is predominantly found in the cytosol), both MAPK4 and 6 shuttle between the cytosol and the nucleus. Nuclear import of both proteins occurs through an active temperature sensitive pathway, while nuclear export depends on XPO1 (Aberg et al, 2006; Julien et al, 2003).
MAPK6 is a short-lived protein with a half-life of 30 minutes in proliferating cells. Turnover is promoted by the conjugation of ubiquitin to the free amino terminal by an unknown ligase and subsequent degradation by the 26 S proteasome (Coulombe et al, 2003; Coulombe et al, 2004). Ubiquitination and degradation of MAPK6 may also occur in the nucleus as well as the cytosol.
Translation of MYC mRNA is negatively regulated by miR-34B and C microRNAs (Kress et al, 2011). miR-34 miRNAs bind and cause degradation of MYC mRNA, resulting in decreased level of MYC protein product (reviewed in Myant and Sansom, 2011; Kostenko et al, 2012).
MYC binds to consensus sites in the MAPKAPK5 promoter to promote transcription. This completes a negative feedback loop controlling MYC expression, as MAPKAPK5 itself negatively regulates MYC protein levels through FOXO3 and miR-34B and C (Kress et al, 2011). This pathway is disrupted in colorectal cancer, leading to aberrant cellular proliferation (Kress et al, 2011; reviewed in Myant and Sansom, 2011; Kostenko et al, 2012).
Binding of MAPK6 or 4 to MAPKAPK5 promotes its redistribution to the cytosol. This depends on a functional protein-protein interaction interface between the two proteins. Cytoplasmic translocation of MAPKAPK5 occurs even in the presence of catalytically inactive MAPK6 or MAPK6 and vice versa, MAPK6 and MAPK4 still provoke nuclear exclusion of kinase inactive MAPKAPK5 (Aberg et al, 2006; Seternes et al, 2004; Kant et al, 2006; reviewed in Kostenko et al, 2012). Phosphorylation of MAPK6 or MAPK4 at S189 or S186 respectively, is required for binding and translocation of MAPKAPK5 to the cytosol (Perander et al, 2008; Deleris et al, 2008; De La Mota-Peynado et al, 2011).
HSBP1, also known as HSP27, is small actin-binding protein with roles in cytoskeletal regulation as well as other processes. HSBP1 is a substrate for MAPKAPK5 both in vitro and in vivo, and phosphorylation of serine residues stimulates forskolin-induced F-actin rearrangements (Sun et al, 2007; Tak et al, 2007; Gerits et al, 2007; New et al, 1998; Seternes et al, 2004; Kostenko et al, 2009a; Kostenko et al, 2009b; Kostenko et al, 2011a; reviewed in Kostenko et al, 2011b; Kostenko et al, 2012). There are divergent reports about the physiological relevance of HSBP1 phosphorylation on actin polymeriztion and cell motility (Lavoie et al, 1995; Lamalice et al, 2007; Katsogiannou et al, 2014; Rousseau et al, 2000; Doshi et al, 2010; Stohr et al, 2012). Actin cytoskeletal rearrangements through the MAPK4 pathway are controlled in part by the IGF2BP1-mediated downregulation of MAPK4 translation which abrogates MAPKAPK5 activity and HSBP1 phosphorylation (Stohr et al, 2012).
MAPKAPK5-dependent phosphorylation of FOXO3 promotes its nuclear localization (Kress et al, 2011). In the nucleus, FOXO3 promotes expression of miR-34B and C and thereby downregulates expression of c-MYC RNA (Kress et al, 2011; reviewed in Myant and Sansom, 2011; Kostenko et al, 2012).
Activated MAPKAPK5 phosphorylates HSP40/DNAJB1 at serines 149, 151 and 171, promoting the ATP hydrolysis activity of the HSP40/HSP70 complex and enhancing the repression of heat shock factor 1 (HSF1) driven transcription by HSP40/DNAJB1 (Kostenko et al, 2014).
The phosphatases CDC14A and CDC14B bind directly to MAPK6 as assessed by yeast two hybrid and by co-immunoprecipitation (Tanguay et al, 2010; Hansen et al, 2008). CDC14 phosphatases are able to reverse the CDK1-dependent phosphorylation of MAPK6 in vitro, and overexpression of WT but not catalytically inactive forms of CDC14A or B in vivo leads to dephosphorylation of T698 (Hansen et al, 2008; Tanguay et al, 2010). These results suggest that CDC14 phosphatases reverse the CDK1-dependent phosphorylation of MAPK6 during mitosis. These reactions are depicted as occuring in the nucleoplasm, but the site of action has not been determined, and CDC14 and MAPK6 colocalize throughout the cell (Hansen et al, 2008).
CDC14A and B bind to MAPK6 and antagonize the CDK1-dependent phosphorylation of the C-terminal extension during mitosis (Tanguay et al, 2010; Hansen et al, 2008).
MAPK6 is hyperphosphorylated by CDK1 at multiple sites in the C-terminal extension, and this phosphorylation is associated with the stabilization of MAPK6 protein in mitosis. Residues S684, S688, T698 and S705 have been identified as in vitro targets of CDK1, and phosphorylation of T698 has also been demonstrated in vivo (Tanguay et al, 2010). The role of hyperphosphorylated MAPK6 during mitosis has not been established, and although the CDK1-dependent phosphorylation of MAPK6 is depicted as occuring in the nucleus, the site of action has also not been determined. CDK1-dependent hyperphosphorylation of the C-terminal tail is reversed by the phosphatases CDC14A and B (Tanguay et al, 2010; Hansen et al, 2008).
