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ERK and p38 MAPK-Activated Protein Kinases: a Family of Protein Kinases with Diverse Biological Functions

Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115

SUMMARY

MAPK FAMILIES

ERK1/2 Module

Properties.

Activation mechanisms.

Substrates and functions.

p38 MAPK Module

Properties.

Activation mechanisms.

Substrates and functions.

JNK Module

Properties.

Activation mechanisms.

Substrates and functions.

DOCKING INTERACTIONS IN THE MAPK CASCADES

Description of Docking Sites

D domains.

DEF domains.

CD and ED motifs.

Docking Interactions with MKs

Properties.

Regulation of docking.

MAPK-ACTIVATED PROTEIN KINASES

RSK Subfamily

Discovery.

Structure and expression.

Activation mechanisms.

Substrates and functions.

(i) Transcriptional regulation by RSK.

(ii) RSK and cell cycle control.

(iii) RSK promotes survival.

(iv) Other targets of RSK.

MSK Subfamily

Discovery.

Structure and expression.

Activation mechanisms.

Substrates and functions.

(i) Transcriptional regulation by MSK.

(ii) MSK mediates the nucleosomal response.

(iii) Other targets of MSK.

MNK Subfamily

Discovery.

Structure and expression.

Activation mechanisms.

Substrates and functions.

MK2 and -3 Subfamily

Discovery.

Structure and expression.

Activation mechanisms.

Substrates and functions.

(i) Regulation of mRNA translation.

(ii) Transcriptional regulation by MK2 and -3.

(iii) Other targets of MK2 and -3.

MK5

Discovery.

Structure and expression.

Activation mechanisms.

Substrates and functions.

CONCLUSIONS AND PERSPECTIVES

ACKNOWLEDGMENTS

REFERENCES

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INTRODUCTION

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Cells recognize and respond to extracellular stimuli by engaging specific intracellular programs, such as the signaling cascade that leads to activation of the mitogen-activated protein kinases (MAPKs). All eukaryotic cells possess multiple MAPK pathways, which coordinately regulate diverse cellular activities running the gamut from gene expression, mitosis, and metabolism to motility, survival and apoptosis, and differentiation. To date, five distinct groups of MAPKs have been characterized in mammals: extracellular signal-regulated kinases (ERKs) 1 and 2 (ERK1/2), c-Jun amino-terminal kinases (JNKs) 1, 2, and 3, p38 isoforms , ß, , and , ERKs 3 and 4, and ERK5 (reviewed in references 25 and 103). Since Saccharomyces cerevisiae possesses six different MAPKs, the relative complexity of the human genome suggests that there are probably several additional vertebrate MAPK subfamilies (118). The most extensively studied groups of vertebrate MAPKs to date are the ERK1/2, JNKs, and p38 kinases.

MAPKs can be activated by a wide variety of different stimuli, but in general, ERK1 and ERK2 are preferentially activated in response to growth factors and phorbol esters, while the JNK and p38 kinases are more responsive to stress stimuli ranging from osmotic shock and ionizing radiation to cytokine stimulation (reviewed in reference 147) (Fig. 1). Although each MAPK has unique characteristics, a number of features are shared by the MAPK pathways studied to date. Each family of MAPKs is composed of a set of three evolutionarily conserved, sequentially acting kinases: a MAPK, a MAPK kinase (MAPKK), and a MAPKK kinase (MAPKKK). The MAPKKKs, which are serine/threonine kinases, are often activated through phosphorylation and/or as a result of their interaction with a small GTP-binding protein of the Ras/Rho family in response to extracellular stimuli (36, 98). MAPKKK activation leads to the phosphorylation and activation of a MAPKK, which then stimulates MAPK activity through dual phosphorylation on threonine and tyrosine residues located in the activation loop of kinase subdomain VIII. Once activated, MAPKs phosphorylate target substrates on serine or threonine residues followed by a proline; however, substrate selectivity is often conferred by specific interaction motifs located on physiological substrates. Furthermore, MAPK cascade specificity is also mediated through interaction with scaffolding proteins which organize pathways in specific modules through simultaneous binding of several components.

View larger version (32K): [in this window] [in a new window]   FIG. 1. Signaling cascades leading to activation of the MKs. Mitogens and cellular stresses lead to activation of the ERK1/2 and p38 cascades, which in turn phosphorylate and activate the five subgroups of MKs.

MAPK FAMILIES

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Activation mechanisms. Typically, cell surface receptors such as tyrosine kinases (RTK) and G protein-coupled receptors transmit activating signals to the Raf/MEK/ERK cascade through different isoforms of the small GTP-binding protein Ras (reviewed in references 19 and 234). Activation of membrane-associated Ras is achieved through recruitment of SOS (son of sevenless), a Ras-activating guanine nucleotide exchange factor. SOS stimulates Ras to change GDP to GTP, allowing it to interact with a wide range of downstream effector proteins, including isoforms of the serine/threonine kinase Raf (63). The exact mechanism of Raf activation is still elusive but is known to require Ras binding as well as multiple phosphorylation events at the membrane (reviewed in reference 28). Regulation of both Ras and Raf is crucial for the proper maintenance of cell proliferation, as activating mutations in these genes lead to oncogenesis (28). Indeed, Ras has been shown to be mutated in 30% of all human cancers, while B-Raf is mutated in 60% of malignant melanomas (reviewed in references 42 and 125). Activated Raf binds to and phosphorylates the dual specificity kinases MEK1 and -2, which in turn phosphorylate ERK1/2 within a conserved Thr-Glu-Tyr (TEY) motif in their activation loop. Amplification through the signaling cascade is such that it is estimated that activation of solely 5% of Ras molecules is sufficient to induce full activation of ERK1/2 (71).

In S. cerevisiae, the MAPK modules are neatly organized by scaffolding proteins that ensure efficiency and specificity in the signaling cascades (226). The yeast scaffolding protein Ste5p selectively binds a MAPKKK (Ste11p), a MAPKK (Ste7p), and a MAPK (Fus3p) and couples them to their upstream activators. In mammals, no homologues of Ste5p have yet been identified, but the scaffolding protein JIP-1 appears to have similar functions in the JNK cascade of vertebrates (reviewed in reference 103). Several proteins have been shown to interact with members of the ERK cascade, including the scaffold proteins MP-1 and KSR and the modulators CNK and RKIP, resulting in stimulation or inhibition of the ERK1/2 cascade (reviewed in reference 148).

