FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2000335680> ?p ?o ?g. }

• W2000335680 endingPage "534" @default.
• W2000335680 startingPage "528" @default.
• W2000335680 abstract "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. 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. cyclin-dependent protein kinase 5 mitogen-activated protein MAP kinase kinase 1 constitutively active MEK1 extracellular signal-regulated kinase glutathioneS-transferase proline-rich domain hemagglutinin dominant negative 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. 1Morgan D.O. Annu. Rev. Cell Dev. Biol. 1997; 13: 261-291Crossref PubMed Scopus (1810) Google Scholar; see also Refs. 2Guidato S. McLoughlin D.M. Grierson A.J. Miller C.C. J. Neurochem. 1998; 70: 335-340Crossref PubMed Scopus (36) Google Scholar and 3Xiong Y. Zhang H. Beach D. Cell. 1992; 71: 505-514Abstract Full Text PDF PubMed Scopus (902) Google Scholar). cdk5 is active mainly in post-mitotic cells such as neurons (4Lew J. Huang Q.Q. Qi Z. Winkfein R.J. Aebersold R. Hunt T. Wang J.H. Nature. 1994; 371: 423-426Crossref PubMed Scopus (540) Google Scholar, 5Tsai L.H. Delalle I. Caviness Jr., V.S. Chae T. Harlow E. Nature. 1994; 371: 419-423Crossref PubMed Scopus (813) Google Scholar), retinal cells (6Hirooka K. Tomizawa K. Matsui H. Tokuda M. Itano T. Hasegawa E. Wang J.H. Hatase O. J. Neurochem. 1996; 67: 2478-2483Crossref PubMed Scopus (22) Google Scholar), and muscle cells (7Philpott A. Porro E.B. Kirschner M.W. Tsai L.H. Genes Dev. 1997; 11: 1409-1421Crossref PubMed Scopus (103) Google Scholar), where its activators p35 (or its truncated form p25) (4Lew J. Huang Q.Q. Qi Z. Winkfein R.J. Aebersold R. Hunt T. Wang J.H. Nature. 1994; 371: 423-426Crossref PubMed Scopus (540) Google Scholar, 5Tsai L.H. Delalle I. Caviness Jr., V.S. Chae T. Harlow E. Nature. 1994; 371: 419-423Crossref PubMed Scopus (813) Google Scholar) and p39 (8Honjyo Y. Kawamoto Y. Nakamura S. Nakano S. Akiguchi I. Neuroreport. 1999; 10: 3375-3379Crossref PubMed Scopus (19) Google Scholar, 9Humbert S. Dhavan R. Tsai L. J. Cell Sci. 2000; 113: 975-983Crossref PubMed Google Scholar, 10Wu D.C. Yu Y.P. Lee N.T. Yu A.C. Wang J.H. Han Y.F. Neurochem. Res. 2000; 25: 923-929Crossref PubMed Scopus (51) Google Scholar, 11Zheng M. Leung C.L. Liem R.K. J. Neurobiol. 1998; 35: 141-159Crossref PubMed Scopus (128) Google Scholar) are specifically expressed. cdk5 has been suggested to play important roles in neurite outgrowth (12Nikolic M. Dudek H. Kwon Y.T. Ramos Y.F. Tsai L.H. Genes Dev. 1996; 10: 816-825Crossref PubMed Scopus (532) Google Scholar, 13Sharma M. Sharma P. Pant H.C. J. Neurochem. 1999; 73: 79-86Crossref PubMed Scopus (59) Google Scholar), neuronal migration (14Chae T. Kwon Y.T. Bronson R. Dikkes P. Li E. Tsai L.H. Neuron. 1997; 18: 29-42Abstract Full Text Full Text PDF PubMed Scopus (664) Google Scholar, 15Ohshima T. Gilmore E.C. Longenecker G. Jacobowitz D.M. Brady R.O. Herrup K. Kulkarni A.B. J. Neurosci. 1999; 19: 6017-6026Crossref PubMed Google Scholar, 16Ohshima T. Ward J.M. Huh C.G. Longenecker G. Veeranna Pant H.C. Brady R.O. Martin L.J. Kulkarni A.B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11173-11178Crossref PubMed Scopus (810) Google Scholar), dopamine signaling in the striatum (17Bibb 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. Nature. 1999; 402: 669-671Crossref PubMed Scopus (491) Google Scholar), exocytosis (18Fletcher A.I. Shuang R. Giovannucci D.R. Zhang L. Bittner M.A. Stuenkel E.L. J. Biol. Chem. 1999; 274: 4027-4035Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 19Matsubara M. Kusubata M. Ishiguro K. Uchida T. Titani K. Taniguchi H. J. Biol. Chem. 1996; 271: 21108-21113Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 20Rosales J.L. Nodwell M.J. Johnston R.N. Lee K.Y. J. Cell. Biochem. 