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Phosphorylation of MEK1 by cdk5/p35 Down-regulates the Mitogen-activated Protein Kinase Pathway*

Open AccessPublished:October 29, 2001DOI:https://doi.org/10.1074/jbc.M109324200
      Cyclin-dependent protein kinase 5 (cdk5), a member of the cdk family, is active mainly in postmitotic cells and plays important roles in neuronal development and migration, neurite outgrowth, and synaptic transmission. In this study we investigated the relationship between cdk5 activity and regulation of the mitogen-activated protein (MAP) kinase pathway. We report that cdk5 phosphorylates the MAP kinase kinase-1 (MEK1) in vivo as well as the Ras-activated MEK1 in vitro. The phosphorylation of MEK1 by cdk5 resulted in inhibition of MEK1 catalytic activity and the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. In p35 (cdk5 activator) −/− mice, which lack appreciable cdk5 activity, we observed an increase in the phosphorylation of NF-M subunit of neurofilament proteins that correlated with an up-regulation of MEK1 and ERK1/2 activity. The activity of a constitutively active MEK1 with threonine 286 mutated to alanine (within a TPXK cdk5 phosphorylation motif in the proline-rich domain) was not affected by cdk5 phosphorylation, suggesting that Thr286 might be the cdk5/p35 phosphorylation-dependent regulatory site. These findings support the hypothesis that cdk5 and the MAP kinase pathway cross-talk in the regulation of neuronal functions. Moreover, these data and the recent studies of Harada et al. (Harada, T., Morooka, T., Ogawa, S., and Nishida, E. (2001) Nat. Cell Biol. 3, 453–459) have prompted us to propose a model for feedback down-regulation of the MAP kinase signal cascade by cdk5 inactivation of MEK1.
      cdk5
      cyclin-dependent protein kinase 5
      MAP
      mitogen-activated protein
      MEK1
      MAP kinase kinase 1
      CA-MEK1
      constitutively active MEK1
      ERK
      extracellular signal-regulated kinase
      GST
      glutathioneS-transferase
      PRD
      proline-rich domain
      HA
      hemagglutinin
      DN
      dominant negative
      NGF
      nerve growth factor
      cdk51 is a member of the cyclin-dependent protein kinase family (cdc2, CDC28, and other generically cyclin-dependent CDKs). Although cdk5 binds to cyclin D, its activity is not regulated by cyclins and there is little evidence that cdk5 is involved in the progression of the cell cycle (for review see Ref.
      • Morgan D.O.
      ; see also Refs.
      • Guidato S.
      • McLoughlin D.M.
      • Grierson A.J.
      • Miller C.C.
      and
      • Xiong Y.
      • Zhang H.
      • Beach D.
      ). cdk5 is active mainly in post-mitotic cells such as neurons (
      • Lew J.
      • Huang Q.Q.
      • Qi Z.
      • Winkfein R.J.
      • Aebersold R.
      • Hunt T.
      • Wang J.H.
      ,
      • Tsai L.H.
      • Delalle I.
      • Caviness Jr., V.S.
      • Chae T.
      • Harlow E.
      ), retinal cells (
      • Hirooka K.
      • Tomizawa K.
      • Matsui H.
      • Tokuda M.
      • Itano T.
      • Hasegawa E.
      • Wang J.H.
      • Hatase O.
      ), and muscle cells (
      • Philpott A.
      • Porro E.B.
      • Kirschner M.W.
      • Tsai L.H.
      ), where its activators p35 (or its truncated form p25) (
      • Lew J.
      • Huang Q.Q.
      • Qi Z.
      • Winkfein R.J.
      • Aebersold R.
      • Hunt T.
      • Wang J.H.
      ,
      • Tsai L.H.
      • Delalle I.
      • Caviness Jr., V.S.
      • Chae T.
      • Harlow E.
      ) and p39 (
      • Honjyo Y.
      • Kawamoto Y.
      • Nakamura S.
      • Nakano S.
      • Akiguchi I.
      ,
      • Humbert S.
      • Dhavan R.
      • Tsai L.
      ,
      • Wu D.C.
      • Yu Y.P.
      • Lee N.T.
      • Yu A.C.
      • Wang J.H.
      • Han Y.F.
      ,
      • Zheng M.
      • Leung C.L.
      • Liem R.K.
      ) are specifically expressed. cdk5 has been suggested to play important roles in neurite outgrowth (
      • Nikolic M.