Substrates and functions. ERK1/2 are distributed throughout quiescent cells, but upon stimulation, a significant population of ERK1/2 accumulates in the nucleus (24, 66, 111). While the mechanisms involved in nuclear accumulation of ERK1/2 remain elusive, nuclear retention, dimerization, phosphorylation, and release from cytoplasmic anchors have been shown to play a role (reviewed in reference 152). ERK1/2 signaling has been implicated as a key regulator of cell proliferation, and for this reason, inhibitors of the ERK pathway are entering clinical trials as potential anticancer agents (97). Two structurally unrelated compounds are commonly used to specifically inhibit the ERK1/2 pathway in cultured cells. Both U0126 and PD98059 are noncompetitive inhibitors of MEK1/2/5 and prevent stimulation-mediated activation of ERK1/2/5 (reviewed in reference 3) (Fig. 1).

Activated ERK1 and ERK2 phosphorylate numerous substrates in all cellular compartments, including various membrane proteins (CD120a, Syk, and calnexin), nuclear substrates (SRC-1, Pax6, NF-AT, Elk-1, MEF2, c-Fos, c-Myc, and STAT3), cytoskeletal proteins (neurofilaments and paxillin), and several MKs (reviewed in reference 25). RSKs, MSKs, and MNKs represent three kinase subfamilies of ERK1/2 substrates. While MSKs and MNKs have been shown to be activated by both the ERK1/2 and p38 pathways, RSK family members are exclusively activated by the ERKs (56). These ERK1/2-activated kinases will be discussed in greater detail below.

Activation mechanisms. MEK3 and MEK6 are activated by a plethora of MAPKKKs which become activated in response to various physical and chemical stresses, such as oxidative stress, UV irradiation, hypoxia, ischemia, and various cytokines, including interleukin-1 (IL-1) and tumor necrosis factor alpha (reviewed in reference 25). MEK3 and MEK6 show a high degree of specificity for p38, as they do not activate ERK1/2 or JNK. MEK4 (also known as MKK4 and Sek1) is a known JNK kinase that possesses some MAPKK activity toward p38, suggesting that MEK4 represents a site of integration for the p38 and JNK pathways (14, 123). While MEK6 activates all p38 isoforms, MEK3 is somewhat selective, as it preferentially phosphorylates the p38 and p38ß isoforms. The specificity in p38 activation is thought to result from the formation of functional complexes between MEK3/6 and different p38 isoforms and the selective recognition of the activation loop of p38 isoforms by MEK3/6 (47). Activation of the p38 isoforms results from the MEK3/6-catalyzed phosphorylation of a conserved Thr-Gly-Tyr (TGY) motif in their activation loop. The structures of inactive and active (phosphorylated) p38 have been solved by X-ray crystallography. The phosphorylated TGY motif and the length of the activation loop were found to differ in ERK2 and JNK, which likely contributes to the substrate specificity of p38 (219, 230).

Substrates and functions. p38 was shown to be present in both the nucleus and cytoplasm of quiescent cells, but upon cell stimulation, the cellular localization of p38 is not well understood. Some evidence suggests that, following activation, p38 translocates from the cytoplasm to the nucleus (156), but other data indicate that activated p38 is also present in the cytoplasm of stimulated cells (6).

A large body of evidence indicates that p38 activity is critical for normal immune and inflammatory responses. p38 is activated in macrophages, neutrophils, and T cells by numerous extracellular mediators of inflammation, including chemoattractants, cytokines, chemokines, and bacterial lipopolysaccharide (143). p38 participates in macrophage and neutrophil functional responses, including respiratory burst activity, chemotaxis, granular exocytosis, adherence, and apoptosis, and also mediates T-cell differentiation and apoptosis by regulating gamma interferon production (143). p38 also regulates the immune response by stabilizing specific cellular mRNAs involved in this process. For instance, with SB203580 and constitutively active forms of p38 and MEK3/6, it has been shown that p38 regulates the expression of many cytokines, transcription factors, and cell surface receptors (143).

While the exact mechanisms involved in p38 immune functions are starting to emerge, activated p38 has been shown to phosphorylate several cellular targets, including cytosolic phospholipase A2, the microtubule-associated protein Tau, and the transcription factors ATF1 and -2, MEF2A, Sap-1, Elk-1, NF-B, Ets-1, and p53 (103). p38 also activates several MKs, including MSK1 and -2, MNK1 and -2, and MK2 and -3, which will be discussed in greater detail below.

Activation mechanisms. Like ERK1/2 and p38, JNK activation requires dual phosphorylation on tyrosine and threonine residues within a conserved Thr-Pro-Tyr (TPY) motif. The MAPKKs that catalyze this reaction are known as MEK4 and MEK7, which are themselves phosphorylated and activated by several MAPKKKs, including MEKK1-4, MLK2 and -3, Tpl-2, DLK, TAO1 and -2, TAK1, and ASK1 and -2 (reviewed in reference 103) (Fig. 1).

Substrates and functions. Like ERK1/2 and p38, the JNKs may relocalize from the cytoplasm to the nucleus following stimulation (127). A well-known substrate for JNKs is the transcription factor c-Jun. Phosphorylation of c-Jun on Ser63 and Ser73 by JNK leads to increased c-Jun-dependent transcription (reviewed in reference 225). Several other transcription factors have been shown to be phosphorylated by the JNKs, such as ATF-2, NF-ATc1, HSF-1, and STAT3 (25, 103). While some cytoplasmic targets of JNK are known, the fact that stimulated JNK does not exhibit exclusive nuclear localization suggests that many other cytoplasmic substrates remain to be identified. Intriguingly, there is currently no known JNK-activated MK.

