Cell. Vol. 68. 3-6, January 10, 1992, Copyright 0 1992 by Cell Press

MAP Kinase by Any Other Name Smells Just as Sweet George Thomas Friedrich Miescher 4002 Base1 Switzerland

Institute

MAP kinases have been implicated in a broad spectrum of biological responses, but most of their notoriety has arisen from their possible role in two cell cycle control points: the GO-G1 transition, and the passage of cells through either meiosis or mitosis. Though more commonly referred to as MAP kinase, the enzyme was first named microtubule-associated protein 2 kinase or MAP2 kinase, because it utilized MAP2 as a substrate in vitro. The discovery of the kinase (Sturgill and Ray, 1986) was a fortuitous event, since at the time an insulin-stimulated kinase was being sought in 3T3-LI adipocyte cell extracts that could catalyze the phosphorylation and inactivation of type 1 phosphatase inhibitor 2. There was no difference in the ability of either extract to phosphorylate inhibitor 2. However, stimulated cell extracts displayed an increased capacity to catalyze the phosphorylation of a high molecular weight protein doublet, subsequently identified as MAP2. Two forms of MAP2 kinase have been purified from fibroblasts having M, 42K and 44K. It is now clear that these enzymes are equivalent to two proteins that earlier had been shown to become heavily phosphorylated at tyrosine following mitogenic stimulation by a number of growth factors and oncogenes (see Cooper, 1989). Over the last two years, the proteins have acquired a number of names and acronyms; it has been suggested that they be referred to as either p42 and p44 MAP kinase or ~42~“~ and ~44~“” (Sturgill and Wu, 1991). As these enzymes may have multiple substrates, at this juncture the names suggested above seem adequate and preserve the historical connection by which they were first identified. Many earlier studies have been recently reviewed (Sturgill and Wu, 1991); therefore, this review will be largely restricted to recent developments. Activation of both forms of MAP kinase requires phosphorylation at both tyrosine and threonine residues (Anderson et al., 1990; Boulton et al., 1991a). Removal of phosphate either from the threonine by phosphatase 2A or from the tyrosine by CD45 results in inactivation of both enzymes (Anderson et al., 1990; Boulton et al., 1991a). In p42mwk both sites of phosphorylation have been recently localized within a short tryptic peptide in subdomain VIII (Payne et al., 1991) just upstream of the conserved AlaPro-Glu motif found in all serine/threonine kinases (Hanks et al., 1988). By aligning this peptide with the cDNA sequence of p42”&, these two sites of phosphorylation can be assigned to Thr-183 and Tyr-185 (Her et al., 1991; Boulton et al., 1991 b). A kinase with 85% identity to p42”qk and containing the two conserved sites of phosphorylation has been cloned from a rat brain cDNA library (Boulton et al., 1990). Based on its M, and its homology to ~42~@, this protein is most likelyp44m@. The cloning of a third member

Minireview

of this family and further evidence for more forms implies that this family of kinases may be quite extensive (Boulton et al., 1991 b). The existence of a large family is also supported by the fact that two very similar kinases have been identified in budding yeast, which are key components of the mating signal transduction pathway. Activation of this pathway by a or a mating factor results in arrest of cell division and the transient differentiation of haploid cells into gamete-like cells. The two gene products, KSSl and FUS3, share 56% identity with one another (about the same value as with p42mwk and ~44~“) and belong to the CDC28/cdc2 family of protein kinases (Boulton et al., 1991 b). KSSl was identified by its ability, when overexpressed, to rescue from Gi arrest ssf2 cells, which are hypersensitive to a factor. The KSS7 gene is not essential for survival, though null mutations clearly lead to perturbation of growth and overexpression in wild-type cells accelerates recovery from Gl arrest. This response is similar to the overexpression of CLN3, whose gene product has homologies with animal cyclins. Indeed, KS% acts positively on CLN3 and requires its expression to exhibit its effects on Gl-arrested cells, indicating that the two gene products act on a common signaling pathway (Courchesne et al., 1989). In contrast, in the presence of a factor, the FUS3 gene product promotes Gl arrest, and its overexpression confers increased a factor sensitivity. Ablation of the functional FUS3 gene product results in both failure to arrest in Gl and sterility. Interestingly, the defect in Gl arrest is suppressed by CLN3 null mutations; possibly FUS3 and KSSl have antagonistic effects on regulating the activity of CLN3 (Elion et al., 1990). It also should be noted that, in fission yeast, the spkl’gene product shares 54% identity with KSSl and FUS3 and exhibits essentially the same phenotype when disrupted as that of FUS3 (Toda et al., 1991). It may be that KSSl, FUS3, and spkl+, as well as p42mwk and ~44~~p”, are all derived from a common precursor. Indeed, all five gene products contain the conserved Thr-183 and Tyr-185 phosphorylation sites found in p42mwk. Experiments addressing the question of whether the yeast mutations can be rescued by either of the mammalian MAP kinase genes are awaited. The early finding that p42mapk can phosphorylate and partially reactivate S6 kinase II in vitro was greeted with great excitement, as it provided evidence for a kinase cascade triggered by a ligand-activated growth factor receptor tyrosine kinase (Sturgill et al., 1988). S6 kinase II is a member of the 90 kd family of serine/threonine kinases initially implicated in the phosphorylation of ribosomal protein S6 and increased protein synthesis during both mitogenesis and meiotic maturation (see Erikson, 1991; Sturgill and Wu, 1991). However, this same enzyme also appears to catalyze the phosphorylation of proteins other than S6. Of potential importance for insulin action is the phosphorylation of the G subunit of phosphatase 1 at a site that stimulates the dephosphorylation and activation of glycogen synthase (Dent et al., 1990).