2000; 78: 151-159Crossref PubMed Scopus (57) Google Scholar, 21Shuang R. Zhang L. Fletcher A. Groblewski G.E. Pevsner J. Stuenkel E.L. J. Biol. Chem. 1998; 273: 4957-4966Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar), differentiation of muscle cells (7Philpott A. Porro E.B. Kirschner M.W. Tsai L.H. Genes Dev. 1997; 11: 1409-1421Crossref PubMed Scopus (103) Google Scholar), and organization of acetylcholine receptors at the neuromuscular junction (22Fu A.K. Fu W.Y. Cheung J. Tsim K.W. Ip F.C. Wang J.H. Ip N.Y. Nat. Neurosci. 2001; 4: 374-381Crossref PubMed Scopus (153) Google Scholar). Although neuronal cytoskeletal proteins were initially identified as the major target substrates (4Lew J. Huang Q.Q. Qi Z. Winkfein R.J. Aebersold R. Hunt T. Wang J.H. Nature. 1994; 371: 423-426Crossref PubMed Scopus (540) Google Scholar, 23Paudel H.K. Lew J. Ali Z. Wang J.H. J. Biol. Chem. 1993; 268: 23512-23518Abstract Full Text PDF PubMed Google Scholar, 24Shetty K.T. Link W.T. Pant H.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6844-6848Crossref PubMed Scopus (202) Google Scholar), the number of cdk5 substrates has expanded considerably (see Table I in Ref. 25Grant P. Sharma P. Pant H.C. Eur. J. Biochem. 2001; 268: 1534-1546Crossref PubMed Scopus (102) Google Scholar). These include DARPP-32, a dopamine and cyclic AMP-regulated phosphoprotein involved in dopamine signaling (17Bibb 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. Nature. 1999; 402: 669-671Crossref PubMed Scopus (491) Google Scholar), NUDEL (a murine homolog of theAspergillus nidulans nuclear migration mutant NudE), a protein involved in neuronal migration and axon transport (26Sasaki S. Shionoya A. Ishida M. Gambello M.J. Yingling J. Wynshaw-Boris A. Hirotsune S. Neuron. 2000; 28: 681-696Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar), and other proteins involved in cross-talk between protein kinases and phosphatases (27Bibb 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. J. Biol. Chem. 2001; 276: 14490-14497Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). 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 (28Nikolic M. Chou M.M. Lu W. Mayer B.J. Tsai L.H. Nature. 1998; 395: 194-198Crossref PubMed Scopus (352) Google Scholar). 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 (16Ohshima T. Ward J.M. Huh C.G. Longenecker G. Veeranna Pant H.C. Brady R.O. Martin L.J. Kulkarni A.B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11173-11178Crossref PubMed Scopus (810) Google Scholar). Although the p35 knockout mice survive longer (14Chae T. Kwon Y.T. Bronson R. Dikkes P. Li E. Tsai L.H. Neuron. 1997; 18: 29-42Abstract Full Text Full Text PDF PubMed Scopus (664) Google Scholar), both cdk5 −/− and p35 −/− mice exhibit similar defects in cortical neuronal migration and affect the development of the nervous system (14Chae T. Kwon Y.T. Bronson R. Dikkes P. Li E. Tsai L.H. Neuron. 1997; 18: 29-42Abstract Full Text Full Text PDF PubMed Scopus (664) Google Scholar, 15Ohshima T. Gilmore E.C. Longenecker G. Jacobowitz D.M. Brady R.O. Herrup K. Kulkarni A.B. J. Neurosci. 1999; 19: 6017-6026Crossref PubMed Google Scholar, 16Ohshima T. Ward J.M. Huh C.G. Longenecker G. Veeranna Pant H.C. Brady R.O. Martin L.J. Kulkarni A.B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11173-11178Crossref PubMed Scopus (810) Google Scholar). 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. 2I. Vincent, personal communication. The antibody cross-reacts with phosphorylated Lys-Ser-Pro (KSP) motifs in neurofilament proteins, tau, and MAPs (29Sternberger L.A. Sternberger N.H. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 6126-6130Crossref PubMed Scopus (1046) Google Scholar), sites that are specifically targeted by proline-directed kinases such as cdk5 and MAP kinases (24Shetty K.T. Link W.T. Pant H.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6844-6848Crossref PubMed Scopus (202) Google Scholar,30Veeranna Amin N.D. Ahn N.G. Jaffe H. Winters C.A. Grant P. Pant H.C. J. Neurosci. 1998; 18: 4008-4021Crossref PubMed Google Scholar). 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 (30Veeranna Amin N.D. Ahn N.G. Jaffe H. Winters C.A. Grant P. Pant H.C. J. Neurosci. 