DOCKING INTERACTIONS IN THE MAPK CASCADES

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DEF domains. Analysis of the Caenorhabditis elegans ETS transcription factor LIN-1 and the scaffold protein KSR-1 revealed the presence of a second class of MAPK docking site that consists of the Phe-Xaa-Phe-Pro sequence, where Xaa is any amino acid and one of the Phe residues can also be a Tyr residue (50, 81, 129). This domain, termed DEF (docking site for ERK and FXFP), has been reported to be recognized only by ERK1/2 and typically lies C-terminal to the phosphoacceptor site. DEF domains are required for efficient substrate phosphorylation by ERK1/2 and have been found in many substrates of the mammalian, C. elegans, and Drosophila melanogaster ERK1/2 orthologs (50, 82, 129). DEF and D domains can sometimes be found on the same protein, such as for Elk-1 and KSR, where these domains may act synergistically to strengthen MAPK interaction.

CD and ED motifs. While it is not clear what region of ERK1/2 interacts with DEF domains, two groups independently identified a conserved C-terminal common docking (CD) motif outside the catalytic domain of ERK, p38, and JNK involved in D domain interactions (166, 200). The CD motif contains acidic and hydrophobic residues that are necessary to establish hydrophobic and electrostatic interactions with the positively charged and hydrophobic residues of D domains, respectively (48, 200). The CD motif has been shown to mediate MAPK interactions with their upstream activators and downstream substrates, suggesting that utilization of the interaction motifs is heavily controlled during the MAPK activation cascade. Interestingly, the Drosophila ERK/Rolled sevenmaker allele is mutated in the CD domain and has been shown to be less sensitive to dephosphorylation, suggesting that the CD domain may also direct binding with presently uncharacterized ERK phosphatases (13).

Other regions of MAPKs have also been shown to play important roles in D domain interactions. Similar to the CD motif, the ERK docking (ED) motif is located opposite the MAPK active center and is thought to regulate binding specificity (202). Exchange of only two residues within the ED site of ERK2 altered its binding specificity to that of p38, rendering ERK2 capable of binding to MK3 (201). The complementary mutations in p38 did not revert the docking specificity of p38 for that of RSK2, suggesting that additional residues must participate in this interaction. Accordingly, another groupidentified a docking groove near the CD and ED motifs of p38 that is required for D domain interactions with MEF2A and MEK3b (21). Mutations of residues within the docking groove disrupted p38 binding to the D domain of MEF2A, suggesting that the homologous residues in ERK1/2 and JNK may also be involved in D domain interaction. Finally, several laboratories have reported a role for the N-terminal domain of MAPKs in docking specificity, but the exact requirement for this region remains to be determined (reviewed in reference 48).

View larger version (42K): [in this window] [in a new window]   FIG. 2. Amino acid alignments of D domains found in MKs. The ERK1/2 and p38 kinase binding regions, termed D domains, are found in most MKs and are required for efficient activation of the MKs by mitogens and cellular stresses. The D domains, shown in boldface, are characterized by a stretch of positively charged residues surrounded by hydrophobic residues. Some D domains mediate specific interaction with ERK1/2 or p38, while others are necessary for interaction with both upstream activators. In some MKs, the D domain region overlaps an NLS sequence (underlined).

Regulation of docking. The regulation of MAPK docking to the MKs is not well understood. Several MKs have been shown to bind to ERK1/2 and/or p38 in quiescent cells and to dissociate following cell stimulation. For instance, RSK family members bind to ERK1/2 in quiescent cells, but following stimulation, the complex transiently dissociates for the duration of ERK1/2 activation (165). In fact, differences have even been noted between different RSK isoforms, with RSK1 having the most regulated interaction with ERK1/2. MSK1 and -2, MK2, and MK5 have been shown to reside in the nucleus of quiescent cells (6, 38, 44, 149, 179). Following stimulation, MK2 and MK5 translocate into the cytoplasm, but their relation to p38 at this point remains elusive (6, 179). Recent evidence indicates that p38 docking to MK5 masks its NLS and thereby promotes its nuclear export (179), suggesting that docking and localization are interrelated for MK5.

The mechanisms involved in complex dissociation are currently unknown, but mutagenesis analysis revealed that autophosphorylation of a residue located near the D domain of RSK1 was required for the regulated release of ERK1/2 (165). MSK1 and MSK2 also contain a homologous residue near their D domains, suggesting that these kinases may also regulate ERK1/2 and p38 docking through autophosphorylation of their C termini. Interestingly, MNK1 has been shown to interact with dephosphorylated ERK2 with greater affinity (221), suggesting that MNK1 activation may also regulate MAPK docking.

MAPK-ACTIVATED PROTEIN KINASES

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The MK family comprises 11 members that are generally activated by mitogens through the ERK1/2 kinase cascade (RSK1, RSK2, RSK3, RSK4, and MNK2), stress stimuli through the p38 kinase cascade (MK2, MK3, and possibly MK5), or both (MSK1, MSK2, and MNK1) (Fig. 1). Of these, the RSKs and MSKs possess two distinct and functional kinase domains within the same polypeptide, termed the N-terminal kinase domain (NTKD) and C-terminal kinase domain (CTKD). Conversely, the MNKs, MK2 and -3, and MK5 have a single kinase domain that is mostly homologous to the CTKD of the RSKs and MSKs (Fig. 3). All of these kinases have similar activation loop sequences that are targeted for phosphor-ylation by either ERK1/2 or p38 (Fig. 4). Based on their sequence homologies, the MKs can be classified into five subgroups, the RSKs, MSKs, MNKs, MK2 and -3, and MK5 (Fig. 5), and each subgroup will be discussed in greater detail below.

View larger version (27K): [in this window] [in a new window]   FIG. 3. Schematic representation of the overall structure of the MKs. While RSK1, -2, -3, and -4 and MSK1 and -2 are composed of two nonidentical kinase domains, the MNKs and MK2, -3, and -5 are single-kinase-domain proteins that display homology to the CTKD of RSKs and MSKs. The NTKDs of the RSKs and MSKs are members of the AGC family of kinases, which also require sequences known as the turn and hydrophobic motif for activity. Also necessary for activation of all MKs are the phosphorylation sites indicated by a black circle. The amino acid composition of each MK and its alternatively spliced variants refers to the human protein. The MK5A isoform has been omitted from this figure because it differs from MK5B only by the lack of two residues.