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Phosphorylation and partial reactivation of S6 kinase II by MAP kinase occurs in partially purified fractions from EGF-stimulated Swiss 3T3 cell fibroblasts (Ahn et al., 1990) and progesterone-stimulated Xenopus oocytes (Haccard et al., 1990). Favoring a role for MAP2 kinase in phosphorylating S6 kinase II in vivo is the fact that MAP2 kinase reaches a peak of activation prior to S6 kinase II in EGF-stimulated Swiss 3T3 cells (Ahn et al., 1990). The partial reactivation of S6 kinase II may reflect the greater complexity of the in vivo tryptic phosphopeptide pattern of the kinase compared with that obtained in vitro (Sturgill and Wu, 1991). It should also be noted that, unlike the 90 kd S6 kinasefamily, the mitogen-activated 70 kd S6 kinase (~70’~~) lies on a signaling pathway distinct from that of ~42”” (Ballou et al., 1991). In addition to S6 kinase II, MAP kinases may phosphorylate other substrates in the cell. The identification of the substrate consensus sequence Pro-X-SerTThr-Pro for MAP2 kinase (Erickson et al., 1990; Alvarez et al., 1991) has made it possible to test other potential substrates. Recent examples include the transcription factors Myc and Jun, which are activated at the GO-G1 transition (Alvarez et al., 1991; Pulver et al., 1991). Because other kinases, including GS3 and ~34*~, phosphorylate serine and threonine residues in proline-rich regions, the challenge will be to establish an unequivocal role for MAP kinase. The phosphorylation of ~42”” at Thr-163 as well as Tyr-185 led to the prediction that integration of two signaling pathways is required for activation of the enzyme (Anderson et al., 1990). Such a model would result in exquisite control of the kinase. However, the two phosphorylation sites reside in subdomain VIII, a common autophosphorylation region in other serine/threonine kinases (Hanks et al., 1988) such that phosphorylation at one or both sites could be self-catalyzed. In support of such a mechanism of activation, if either ~42~‘~, ~44~w’, or both enzymes are expressed in bacteria, the kinase autophosphorylates at tyrosine and threonine when incubated in vitro with ATP (Crews et al., 1991; Seger et al., 1991; Wu et al., 1991). In all three cases the level of activity is extremely low compared with the enzyme purified from animal cells. In the only case where the sites of phosphorylation were mapped, the double-phosphorylated peptide was not detected, only the peptide single-phosphorylated at Tyr-185 (Wu et al., 1991) leaving open the question of how Thr-183 becomes phosphorylated. The basal rate of autophosphorylation appears much too slow to account for the rapid activation of MAP kinase activity described in mammalian cells treated with mitogens, implying that other upstream factors are required for regulating MAP kinase activity. These factors could be either effector molecules that accelerate the autophosphorylation reaction or kinases that independently phosphorylate and activate the enzyme. Such factors have been described in EGF-stimulated Swiss 3T3 cells (Ahn et al., 1991) and NGF-stimulated PC12 cells (Gomez and Cohen, 1991) based on their ability to raise basal MAP kinase activity or reactivate the phosphatase-inactivated