1998; 18: 4008-4021Crossref PubMed Google Scholar) prompted us to examine the relationship between cdk5/p35 and MAP kinase activities in vitro andin vivo. The MAP kinases mediate a wide range of cellular functions via a variety of signal transduction pathways (31Pearson G. Robinson F. Beers Gibson T. Xu B. Karandikar M. Berman K. Cobb M.H. Endocr. Rev. 2001; 22: 153-183Crossref PubMed Scopus (3564) Google Scholar, 32Schaeffer H.J. Weber M.J. Mol. Cell. Biol. 1999; 19: 2435-2444Crossref PubMed Scopus (1407) Google Scholar). 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 (33Cowley S. Paterson H. Kemp P. Marshall C.J. Cell. 1994; 77: 841-852Abstract Full Text PDF PubMed Scopus (1854) Google Scholar). 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 (31Pearson G. Robinson F. Beers Gibson T. Xu B. Karandikar M. Berman K. Cobb M.H. Endocr. Rev. 2001; 22: 153-183Crossref PubMed Scopus (3564) Google Scholar), and/or associations with shared scaffolding proteins (34Whitmarsh A.J. Davis R.J. Trends Biochem. Sci. 1998; 23: 481-485Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar). 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. All fine chemicals were purchased from Sigma unless indicated. [γ-32P]ATP and [32P]orthophosphate were purchased from Amersham Pharmacia Biotech. The glutathione-Sepharose beads were a product of Sigma Life Sciences. Roscovitine was a product of BioMol. A constitutively active mutant (CA-MEK1) (engineered by deleting residues 32–51 from the N terminus of MEK1 and by mutating its Ser218 and Ser222 to Glu and Asp, respectively (35Mansour S.J. Matten W.T. Hermann A.S. Candia J.M. Rong S. Fukasawa K. Vande Woude G.F. Ahn N.G. Science. 1994; 265: 966-970Crossref PubMed Scopus (1263) Google Scholar)) or a T286A mutant was used for cell transfection (as HA tag) as well as for bacterial protein expression (His6 and/or GST-tagged) purposes. The mutant (T286A) was created in a plasmid encoding CA-MEK1 using a Quick Change site-directed mutagenesis kit (Stratagene). CA-MEK1(K97M) was engineered by mutating Lys97 to Met in the CA-MEK1 plasmid. This was used as a template for making T286A(K97M) by mutating Thr286 to Ala. CA-MEK1 and its variant proteins (T286A, CA-MEK1(K97M), and T286A(K97M)) were bacterially expressed with His6 tag (35Mansour S.J. Matten W.T. Hermann A.S. Candia J.M. Rong S. Fukasawa K. Vande Woude G.F. Ahn N.G. Science. 1994; 265: 966-970Crossref PubMed Scopus (1263) Google Scholar), and cdk5 and p35 were expressed as GST fusion proteins as described previously (4Lew J. Huang Q.Q. Qi Z. Winkfein R.J. Aebersold R. Hunt T. Wang J.H. Nature. 1994; 371: 423-426Crossref PubMed Scopus (540) Google Scholar). cdk5 and cdk5 dominant negative (DN) constructs for cell transfections were in pcDNA3.1His vector and were expressed as His6 tag proteins (gift from Dr. Li-Huei Tsai, Harvard Medical School). The CMV-p35 plasmid was a gift from Dr. Li Tsai (Harvard Medical School). Raf-activated MEK1 (GST fused at the N terminus and His6 fused at the C terminus) was purchased from Upstate Biotech Industries. Cortices from 18-day-old rat embryos were dissected, and the cortical neuronal cell cultures were grown on polylysine-treated 6-well cell culture dishes. The cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum for 7–8 days before drug treatment. PC12 cells were cultured in Dulbecco's modified Eagle's medium containing 12.5% fetal horse serum and 2.5% fetal bovine serum, and NIH 3T3 cells were cultured in 10% fetal bovine serum as described earlier (33Cowley S. Paterson H. Kemp P. Marshall C.J. Cell. 1994; 77: 841-852Abstract Full Text PDF PubMed Scopus (1854) Google Scholar). The cells were serum-starved by culturing in medium containing 1% fetal bovine serum for 16 h prior to both transfection and roscovitine inhibition experiments. cDNA encoding HA-tagged CA-MEK1 or its variant, T286A, His6-tagged cdk5, or its kinase defective mutant cdk5(DN) and CMV-p35 (4Lew J. Huang Q.Q. Qi Z. Winkfein R.J. Aebersold R. Hunt T. Wang J.H. Nature. 