View larger version (115K): [in this window] [in a new window]   FIG. 4. Alignment of the amino acid sequences of the single-kinase domain MKs (MNK1 and -2, MK2 and -3 and MK5) and the CTKDs of RSK1 and MSK1. The sequences comprising the kinase domain and its subregions are boxed and reveal the region of highest homology. The conserved activation loop threonine residue within the sequence Leu-Xaa-Thr-Pro is shown by an arrow, and the D domain region is identified by a line.

View larger version (12K): [in this window] [in a new window]   FIG. 5. Phylogenetic tree of MK family members. The root of the tree is marked with a circle. All sequences used for the construction of this tree were human, and alternatively spliced isoforms of MNK2, MK2, and MK5 were also included in the analysis. The relative similarities between all MKs reflected by this tree suggest that the MKs comprise five groups, the RSK, MSK, MNK, MK2 and -3, and MK5 subfamilies. The Clustal X program was used to generate the multiple alignment on which the tree was based.

RSK1, RSK2, RSK3, and ERK1/2 are usually present in the cytoplasm of quiescent cells, but upon stimulation, a significant portion of these proteins translocate to the nucleus of activated HeLa, COS-7, and HEK293 cells (24, 111, 159, 213, 247). RSK3 is the only human isoform to possess a potential NLS (Lys-Lys-Xaa₁₀-Leu-Arg-Arg-Lys-Ser-Arg) (Fig. 3), whereXaa₁₀ is a stretch of any 10 amino acids, but it remains unknown whether this domain is functional or if other regions are required for nuclear translocation of the RSKs. The small death effector domain protein PEA-15 has been shown to inhibit RSK2 nuclear translocation, but the biological function of this interaction remains unknown (213). Activated RSK2 can also be found in the cytoplasm of stimulated cells, suggesting that RSK2 substrates may exist in both nuclear and cytoplasmic compartments (24). It appears likely that the RSKs possess protein substrates in all cellular compartments, including the cytosol, nucleus, and plasma membranes, and that their localization is regulated via multiple mechanisms.

Analysis of the expression of the RSK isoforms has revealed differential expression patterns. Northern analysis of rsk1 expression showed that this isoform is present in most tissues but is expressed at higher levels in the kidney, lung, and pancreas (243). Analysis of rsk2 mRNA revealed that the alternative use of two different polyadenylation sites gave rise to two transcripts of 3.5 and 8.5 kb (243). The rsk2 and rsk3 transcripts and proteins are expressed in most tissues, including heart, brain, placenta, liver, kidney, and pancreas, with predominant expression in skeletal muscle (43, 243, 247). The rsk4 transcript is also expressed ubiquitously but was shown to be present in high levels in fetal tissues and in adult brain and kidney (241). Northern analysis also revealed the presence of two secondary rsk4 transcripts (5 and 9 kb), but whether these transcripts result from alternative splicing or alternative polyadenylation remains unknown (241). Interestingly, the reported mouse and rat RSK4 sequences contain an additional N-terminal region that is not found in human RSK4 or in other RSK family members. Since human RSK4 was originally identified by positional cloning, this suggests that human RSK4 may also contain this additional domain at its N terminus. However, the function of this potentially novel domain remains to bedetermined.

Activation mechanisms. All RSK isoforms, including the C. elegans and D. melanogaster homologues of RSK, contain six phosphorylation sites that have been shown in RSK1 and RSK2 to be responsive to mitogen stimulation (35) (Fig. 6). Mutational analysis revealed that four of these sites (Ser221, Ser363, Ser380, and Thr573 in human RSK1) are essential for RSK1 activation (35) (Fig. 7). Of these, Ser363 and Ser380 are located within sequences conserved among most AGC kinases, the turn motif and hydrophobic motif, respectively (138). Phosphorylation of the turn motif in RSK1 and RSK2 is essential for kinase activity and is thought to be mediated by ERK1/2 because it is located within a proline-directed phosphorylation site. However, results from our group indicate that this site is not efficiently phosphorylated by ERK1 in vitro (159), and alanine substitution of Pro364 only slightly reduces mitogen-stimulated RSK1 activation (P. Roux, unpublished results), suggesting that another kinase that does not require a proline at position +1 is able to phosphorylate this site in vivo. Interestingly, protein kinases A and C have been shown to autophosphorylate on the homologous site in vivo (4), and this site is modified by a heterologous kinase in Akt (5, 206). It is currently unclear which kinase(s) is responsible for phosphorylating the turn motif in RSK isoforms, and further studies are needed to understand the regulation of this site. Interestingly, the turn motif in protein kinase C has been shown to act as a phosphorylation-dependent docking site for heat shock protein 70 (HSP70) (61), suggesting that Ser363 may have a similar function during RSK1 activation.

View larger version (27K): [in this window] [in a new window]   FIG. 6. Signaling cascades leading to activation of RSKs and MSKs. RSK1, -2, -3, and -4 are activated by two inputs originating from the ERK1/2 and PDK1 enzymes. Similarly, MSK1 and -2 are activated by ERK1/2 but also by stimuli that activate the p38 kinases. Despite their relatively high homology, the RSKs and MSKs appear to have different biological functions.

View larger version (46K): [in this window] [in a new window]   FIG. 7. Schematic representation of RSK1 compared to the related MSK1. The known phosphorylation sites on RSK1 and MSK1 are indicated by solid (essential site) and open (nonessential site) circles. The amino acid alignment and phosphorylated residues of RSK1 and MSK1 refer to the human proteins. While the kinases responsible for the phosphorylation of most sites within RSK1 and MSK1 are known, the identity of the kinase(s) responsible for the phosphorylation of several other essential sites remains unknown.