      • Dudek H.
      • Kwon Y.T.
      • Ramos Y.F.
      • Tsai L.H.
      ,
      • Sharma M.
      • Sharma P.
      • Pant H.C.
      ), neuronal migration (
      • Chae T.
      • Kwon Y.T.
      • Bronson R.
      • Dikkes P.
      • Li E.
      • Tsai L.H.
      ,
      • Ohshima T.
      • Gilmore E.C.
      • Longenecker G.
      • Jacobowitz D.M.
      • Brady R.O.
      • Herrup K.
      • Kulkarni A.B.
      ,
      • Ohshima T.
      • Ward J.M.
      • Huh C.G.
      • Longenecker G.
      • Veeranna
      • Pant H.C.
      • Brady R.O.
      • Martin L.J.
      • Kulkarni A.B.
      ), dopamine signaling in the striatum (
      • Bibb J.A.
      • Snyder G.L.
      • Nishi A.
      • Yan Z.
      • Meijer L.
      • Fienberg A.A.
      • Tsai L.H.
      • Kwon Y.T.
      • Girault J.A.
      • Czernik A.J.
      • Huganir R.L.
      • Hemmings Jr., H.C.
      • Nairn A.C.
      • Greengard P.
      ), exocytosis (
      • Fletcher A.I.
      • Shuang R.
      • Giovannucci D.R.
      • Zhang L.
      • Bittner M.A.
      • Stuenkel E.L.
      ,
      • Matsubara M.
      • Kusubata M.
      • Ishiguro K.
      • Uchida T.
      • Titani K.
      • Taniguchi H.
      ,
      • Rosales J.L.
      • Nodwell M.J.
      • Johnston R.N.
      • Lee K.Y.
      ,
      • Shuang R.
      • Zhang L.
      • Fletcher A.
      • Groblewski G.E.
      • Pevsner J.
      • Stuenkel E.L.
      ), differentiation of muscle cells (
      • Philpott A.
      • Porro E.B.
      • Kirschner M.W.
      • Tsai L.H.
      ), and organization of acetylcholine receptors at the neuromuscular junction (
      • Fu A.K.
      • Fu W.Y.
      • Cheung J.
      • Tsim K.W.
      • Ip F.C.
      • Wang J.H.
      • Ip N.Y.
      ). Although neuronal cytoskeletal proteins were initially identified as the major target substrates (
      • Lew J.
      • Huang Q.Q.
      • Qi Z.
      • Winkfein R.J.
      • Aebersold R.
      • Hunt T.
      • Wang J.H.
      ,
      • Paudel H.K.
      • Lew J.
      • Ali Z.
      • Wang J.H.
      ,
      • Shetty K.T.
      • Link W.T.
      • Pant H.C.
      ), the number of cdk5 substrates has expanded considerably (see Table I in Ref.
      • Grant P.
      • Sharma P.
      • Pant H.C.
      ). These include DARPP-32, a dopamine and cyclic AMP-regulated phosphoprotein involved in dopamine signaling (
      • Bibb J.A.
      • Snyder G.L.
      • Nishi A.
      • Yan Z.
      • Meijer L.
      • Fienberg A.A.
      • Tsai L.H.
      • Kwon Y.T.
      • Girault J.A.
      • Czernik A.J.
      • Huganir R.L.
      • Hemmings Jr., H.C.
      • Nairn A.C.
      • Greengard P.
      ), NUDEL (a murine homolog of theAspergillus nidulans nuclear migration mutant NudE), a protein involved in neuronal migration and axon transport (
      • Sasaki S.
      • Shionoya A.
      • Ishida M.
      • Gambello M.J.
      • Yingling J.
      • Wynshaw-Boris A.
      • Hirotsune S.
      ), and other proteins involved in cross-talk between protein kinases and phosphatases (
      • Bibb J.A.
      • Nishi A.
      • O'Callaghan J.P.
      • Ule J.
      • Lan M.
      • Snyder G.L.
      • Horiuchi A.
      • Saito T.
      • Hisanaga S.
      • Czernik A.J.
      • Nairn A.C.
      • Greengard P.
      ). cdk5 also modulates protein kinase reactions such as the small GTPase-Rac dependent phosphorylation of p21-activated kinase, which results in modification of the actin cytoskeleton (
      • Nikolic M.
      • Chou M.M.
      • Lu W.
      • Mayer B.J.
      • Tsai L.H.