1994; 371: 423-426Crossref PubMed Scopus (540) Google Scholar) were transfected in NIH 3T3 or PC12 cells using LipofectAMINE PLUS (Invitrogen) following the manufacturer's instructions. Briefly, 2 μg of each plasmid DNA was used in 35-mm collagen-coated dishes to perform transfections for 60 h. In some experiments cells were treated with NGF (50 ng/ml) 12 h after transfection for every 24 h. Subsequently, the cells were lysed and used for immunoblotting after normalizing the protein using anti-phospho-ERK1/2 or anti-ERK1/2 antibodies (New England Biolabs). The phosphorylated form of ERK1/2 is indicated as pp-ERK1/2 in the figures, whereas ERK1/2 indicates total amount of ERK1/2. For in vivo labeling experiments, the cells were incubated in phosphate-deficient Dulbecco's modified Eagle's medium 2 h prior to incubation in [32P]orthophosphoric acid (0.2 mCi/ml) for 3 h. The cell lysates were prepared in a buffer containing 50 mm Tris, pH 7.5, 1 mm EDTA, 0.1% Nonidet P-40, 50 μm β-glycerophosphate, 50 μm sodium fluoride, 0.1 μm sodium vanadate, and protease inhibitor mixture (Roche Molecular Biochemicals). An enhanced chemiluminescence (Amersham Biosciences, Inc. or Pierce) method was used for immunoblotting following manufacturer's protocol in all experiments. Anti-HA antibody (Roche Molecular Biochemicals) was used to immunoprecipitate CA-MEK1 or T286A as described earlier (30Veeranna Amin N.D. Ahn N.G. Jaffe H. Winters C.A. Grant P. Pant H.C. J. Neurosci. 1998; 18: 4008-4021Crossref PubMed Google Scholar). cdk5 kinase assays were performed in a total volume of 50 μl by incubating a preformed complex of bacterially expressed GST-cdk5 and GST-p25, a truncated form of p35 (4Lew J. Huang Q.Q. Qi Z. Winkfein R.J. Aebersold R. Hunt T. Wang J.H. Nature. 1994; 371: 423-426Crossref PubMed Scopus (540) Google Scholar, 5Tsai L.H. Delalle I. Caviness Jr., V.S. Chae T. Harlow E. Nature. 1994; 371: 419-423Crossref PubMed Scopus (813) Google Scholar) and 1 μg of either Raf-phosphorylated or unphosphorylated GST-MEK1-His6 (N terminus tagged with GST and C terminus tagged with His6) (Upstate Biotech Industries) or bacterially expressed CA-MEK1 or its variants, in a buffer containing 20 mm Tris, pH 7.4, 1 mmEDTA, 10 mm MgCl2, 10 μm sodium fluoride, 10 μm β-glycerophosphate, 1 μmsodium vanadate, protease inhibitor mixture (Roche Molecular Biochemicals), 100 μm [γ-32P]ATP for 60–90 min at 30 °C. The reaction was stopped by boiling the samples in Laemmli's sample buffer. The phosphate incorporation was detected by autoradiography of the protein gels. A similar procedure was used for assessing ERK2 activity using myelin basic protein or a synthetic KSPXK peptide derived from NF-H (VKSPAKEKAKSPEK) (30Veeranna Amin N.D. Ahn N.G. Jaffe H. Winters C.A. Grant P. Pant H.C. J. Neurosci. 1998; 18: 4008-4021Crossref PubMed Google Scholar) as the substrate. The reaction mixture was spotted on phospho-cellulose paper (Whatman), and the phosphate incorporation was measured by scintillation counting as described previously. To examine the effect of cdk5/p25 phosphorylation on MEK1 activity, similar kinase assays were performed using unlabeled ATP (1 mm). GST-Sepharose was used to concentrate MEK1 because it was GST-fused, and then the fusion protein-coupled Sepharose beads were used to phosphorylate 1 μg of bacterially expressed GST-ERK2 as described above for the cdk5 assays. The reaction mixture was immunoblotted using anti-phospho-ERK1/2 antibody (New England Biolabs) to assess the MEK1 activity. When radiolabeled [γ-32P]ATP was used, the phosphate incorporation was observed by autoradiography of the protein gels or scintillation counting as described above. The p35 −/− mice were created as described earlier (14Chae T. Kwon Y.T. Bronson R. Dikkes P. Li E. Tsai L.H. Neuron. 