The sequence of events during activation of the RSKs remains elusive, but the mechanisms of activation are starting to emerge and appear to require well-ordered phosphorylation events (Fig. 7). Upon mitogen stimulation, activated ERK1/2 is thought to phosphorylate RSK1 on Thr573, which is partly required for CTKD activation, and possibly also on Ser363 (turn motif) and Thr359 (site with no associated function) (35, 165). As mentioned above, the role of ERK1/2 in activation of the RSKs may also be to bring the RSK isoforms into proximity with membrane-associated kinases that phosphorylate Ser363 and/or Ser380 (159; P. Roux, unpublished results). The activated CTKD then phosphorylates the hydrophobic motif (Ser380) of the RSKs, which creates a docking site for PDK1, the enzyme that phosphorylates the activation loop site (Ser221) and thereby activates the NTKD. While the CTKD has definitely been shown to phosphorylate Ser380 (29, 217), the involvement of the NTKD or other kinases in the phosphorylation of this site remains unknown. Interestingly, the homologous site in Akt has been shown to be potentially regulated by autophosphorylation (206) and by a heterologous kinase localized to lipid rafts (78), suggesting that the NTKD of the RSKs or a membrane-associated kinase may also phosphorylate the RSK isoforms at this site. Consistent with this, we have found that a CTKD-inactive mutant of RSK1 is still partially responsive to mitogen and phosphorylated at Ser380, agreeing with the idea that the hydrophobic motif may be regulated by multiple inputs (P. Roux, unpublished results).

The importance of the hydrophobic motif in activation of the RSKs was recently underscored by the identification of a phosphate-binding pocket in the kinase domain of many AGC kinases, which is used for intramolecular interaction with their own phosphorylated hydrophobic motif (55). This interaction was shown to induce a synergistic stimulation of RSK2 catalytic activity, whereas mutation of the phosphate-binding pocket led to a reduction in the overall kinase activity of RSK2. PDK1 also contains such a binding motif in its kinase domain but does not have a hydrophobic motif. Using embryonic stem cells with a knock-in mutation in the phosphate-binding pocket of PDK1, Alessi and colleagues have recently shown that the binding pocket in PDK1 is essential for activation of RSK2, S6K1, and SGK, suggesting that PDK1 uses this motif to interact with the phosphorylated hydrophobic motif of these kinases (34).

Finally, RSK1 activity has been shown to be regulated through its interaction with 14-3-3ß (20). 14-3-3 proteins interact with a wide variety of cellular proteins, including protein kinases, receptor proteins, enzymes, and small G proteins, generally through a phosphoserine-containing motif (240). 14-3-3ß binds to phosphorylated Ser154 of RSK1 and thereby negatively regulates its catalytic activity (20). Mutation of Ser154 to alanine increased both basal and serum-stimulated RSK1 activity, indicating that 14-3-3ß normally represses RSK1 activity. Because the RSKs have been shown to interact with their upstream activators (ERK1/2 and PDK1), various downstream substrates, and regulatory proteins such as 14-3-3ß and PEA-15, they appear to function as scaffold proteins that allow multiple proteins to come together and form a signaling network.

Substrates and functions. As mentioned above, RSK1 was first discovered as a kinase that phosphorylated the ribosomal subunit protein S6. However, several lines of evidence indicated that S6 is not a major physiological target of the RSKs (30; reviewed in reference 120). Indeed, several groups have shown that S6K1 and S6K2 were the physiological S6 kinases in somatic cells, because treatment of cells with the mTOR (mammalian target of rapamycin) inhibitor rapamycin was shown to fully inhibit mitogen-induced S6 phosphorylation without affecting the activity of the RSKs (30, 87). Thus, it is generally thought that the RSKs may only modulate S6 phosphorylation under certain specific cellular circumstances (120).

The important physiological roles played by the RSKs have been underscored by the discovery that defects in the rsk2 gene are the cause of Coffin-Lowry syndrome (Table 1). Coffin-Lowry syndrome is an X-linked dominant disorder characterized by psychomotor and growth retardation and facial, hand, and skeletal malformations (84, 210). Numerous mutations have now been identified in the rsk2 gene, most of which are predicted or have been shown to result in truncated or inactive RSK2 proteins (40, 83). Fibroblasts derived from Coffin-Lowry syndrome patients have been useful in determining the function of RSK2 with respect to this human disease; however, differences found between these cells and RSK2-deficient mouse fibroblasts suggest that Coffin-Lowry syndrome may be a multivariable disease and may not be the ideal system with which to study RSK2 function. Both the rsk2 and rsk4 genes are located on chromosome X, and recent data also implicate rsk4 in nonspecific X-linked mental retardation (Table 1), but definitive evidence remains to be provided for rsk4 (241).

The substrate specificity of RSK1 for target phosphorylation has been determined with synthetic peptide libraries and found to require the minimum sequences Arg/Lys-Xaa-Arg-Xaa-Xaa-pSer/Thr or Arg-Arg-Xaa-pSer/Thr, where pSer is phosphoserine/phosphothreonine(110). These analyses also revealed that RSK1 prefers to phosphorylate Ser rather than Thr residues by a factor of about 5, and consistent with this, the majority of RSK1 and RSK2 substrates found to date are phosphorylated on Ser residues. A number of RSK functions can be deduced from the nature of their substrates, and data from many groups point towards roles for the RSKs in transcriptional regulation, cell cycle regulation, and cell survival (Fig. 8). Although more substrates have been identified for RSK2, most studies have not determined whether the other RSK isoforms could also phosphorylate the same targets, indicating that many currently known substrates may be shared by different RSK family members.

View larger version (66K): [in this window] [in a new window]   FIG. 8. Substrates of the MKs. Upon activation, the RSKs, MSKs, MNKs, and MK2, -3, and -5 phosphorylate several substrates and regulate many biological responses, including mRNA translation, cell proliferation and survival, and the nuclear response to stress and mitogen stimulation. The list of substrates indicated in this figure is not exhaustive but emphasizes the many important substrates identified to date.

Several kinases phosphorylate CREB at Ser133, including protein kinase A, Akt, and MSK1/2, but a controversial report taking advantage of Coffin-Lowry syndrome patient-derived fibroblasts indicated that RSK2 was the major CREB kinase activated by epidermal growth factor (39). In these cells, epidermal growth factor failed to increase transcription of the c-fos gene, which was suggested to result from altered CREB-mediated transcription (39). To gain better insight into the physiological role of RSK2, mice deficient in RSK2 that were generated by homologous recombination yielded slightly different results (15). While transcription of the c-fos gene was also altered in fibroblasts derived from RSK2-null mice, CREB phosphorylation was found to be normal following platelet-derived growth factor and IGF-1stimulation (15). Although stimulation of Elk-1 phosphorylation was unaltered in either human or mouse RSK2-deficient cells, the transcriptional activity of both Elk-1 and serum response factor was reduced in mouse cells, suggesting that altered c-fos transcription may in fact result from defects in the activation of Elk-1 and SRF (15).