enzyme. In either case, activation or reactivation requires the presence of ATP. The activation factor from 3T3 cells has been resolved into two components on anion exchange columns, and both exhibit almost identical behavior on sizing columns, suggesting they may be highly related (Ahn et al., 1991). The activating factor from PC1 2 cells behaves as a single component, but on sizing columns exhibits a relative molecular mass similar to that described in 3T3 cells. Surprisingly, the factor in PC12 cells is inactivated by the serinel threonine type 2A phosphatase and not by any of three distinct tyrosine-specific phosphatases, arguing that, if MAP2 kinase lies on a signaling pathway triggered by ligand activation of its tyrosine kinase receptor, at least one other kinase must reside on the pathway. The finding in PC1 2 cells is consistent with the activation of the 3T3 effector molecule by TPA, presumably operating through protein kinase C, which is specific for serine/threonine. Purification of the effector protein from both sources should determine whether they are indeed identical molecules. The basis for not referring to the effector molecule as a kinase is derived from studies that have tested the ability of this factor to catalyze the phosphorylation of avast number of substrates, with no success (Ahn et al., 1991). That the factor may act as an activator molecule accelerating the rate of autophosphorylation is tantalizing, especially in view of the similarity of MAP kinases to the cdc2KDC28 family of protein kinases (Boulton et al., 1990, 1991 b; Wu et al., 1991). With only the data presently available, however, one has to do a careful balancing act to support this view. To determine whether the effector molecule is a kinase, one could test whether its activity can be abolished with an ATP affinity ligand such as 5’-pfluorosulfonylbenzoyl adenosine, a successful approach used in the identification of other kinases. Even if effector-stimulated autophosphorylation leads to activation of the kinase, it does not exclude the possibility that other kinases independently phosphorylate and activate MAP kinase through the same sites. p44”w containing an epitope tag has been stably transfected into Chinese hamster lung fibroblasts (Meloche et al., 1991). Such a tag allows resolution of the transfected MAP kinase from the endogenous enzyme. Using this approach, the basal level of activity of the transfected kinase has been shown to be stimulated up to 25fold by various mitogens. By testing exogenous MAP kinases in which critical amino acids have been mutated, such as the lysine downstream of the Gly-X-Gly-X-X-Gly motif in subdomain I or Thr-183 and Tyr-185, it has been possible to demonstrate that the activation stateof the MAP kinase is modulated by other protein kinases. The ability of wild-type and mutant Xenopus p42mapk homologs to become phosphorylated during meiotic maturation has been tested by injecting their mRNAs into Xenopus oocytes (Posada and Cooper, 1991). ~42~“~ mutants lacking kinase activity still become phosphorylated, as do translation products mutated at the sites equivalent to Thr-183 and Tyr-185. More importantly, Xenopus egg extracts depleted of endogenous p42”@ still

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cells, as suggested by the studies of Gotoh et al. (1991a) in Xenopus. It may seem at odds that MAP kinase is activated during mitosis or meiosis and as well during the GO-G1 transition. However, the same is true for S6 kinase II (see Erikson, 1991; Sturgill and Wu, 1991), suggesting that these enzymes could be directing common metabolic events in both systems. Indeed, these systems are alike in that exit from mitosis may have common features with reentry of quiescent cells into the cell cycle. However, until recently, no ~34~ kinase has been detected at the GO-G1 boundary, though a recent report suggests that such an enzyme may exist (Hall et al., 1991). Obviously, one point of emphasis in the future will be the elucidation of the interplay between the growth factor receptor signaling pathway leading to MAP kinase activation and the possible role played by the ~34~~ kinase family in this process (see figure).

MAP Kinase Effector

MAP Ktnasz

References MAP Proteins

Possible

Pathways

Asterisks

indicate

Involved

in MAP Kinase

the activated

forms

S6 Kmax II

Activation

of kinases.

catalyze this reaction in vitro. Once again, it should be noted that modulation of MAP kinase activity by other kinases does not exclude a role for effector-stimulated autophosphorylation. The possible signaling pathways leading to MAP kinase activation are summarized in the accompanying figure. As just mentioned, MAP kinase becomes activated during the completion of meiotic maturation (Posada et al., 1991; Gotoh et al., 1991a) and the ensuing mitotic cycles (Gotoh et al., 1991a), though the latter result is under contention (Ferrell et al., 1991). A number of nuclear events in meiosis and mitosis are triggered by the activation of maturation promoting factor (~34~~ kinase from Xenopus and starfish eggs) and the apparent mammalian counterpart of cdc2. Injection of ~34~~ kinase into immature oocytes can mimic these events while leading to precocious maturation. It also induces the activation of MAP kinase (Gotoh, 1991 b) and can catalyze the activation of S6 kinase II (Haccard et al., 1990). Furthermore, MAP kinase from Xenopus eggs as well as purified murine MAP kinase are capable of inducing microtubule reorganization in vitro. It may be that MAP kinase is involved in the regulation of the more rapid turnover of tubulin that is observed in extracts of metaphase versus interphase cells. The two cDNA clones for MAP kinase that have been isolated from either a Xenopus ovary or oocyte cDNA library are almost identical and may simply represent an allelic variation in different genes (Posada et al., 1991; Gotoh et al., 1991 b). By comparing their sequences with those reported for mammalian MAP kinase, they are very close homologs of ~42”” (Her et al., 1991). It will now be important to establish whether MAP kinase activities are regulated during mitosis in somatic

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MAP kinase by any other name smells just as sweet.

Cell. Vol. 68. 3-6, January 10, 1992, Copyright 0 1992 by Cell Press MAP Kinase by Any Other Name Smells Just as Sweet George Thomas Friedrich Miesch...
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