      ). By virtue of phosphorylating these diverse substrates, cdk5 plays a multifunctional role in the nervous system.
      It has been demonstrated that the absence of cdk5 in cdk5 −/− mice results in embryonic lethality (
      • Ohshima T.
      • Ward J.M.
      • Huh C.G.
      • Longenecker G.
      • Veeranna
      • Pant H.C.
      • Brady R.O.
      • Martin L.J.
      • Kulkarni A.B.
      ). Although the p35 knockout mice survive longer (
      • Chae T.
      • Kwon Y.T.
      • Bronson R.
      • Dikkes P.
      • Li E.
      • Tsai L.H.
      ), both cdk5 −/− and p35 −/− mice exhibit similar defects in cortical neuronal migration and affect the development of the nervous system (
      • Chae T.
      • Kwon Y.T.
      • Bronson R.
      • Dikkes P.
      • Li E.
      • Tsai L.H.
      ,
      • Ohshima T.
      • Gilmore E.C.
      • Longenecker G.
      • Jacobowitz D.M.
      • Brady R.O.
      • Herrup K.
      • Kulkarni A.B.
      ,
      • Ohshima T.
      • Ward J.M.
      • Huh C.G.
      • Longenecker G.
      • Veeranna
      • Pant H.C.
      • Brady R.O.
      • Martin L.J.
      • Kulkarni A.B.
      ). We observed that in cdk5 −/− mice brain stem neurons showed ballooning and hyperphosphorylation of cytoskeletal proteins as detected by the SMI31 antibody (see Fig. 1). Similar observations were obtained from p35 (−/−) mice.
      I. Vincent, personal communication.
      2I. Vincent, personal communication.
      The antibody cross-reacts with phosphorylated Lys-Ser-Pro (KSP) motifs in neurofilament proteins, tau, and MAPs (
      • Sternberger L.A.
      • Sternberger N.H.
      ), sites that are specifically targeted by proline-directed kinases such as cdk5 and MAP kinases (
      • Shetty K.T.
      • Link W.T.
      • Pant H.C.
      ,
      • Veeranna
      • Amin N.D.
      • Ahn N.G.
      • Jaffe H.
      • Winters C.A.
      • Grant P.
      • Pant H.C.
      ). The data suggested that in the absence of cdk5 activity, other proline-directed protein kinases were up-regulated. The findings that KSP motifs in rat NF proteins (particularly, NF-M) are preferentially phosphorylated by ERK1/2 (
      • Veeranna
      • Amin N.D.
      • Ahn N.G.
      • Jaffe H.
      • Winters C.A.
      • Grant P.
      • Pant H.C.
      ) prompted us to examine the relationship between cdk5/p35 and MAP kinase activities in vitro andin vivo.
      Figure thumbnail gr1
      Figure 1A, immunohistochemical staining of 18-day-old embryonic brain stem sections from cdk5 −/− and cdk5 +/+ mice with SMI31 antibody. Note the intense immunostaining of hyperphosphorylated cytoskeletal proteins in large brain stem neurons of cdk5 −/− mice (arrows). These neurons in cdk5 +/+ mice were not immunostained (arrowheads). B, immunoblot analysis of phosphorylated cytoskeletal protein preparations from 4-week-old p35 −/− and wild type mice brains using phosphoepitope-specific SMI-31 antibody. The SMI-31 immunoreactivity of NF-M, particularly from the cortex and cerebella in p35 −/− mice, was severalfold higher than in p35 +/+ mice.
      The MAP kinases mediate a wide range of cellular functions via a variety of signal transduction pathways (
      • Pearson G.
      • Robinson F.
      • Beers Gibson T.
      • Xu B.
      • Karandikar M.
      • Berman K.
      • Cobb M.H.
      ,
      • Schaeffer H.J.
      • Weber M.J.
      ). In one well studied pathway, the binding of GTP to Ras protein initiates a phosphorylation cascade through Raf-1 and MEK1/2 (MAPK kinase), which results in stimulation of the MAP kinases, ERK1/2. Upon stimulation, ERKs are known to phosphorylate a variety of cytosolic substrates and are also translocated into the nucleus where they initiate the transcription of immediate early genes (
      • Cowley S.
      • Paterson H.
      • Kemp P.
      • Marshall C.J.
      ). The Ras-Raf-MEK-ERK pathway is stimulated by various growth factors and extracellular stimuli and plays important roles in cell survival, differentiation, and proliferation. This pathway interacts (cross-talks) with other signal transduction cascades, either because of overlapping substrate specificity, shared regulatory sites (
      • Pearson G.