Differences between the human and mouse models may be due to species-specific compensation for the loss of RSK2 by other RSK isoforms, and for this reason, mice deficient in RSK2 may not be the ideal model for Coffin-Lowry syndrome. Unlike Coffin-Lowry syndrome patients, RSK2-deficient mice do not display any major cerebral abnormalities, suggesting that Coffin-Lowry syndrome may result from several mutations affecting normal brain development, which include RSK2 inactivation (15). However, it was recently demonstrated that RSK2-deficient mice may have impaired learning abilities (43), but the exact role of RSK2 in mouse brain development and whether these mice can be used as a partial model for Coffin-Lowry syndrome remains to be determined.

RSK1 also interacts with the ETS transcription factor ER81 and enhances ER81-dependent transcription by phosphorylating Ser191 and Ser216 (236). ER81 performs many essential functions in homeostasis, signaling response, and development, implying that RSK1 is also involved in these processes. The transcription initiator factor TIF-1A also becomes phosphorylated by ERK1/2 and RSK2 following serum stimulation on two Ser residues that are important for TIF-1A function (245). TIF-1A is required for RNA polymerase I transcription and rRNA synthesis, suggesting that RSK2 and ERK1/2 regulate transcription initiation during growth-promoting conditions. Estrogen receptor is an ERK1/2 substrate that becomes activated following stimulation with growth factors. RSK1 was also shown to associate with and to phosphorylate estrogen receptor on Ser167, which increases estrogen receptor -mediated transcription (90). Another transcription factor, microphthalmia (Mi), has been shown to be phosphorylated by the RSKs (237) and has been linked to malignant melanoma (145). As mentioned above, the MAPKKK B-Raf was shown to be activated in 60% of melanomas (125), suggesting that the RSKs may be potential therapeutic targets for the treatment of melanomas.

Finally, stimulation of the ERK pathway promotes the interaction between RSK1 and the transcriptional coactivator CREB-binding protein (CBP) (130). CBP and its paralog p300 are large molecules that facilitate complex formation between different components of the transcriptional machinery. RSK1 interaction with CBP was found to modulate its function (130, 220), but the exact outcome of this interaction remains to be determined. Ectopic expression of the RSK1 binding region of CBP in PC12 cells inhibits NGF-mediated transcription of c-fos and neurite outgrowth, suggesting that RSK1 binding to CBP is important for these processes (130). Interestingly, CBP and p300 are known to associate with several transcription factors also known to be RSK1 and RSK2 substrates, such as CREB, c-Fos, c-Jun, ER81, and NF-B, suggesting that RSK1 binding to CBP may provide a second mechanism of transcriptional control.

(ii) RSK and cell cycle control. In addition to contributing to the immediate-early gene response during the G₀/G₁ phase of the cell cycle, a recent report demonstrated that RSK1 and RSK2 may promote G₁-phase progression through the phosphorylation of the cyclin-dependent kinase (CDK) inhibitor p27^kip1. Phosphorylation of p27^kip1 by RSK1 and RSK2 was found to promote its association with 14-3-3 and prevent its translocation to the nucleus (59). Under growth arrest conditions, nuclear p27^kip1 negatively regulates G₁ progression by sequestering and inactivating cyclin E- or cyclin A-CDK2 complexes. Through phosphorylation of Thr198, which is also recognized by Akt (58), RSK1- and RSK2-mediated inhibition of p27^kip1 nuclear translocation may promote G₁ progression within mammalian cells.

ERK1/2 and the RSKs may also regulate progression through the G₂ phase of the cell cycle. The role of ERK1/2 in cell cycle regulation has been demonstrated by many groups with the preferred model of Xenopus oocyte maturation (reviewed in reference 131). Immature oocytes are usually arrested in the G₂ phase of the first meiotic cell division. Addition of progesterone induces synthesis of the MAPKKK c-Mos, which in turn activates the MAPK cascade leading to M-phase entry and subsequent maturation to an unfertilized egg. M-phase entry is controlled in part by Cdc2, which is a CDK normally kept in check by dual phosphorylation on both Thr14 and Thr15 by the inhibitory kinase Myt1. RSK2 has been shown to be the prominent RSK isoform in Xenopus oocytes (8), and with this model system, RSK2 has been shown to contribute to the control of the meiotic cell cycle at several critical points (174).

One mechanism by which RSK2 participates in the progression of oocytes through the G₂/M phase of meiosis I is through phosphorylation and inhibition of the Myt1 kinase (144). The importance of RSK2 is such that constitutively activated RSK2 can mediate meiosis I entry even in the absence of progesterone or MAPK activation (67). RSK2 regulates meiosis I entry by binding to and phosphorylating the C terminus of Myt1, reducing its ability to inhibit the kinase activity of Cdc2/cyclin B1 complexes (144). It remains unknown whether this mechanism is conserved in other species, but recent efforts demonstrated that Akt can also act as a Myt1 kinase in starfish oocytes (142). RSK2 may also be important for progression of mammalian somatic cells through the G₂/M phase of mitosis because ERK1/2 activity was shown to be required in synchronized NIH 3T3 fibroblasts (235).

Another way by which RSK2 can modulate the meiotic cell cycle in Xenopus laevis is through mediating MAPK-mediated metaphase II arrest, an activity known as cytostatic factor. Two groups have found that Xenopus RSK1 and RSK2 are essential for cytostatic factor by showing that activated RSK2 causes cytostatic factor even when MAPK is inactive and that depletion of RSK2 from oocyte extracts removes cytostatic factor activity (9, 68). RSK1 was later shown to phosphorylate and activate the kinase Bub1 in vitro, a mediator of anaphase-promoting complex inhibition (177), suggesting that RSK1-mediated Bub1 activation contributes, at least in part, to metaphase II arrest (211).