MAPK6 expression is stimulated in response to cytokines through JUN-mediated transcriptional activation. Phosphorylated JUN binds to a canonical JUN-binding site in the MAPK6 gene as assessed by ChIP, and stimulates transcription in response to TNFalpha (Wang et al, 2014).
MAPK6 interacts with Cyclin D3 (CCND3) through its C-terminal extension, and this interaction is stabilized by overexpression of CDC14 phosphatase (Sun et al, 2006; Hansen et al, 2008). Although the physiological relevance of the interaction between MAPK6 and CCND3 is not known, both proteins regulate cell cycle entry and MAPK6 is stabilized during differentiation and upon inhibition of proliferation (Coulombe et al, 2003; Bartkova et al, 1998; Julien et al, 2003).
SEPT7 forms a ternary complex with MAPK6 and MAPKAPK5 that contributes to neuronal development through the phosphorylation of the Binders of RHO GTPase (BORG) proteins (Brand et al, 2012). Septins are cytoskeletal GTP-binding proteins that form filaments and contribute to processes such as cytokinesis, cell polarity and cell division, among others (reviewed in Spiliotis and Nelson, 2006). Interaction of septin proteins with the CDC42 effector proteins 2, 3 and 5 (CDC42EP2, 3, 5; also known as BORG1, 2 and 3) inhibits formation of septin filaments (Jouberty et al, 2001).
MAPK6 and MAPKAPK5 directly phosphorylate the septin regulating proteins CDC42EP2, 3 and 5 (also known as BORG1, 2 and 3 for Binders of Rho GTPases) in vitro. BORG/CDC42EP proteins interact with septins through the septin GTPase domain and inhibit filament formation. This effect of the BORG proteins on septin filamentation is itself inhibited by CDC42 (Jouberty et al, 2001; reviewed in Spiliotis and Nelson, 2006). The interaction between SEPT7 and the CDC42EP proteins may facilitate their recruitment to the ternary MAPK6:MAPKAPK5:SEPT7 complex for phosphorylation, although the significance of this phosphorylation is not yet clear (Brand et al, 2012).
MAPKAPK5 phosphorylates FOXO1 at S215. This phosphorylation is essential for the FOXO1-dependent activation of RAG gene transcription during B-cell development and promotes the direct binding of FOXO1 to the RAG gene promoter (Chow et al, 2013).
MAPK6 and MAPKAPK5 bind to the GDP-exchange factor KALRN, also known as Kalirin-7 or KAL7 (Brand et al, 2012). CaMKII-dependent phosphorylation of KALRN7 results in activation of PAK kinases in dendritic spines, potentially establishing a positive feedback loop that governs the MAPK6:MAPKAPK5 module in neuronal developement (Penzes et al, 2003; Xie et al, 2007).
After phosphorylation by MAPKAPK5, p-S215 FOXO1 binds to the RAG gene promoter to promote transcription (Lin et al, 2010; Ochiai et al, 2012; Chow et al, 2013).
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MAPK6:p-T182
MAPKAPK5PAK1, PAK2 and PAK3 are direct effectors of RAC1 and CDC42 GTPases. RAC1 and CDC42 bind to the CRIB domain. This binding induces a conformational change that disrupts inactive PAK homodimers and relieves autoinhibition of the catalytic carboxyl terminal domain (Manser et al. 1994, Manser et al. 1995, Zhang et al. 1998, Lei et al. 2000, Parrini et al. 2002; reviewed by Daniels and Bokoch 1999, Szczepanowska 2009). Autophosphorylation of a conserved threonine residue in the catalytic domain of PAKs (T423 in PAK1, T402 in PAK2 and T436 in PAK3) is necessary for the kinase activity of PAK1, PAK2 and PAK3. Autophosphorylation of PAK1 serine residue S144, PAK2 serine residue S141, and PAK3 serine residue S154 disrupts association of PAKs with RAC1 or CDC42 and enhances kinase activity (Lei et al. 2000, Chong et al. 2001, Parrini et al. 2002, Jung and Traugh 2005, Wang et al. 2011). LIMK1 is one of the downstream targets of PAK1 and is activated through PAK1-mediated phosphorylation of the threonine residue T508 within its activation loop (Edwards et al. 1999). Further targets are the myosin regulatory light chain (MRLC), myosin light chain kinase (MLCK), filamin, cortactin, p41Arc (a subunit of the Arp2/3 complex), caldesmon, paxillin and RhoGDI, to mention a few (Szczepanowska 2009).
Class II PAKs also have a CRIB domain, but lack a defined autoinhibitory domain and proline-rich regions. They do not require GTPases for their kinase activity, but their interaction with RAC or CDC42 affects their subcellular localization. Only conventional PAKs will be annotated here.
MAPK6:p-T182
MAPKAPK5MAPK6:p-T182
MAPKAPK5FOXO3:MIR34B,C
genesNOCA3:ETV4:MMP2,10
genesAnnotated Interactions
MAPK6:p-T182
MAPKAPK5MAPK6:p-T182
MAPKAPK5MAPK6:p-T182
MAPKAPK5MAPK6:p-T182
MAPKAPK5MAPK6:p-T182
MAPKAPK5MAPK6:p-T182
MAPKAPK5FOXO3:MIR34B,C
genesFOXO3:MIR34B,C
genesNOCA3:ETV4:MMP2,10
genes