      • Robinson F.
      • Beers Gibson T.
      • Xu B.
      • Karandikar M.
      • Berman K.
      • Cobb M.H.
      ), and/or associations with shared scaffolding proteins (
      • Whitmarsh A.J.
      • Davis R.J.
      ).
      To explore the nature of interactions between cdk5 and the MAP kinase signaling cascade, we studied the effect of cdk5 on MEK1 activityin vitro and in vivo. In this report we provide evidence that cdk5 regulates the MAP kinase pathway in a negative manner via phosphorylation of MEK1.

      DISCUSSION

      MEK1 occupies a central position in the network of interactive signaling cascades in all cells. Its target specificity, extent of activation, and localization in cells are controlled by complex formation with other kinases and non-kinase scaffolding proteins (
      • Whitmarsh A.J.
      • Davis R.J.
      ,). Signal cascades may be regulated positively or negatively by a variety of factors including cross-talk interactions between components of specific signaling pathways (
      • Hucho F.
      • Buchner K.
      ,
      • Xia Z.
      • Dickens M.
      • Raingeaud J.
      • Davis R.J.
      • Greenberg M.E.
      ). For example, the Rho family of G-proteins may cooperate with Raf-1 to activate the Erk pathway (
      • Frost J.A.
      • Steen H.
      • Shapiro P.
      • Lewis T.
      • Ahn N.
      • Shaw P.E.
      • Cobb M.H.
      ). Our results, on the other hand, show that a neuronal-specific cdk5/p35 complex phosphorylated MEK1 in vitro and in vivo, which resulted in a reduction of MEK1 activity. The cdk5/p35-mediated decrease in MEK1 activity down-regulated the MAP kinase pathwayin vivo. The data also suggest that for cdk5 to down-regulate MEK1, the latter must be in an activated state, phosphorylated at Ser218 and Ser222 in the T-loop by Raf. It implies that cdk5 regulation occurs only after the MAP kinase cascade has been stimulated by cellular signals that interact with diverse surface receptors (
      • Pearson G.
      • Robinson F.
      • Beers Gibson T.
      • Xu B.
      • Karandikar M.
      • Berman K.
      • Cobb M.H.
      ,
      • Widmann C.
      • Gibson S.
      • Jarpe M.B.
      • Johnson G.L.
      ). Furthermore, phosphorylation of the Thr 286 residue in the PRD of MEK1 inhibited MEK1 and ERK activity, suggesting it as the putative site of cdk5/p35 phosphorylation. This has led us to propose that a conformational change induced by the Raf activation of MEK1 may be required for phosphorylation of Thr286 by cdk5. It is possible that the conformational change in MEK1 caused by cdk5 phosphorylation of Thr 286 is also unfavorable for MEK1 interaction with ERK1/2, thereby inhibiting the phosphorylation and activation of the latter. This, in turn, would switch off the MAP kinase signaling cascade in stimulated cells.
      A related cyclin-activated kinase, p34cdc2, active during the cell division cycle, also inactivates MEK1 by phosphorylation in vivo and in vitro at sites Thr286 and Thr292 in the PRD (
      • Rossomando A.J.
      • Dent P.
      • Sturgill T.W.
      • Marshak D.R.
      ). It was suggested that this phosphorylation might act as a feedback regulator to shut down the cell cycle. It appears, therefore, that the PRD may be a critical domain for the regulation of MEK1 catalytic activity in both proliferating and terminally differentiated cells such as neurons.
      The PRD of MEK1 seems to be principally involved in modulating the efficient activation of ERK1/2 in the MAP kinase cascade (
      • Dang A.
      • Frost J.A.
      • Cobb M.H.
      ). On the one hand, deletion of the PRD domain residues (265) has no effect on MEK1 binding to Raf or its activity in vitro (
      • Dang A.
      • Frost J.A.
      • Cobb M.H.
      ). On the other hand, a similar PRD deletion in MEK1 (residues 270–307) blocked MEK1-Raf binding and decreased MEK1 activity in response to growth factors (
      • Catling A.D.
      • Schaeffer H.J.
      • Reuter C.W.
      • Reddy G.R.
      • Weber M.J.
      ). The contradictory results could be attributed to a difference in the residues, i.e. residues between 301 and 307 may be essential for PRD activity.