Finally, RSK2 also modulates the meiotic cell cycle through phosphorylation of histone H3, a process that requires the activation of the MAPK-RSK pathway during oocyte maturation (173). MAPK-mediated histone H3 phosphorylation has been shown to contribute to chromatin remodeling during cell cycle progression and also results in increased transcriptional regulation of several genes (31). Although RSK1 and RSK2 can phosphorylate histone H3 in vitro (173), it remains unknown whether the RSKs or another H3 kinase, such as aurora B, can directly phosphorylate histone H3 in this system. In mammalian cells, RSK2 has been shown to mediate mitogen-stimulated phosphorylation of histone H3. Fibroblasts derived from Coffin-Lowry syndrome patients and RSK2-deficient mouse embryonic stem cells display attenuated histone H3 phosphorylation in response to epidermal growth factor (168). However, recent evidence coming from the use of MSK1 and MSK2 knockout cells is currently casting some doubt on the physiological role of RSK2 in histone H3 phosphorylation, and recent attempts to reproduce the experiments in fibroblasts from Coffin-Lowry syndrome patients have failed (190). Indeed, a completely normal histone H3 phosphorylation response was seen in fibroblasts from Coffin-Lowry syndrome patients, which compromises the major piece of evidence supporting the role of RSK2 in mitogen-stimulated histone H3 phosphorylation (190).

(iii) RSK promotes survival. RSK1 and RSK2 have also been shown to regulate survival in proliferating as well as in differentiated cells. For example, RSK2 has been found to promote the survival of primary cortical neurons through both transcription-dependent and -independent mechanisms (12). Neurotrophic factor-stimulated RSK2 phosphorylates the proapoptotic protein Bad on Ser112, thereby repressing its death-promoting activity (12). Similar regulation of Bad phosphorylation on Ser112 was seen in a hematopoietic cell line, where RSK1-mediated survival required Bad phosphorylation and inactivation (184). RSK2-mediated phosphorylation of the transcription factor CREB on Thr133 was also found to promote the survival of primary cortical neurons through increased transcription of survival-promoting genes (12, 65, 239).

RSK1 promotes the survival of hepatic stellate cells by phosphorylating mouse C/EBPß on Thr217 in response to the hepatotoxin CCI₄ (17).Phosphorylation of Thr217 was suggested to create a functional XEVD caspase-inhibitory box (K-phospho-T²¹⁷-VD) that binds and inhibits caspases 1 and 8, and consistent with this, the cell-permeating tetrapeptide KE²¹⁷VD was also shown to inhibit CCI₄-mediated apoptosis of stellate cells (17). Interestingly, phosphorylation of this site was also shown to promote cell proliferation in response to transforming growth factor alpha (18), suggesting that RSK1 may be involved in this process. Two other studies indicated that RSK1 may also promote survival through the activation of the transcription factor NF-B (64, 175). NF-B comprises a family of heterodimeric transcription factors that are key regulators of a broad range of genes involved in inflammatory responses, proliferation, and apoptosis. RSK1 was shown to phosphorylate the NF-B inhibitor IB on Ser32 (64, 175), suggesting that RSK1 may modulate NF-B-dependent survival and proliferation.

(iv) Other targets of RSK. In addition to transcriptional regulators and cell cycle controllers, the RSKs have been shown to phosphorylate many other targets involved in diverse cellular processes. Upon metabotropic glutamate receptor activation, RSK1 phosphorylates several protein targets within polyribosomes, including the known RSK1 substrate glycogen synthase 3ß (GSK3ß) (1, 196). Phosphorylation of GSK3ß on Ser9 by RSK1 inhibits its kinase activity and results in increased protein translation through the release of inhibition of the GSK3ß-regulated translation initiation factor eIF2B (reviewed in reference 33). RSK2 also phosphorylates another downstream substrate, the Na⁺/H⁺ exchanger isoform 1 (NHE-1) (198). NHE-1 is a key member of a family of exchangers that regulate intracellular pH and cell volume (197). RSK2-mediated phosphorylation of NHE-1 on Ser703 was found to regulate mitogen-dependent Na⁺/H⁺ exchange and intracellular pH (198).

The tumor suppressor LKB1 (also known as STK11), which is mutated in Peutz-Jeghers syndrome, represents another phosphorylation target of RSK2 (167). LKB1 was recently shown to phosphorylate and activate at least 12 protein kinases of the AMP-activated protein kinase subfamily (115, 181). Phosphorylation of LKB1 on Ser431 by RSK2 did not affect its activity, membrane association, or prenylation but was found to be necessary for LKB1-mediated growth suppression through unknown mechanisms (167). As LKB1 is likely an important player regulating cell proliferation, it will be interesting to determine the mechanisms involved in RSK2-mediated modulation of its function.

RSK2 was also shown to phosphorylate the cell adhesion molecule L1 on Ser1152 (232). L1 is a phosphoprotein that becomes hyperphosphorylated during periods of high neuronal activity, suggesting the involvement of RSK2 in neurite outgrowth. Recently, RSK1 was suggested to play a role in membrane ruffling, as the cytoskeleton-associated protein filamin A was found to be phosphorylated on Ser2152 by RSK1 (233). Ser2152 was previously shown to be phosphorylated by PAK1 and to be necessary for membrane ruffling in response to PAK1 activation (212), suggesting that RSK1 may play a similar role in cytoskeletal reorganization.

Proper regulation of the Ras/ERK pathway through phosphorylation-mediated negative feedback has been demonstrated to occur at many levels, including Ras, Raf-1, and MEK1 and -2 (19, 28). Despite having lower ERK1/2 protein levels, fibroblasts from RSK2-deficient mice display higher and more sustained phosphorylation of ERK1/2 in response to exercise and insulin (43), suggesting that RSK2 inhibits the ERK1/2 signaling cascade. A possible mechanism by which RSK2 can do this is through phosphorylation of SOS (and possibly Raf-1) in response to epidermal growth factor treatment (41, 94). RSK2 would therefore prevent further activation of the ERK1/2 pathway through phosphorylation and inactivation of its upstream activators. Interestingly, epidermal growth factor-mediated stimulation of Akt is also higher in RSK2-deficient cells (43), suggesting that Akt may compensate for the loss of RSK2 or that RSK2 is also involved in feedback inhibition of the phosphatidylinositol 3-kinase/Akt pathway through an unknownmechanism.

Structure and expression. MSK1 and MSK2 share the major structural characteristics of the RSKs, including two distinct catalytic domains, a linker region of about 100 amino acids, and relatively similar C- and N-terminal regions (Fig. 3). Analogous to the RSKs, the NTKD of MSK1 and -2 is considered to be mainly responsible for substrate phosphorylation, and the CTKD is involved in MSK1 and -2 activation through autophosphorylation of the kinase. Both MSK isoforms contain a D domain (Leu-Ala-Lys-Arg-Arg-Lys) in their C terminus that lies within a region found to be necessary for MSK2 docking to ERK1/2 and p38 (Fig. 2) (207).

Unlike the RSKs, MSK1 and -2 are usually present in the nuclei of quiescent cells (38, 149, 207). The C-terminal region of the MSKs contains a functional bipartite NLS (Lys-Arg-Xaa₁₄-Lys-Arg-Arg-Lys-Gln-Lys in MSK2) (Fig. 2) which confers on MSK1 and MSK2 an almost exclusive nuclear localization (38, 149). Although it is unknown whether the MSKs translocate to different cellular locations following activation, expression of MSK2 was found to regulate the location of ectopically expressed p38 and ERK1 (149). For instance, in the absence of MSK2, both p38 and ERK1 localized to the nucleus and cytoplasm, but upon expression of MSK2 constructs containing an intact D domain, p38 and ERK1 shifted to a more nuclear localization (149). These results indicate that MSK1 and MSK2 may control the cellular localization of their upstream activators, ERK1/2 and p38, a finding that was also observed with the closely related kinase MK2 (6).

Analysis of MSK1 and MSK2 expression in different tissues revealed that they are ubiquitously expressed, with predominant expression of the msk1 and msk2 mRNAs in the brain, heart, placenta, and skeletal muscles (38). Interestingly, the msk2 gene maps to the BBS1 locus on chromosome 11 (248). Bardet-Beidl syndrome (BBS) is a genetically heterogeneous disorder characterized primarily by retinal dystrophy, obesity, polydactyly, renal malformations, and learning disabilities (92). Although MSK2 inactivation has not yet been shown to contribute to Bardet-Beidl syndrome, it is worth noting that the clinical symptoms are reminiscent of Coffin-Lowry syndrome, suggesting that inactivation of RSK2 and MSK2 may contribute to similar disorders.

Activation mechanisms. MSK1 and MSK2 are potently activated in vivo by ERK1/2 and p38 but not JNK (38, 149), indicating that both mitogens and stress stimuli lead to the activation of MSK1 and -2. Indeed, specific inhibitors of p38 and p38ß (SB203580) and MEK1 and -2 (U0126) block MSK1 and -2 activation, depending on the origin of the stimuli (Fig. 6). Although the site-specific and sequential phosphorylation events during MSK1 and -2 activation have not yet been fully characterized, the four key phosphorylation sites present in RSKs are conserved in MSK1 and -2 (Fig. 7), suggesting that they have similar activation mechanisms (56). Indeed, changing all four residues to Ala inactivates MSK2 in response to MEK1 and MEK6 activation (208). Two of the four essential sites in MSK1 and -2 (Ser360 within the turn motif and Thr581 in the CTKD activation loop of MSK1) are followed by a proline residue, suggesting that they are likely ERK1/2 and p38 phosphorylation sites (Fig. 7). The two other phosphorylation sites are homologous to most AGC kinases and lie within the hydrophobic motif (Ser376) and the NTKD activation loop (Ser212) of MSK1. Inactivation of either kinase domain through mutation of conserved residues completely blocks the N-terminal kinase activity of MSK1 and -2 (38, 149, 208). Similar mutations in the CTKD of RSK1 and RSK2 have been shown to only partly reduce the activity of RSK1 and RSK2 against exogenous substrates (10, 29, 165), indicating that, unlike the RSKs, MSK1 and -2 activation critically requires CTKD activation (29). Since RSK1 translocates to the plasma membrane for full activation (159), the different requirements of these kinases for CTKD activity could be explained by their different cellular localizations.

During activation of the RSKs, phosphorylation of the hydrophobic motif creates a docking site for PDK1, allowing it to phosphorylate the activation loop of the NTKD. These phosphorylation sites are also conserved within MSK1 and MSK2 (Fig. 7), implying similar activation mechanisms. However, recent data indicate that MSK1 becomes fully activated in PDK1-null embryonic stem cells (229), suggesting that a kinase other than PDK1 phosphorylates the activation loop of MSK1 and -2. The high homology between the activation loop sequences of the RSKs and MSKs suggests that the kinase responsible for the phosphorylation of this site may be similar or related to PDK1. Although it is possible that the MSKs regulate their own activation loop through autophosphorylation, it seems more likely that an unknown PDK1-like kinase phosphorylates this site in vivo. Analogous to the RSKs, the MSKs have a phosphate-binding pocket within the NTKD that mediates intramolecular interaction with the phosphorylated hydrophobic motif (34, 55). This pocket was shown to be essential for MSK1 kinase activation, which underscores the importance of hydrophobic motif phosphorylation in MSK1 and -2 activation. It is clear that further experiments will be necessary to characterize the regulation of the hydrophobic motif in MSK1 and -2 and to identify the kinase responsible for the phosphorylation of this site.

According to their homology to the RSKs, the MSKs may also contain two additional phosphorylation sites. Based on work from our group (165), the role of the site located near the C terminus of MSK2 (Ser737) may be to regulate its association with ERK1/2 and p38. Although the D domain of MSK1 and -2 is located near the C terminus, MSK1 and MSK2 have an additional 20 and 50 amino acids C-terminal to the docking site, respectively, suggesting that these regions may contain additional elements necessary for ERK1/2 and p38 association (Fig. 7).

Substrates and functions. The substrate specificity of the MSKs resembles that of the RSKs, with the minimal consensus substrate sequence Arg-Xaa-Xaa-pSer/Thr (38), indicating that the MSKs and RSKs may have similar targets. MSK1 and MSK2 are localized in the nucleus of quiescent and activated cells (38, 149), which suggests that they may preferentially phosphorylate nuclear substrates. Consistent with this, studies from newly engineered MSK1-, MSK2-, and MSK1/MSK2-deficient mice indicated that MSK1 and -2 play active roles in transcriptional