      Formation of the Raf-MEK1 complex seems to be essential for downstream signaling, and complex formation is modulated by phosphorylation of sites in the PRD domain. For example, phosphorylation of Ser298 by p21-activated kinase 1 enhances activation of MEK1 by promoting MEK1-Raf binding (
      • Frost J.A.
      • Steen H.
      • Shapiro P.
      • Lewis T.
      • Ahn N.
      • Shaw P.E.
      • Cobb M.H.
      ). This is consistent with the observation that mutation of sites Ser298 and Thr292 to Ala inhibited MEK-Raf binding. Evidently, conformational changes induced by the additional negativity of the phosphate groups favor Raf binding and MEK1 activation. Our results suggest that phosphorylation of Thr286 by neuronal-specific cdk5/p35 inhibits MEK activity. It may do so by virtue of a conformational change in MEK1 that may affect binding of the activated Raf-MEK1 complex to other proteins essential for downstream activation of ERK1/2. Apparently, different conformational changes may be induced upon phosphorylation of different residues in the PRD domain.
      Our model of cdk5/p35 down-regulation of the MAP kinase signaling cascade is shown in Fig. 6 on left side of the diagram. The cdk5 cross-talk inhibition of the cascade is targeted at Raf-activated MEK1, an event occurring shortly after receptor activation. We suggest that transient increases in activated MEK1 are modulated by cdk5 phosphorylation of MEK1 in the Raf-MEK1 complex. Because cdk5 activity depends, in part, on its regulator, p35, the extent of cdk5 inhibition is limited by the availability of p35. This model is consistent with recent data showing the activation of cdk5/p35 by ERK in NGF-stimulated PC12 cells (
      • Harada T.
      • Morooka T.
      • Ogawa S.
      • Nishida E.
      ) (NGF to ERK1/2 in Fig. 6). It has been well established that NGF stimulates the MAP kinase cascade with the peak of ERK and MEK1 activity attained rapidly, within 10–20 min (see Fig. 1C) (
      • Harada T.
      • Morooka T.
      • Ogawa S.
      • Nishida E.
      ). They have also shown that ERK activation induces a transcription factor, EGR-1, that initiates p35 transcription and activation of cdk5 within 1–2 h (Fig.1) (
      • Harada T.
      • Morooka T.
      • Ogawa S.
      • Nishida E.
      ). What is most striking is that the increasing cdk5/p35 activity correlates directly with the subsequent decline in ERK and MEK1 activation as if cdk5/p35 is acting as a feedback regulator or switch to shut down the signaling cascade by phosphorylating and inactivating the Raf-MEK1 complex. It is significant that the data in Fig.1B (
      • Harada T.
      • Morooka T.
      • Ogawa S.
      • Nishida E.
      ) showing an early decrease in activated MEK1 preceding the decline in ERK activation are consistent with our model.
      Figure thumbnail gr6
      Figure 6A model of cdk5/p35 feedback regulation of the MAP kinase cascade in PC12 cells based on our data and the data reported in Ref.
      • Harada T.
      • Morooka T.
      • Ogawa S.
      • Nishida E.
      on the transcription of p35 in PC12 cells by NGF-induced ERK activation of the transcription factor Egr1. The cdk5/p35 feedback inhibitory loop is shown on the left sideof the figure in bolder arrows, targeted at activated MEK-1. Inhibition is limited by the availability of cdk5/p35, which is in turn dependent on levels of p35. As seen in Fig. B (
      • Harada T.
      • Morooka T.
      • Ogawa S.
      • Nishida E.
      ), within 5–10 min, NGF induces a rapid activation of the ERK 1/2 pathway, which induces the active transcription factor EGR-1 followed by transcription and up-regulation of p35. This persists for about an hour until phospho-ERK 1/2 and phospho-MEK1 begin to decline. The timing of this decline (1–3 h) coincides with increased expression of p35 and cdk5 activity. We suggest that cdk5/p35 phosphorylation and inhibition of MEK1 activity is a feedback switch responsible for down-regulating the MAP kinase cascade.

      Acknowledgments

      We are thankful to Drs. R. W. Albers and H. Gainer for critical discussions and suggestions with the manuscript. DNA sequencing support by Jim Neagle (NINDS DNA sequencing facility at the National Institutes of Health) is appreciated. We would also like to thank Drs. T. Oshima and T. Tanaka for providing p35(−/−) mice when they were at NIDCR, NIH, Bethesda, MD.

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