1997; 18: 29-42Abstract Full Text Full Text PDF PubMed Scopus (664) Google Scholar,36Ohshima T. Ogawa M. Veeranna Hirasawa M. Longenecker G. Ishiguro K. Pant H.C. Brady R.O. Kulkarni A.B. Mikoshiba K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2764-2769Crossref PubMed Scopus (114) Google Scholar). Lysates were prepared from the cerebral cortex and cerebellar tissues of 3–4-week-old p35 −/− mice as described above for PC12 cells. Cytoskeletal extracts containing NF proteins were prepared according to previously published procedures (30Veeranna Amin N.D. Ahn N.G. Jaffe H. Winters C.A. Grant P. Pant H.C. J. Neurosci. 1998; 18: 4008-4021Crossref PubMed Google Scholar). The protein levels were normalized, and cdk5 and MEK1 were immunoprecipitated by using anti-cdk5 (C-8, Santa Cruz) and anti-MEK1/2 (New England Biolabs) antibodies, respectively. Bacterially expressed GST-ERK2 was used as the substrate for MEK1 assays, whereas VKSPAKEKAKSPEK, a synthetic KSPXK peptide derived from the sequence of neurofilament-H, was used for cdk5/p35 assays as described previously (30Veeranna Amin N.D. Ahn N.G. Jaffe H. Winters C.A. Grant P. Pant H.C. J. Neurosci. 1998; 18: 4008-4021Crossref PubMed Google Scholar). The lysates were immunoblotted using phospho-ERK1/2 or ERK1/2 antibodies. It has been observed that neurofilament and microtubule-associated proteins are hyperphosphorylated in neurons of cdk5 −/− mice (16Ohshima T. Ward J.M. Huh C.G. Longenecker G. Veeranna Pant H.C. Brady R.O. Martin L.J. Kulkarni A.B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11173-11178Crossref PubMed Scopus (810) Google Scholar). Because cdk5 activity is dependent on p35, we examined whether p35 −/− mice would show hyperphosphorylation of neurofilament proteins. Unlike cdk5 −/− mice, the p35 −/− mice are viable after birth, and therefore, the phosphorylation levels of neurofilament proteins were easily followed by SMI31 antibody. Fig. 1Ashows the ballooning and accumulation of hyperphosphorylated anti-SMI31 epitope immunoreactive proteins in the brain stem neurons of cdk5 −/− mice. To further verify this observation, the cytoskeletal protein fraction from the cortex and cerebella of 3–4-week-old p35 −/− and +/+ wild type mice were analyzed by immunoblotting with SMI-31 antibody, which specifically recognizes phosphorylated KSP sites on neurofilament and microtubule-associated proteins. As shown in Fig.1B, the immunoreactivity of NF-M to SMI31 in the cortex of p35 −/− mice was severalfold higher than in the wild type mice. However, in contrast to the observations in the cortex, the NF-M from the control and p35 −/− mice cerebella showed fewer significant differences in immunoreactivity to SMI-31. However, the intensity of immunoreactivity of NF-H to SMI-31 in wild type and knockout mice was very similar. It should be noted that the rodent NF-M is a preferred substrate for ERK1/2 phosphorylation as compared with cdk5 (30Veeranna Amin N.D. Ahn N.G. Jaffe H. Winters C.A. Grant P. Pant H.C. J. Neurosci. 1998; 18: 4008-4021Crossref PubMed Google Scholar), therefore suggesting that in p35 −/− mice the absence of cdk5 activity might have up-regulated ERK1/2. It has been reported earlier that the brain extracts from p35 −/− mice exhibited insignificant levels of cdk5 activity (14Chae T. Kwon Y.T. Bronson R. Dikkes P. Li E. Tsai L.H. Neuron. 1997; 18: 29-42Abstract Full Text Full Text PDF PubMed Scopus (664) Google Scholar). It is possible that the cerebellum might contain higher levels of p39, a cdk5 activator present in both wild type and mutant mice that could possibly compensate for the absence of p35 (10Wu D.C. Yu Y.P. Lee N.T. Yu A.C. Wang J.H. Han Y.F. Neurochem. Res. 2000; 25: 923-929Crossref PubMed Scopus (51) Google Scholar, 11Zheng M. Leung C.L. Liem R.K. J. Neurobiol. 1998; 35: 141-159Crossref PubMed Scopus (128) Google Scholar). These data suggested that cdk5 activity in +/+ mouse cortex might inhibit the activity of other proline-directed kinases like ERK1/2 that are known to preferentially phosphorylate NF-M. Because MEK1 is a key regulator in the MAP kinase pathway, we immunoprecipitated MEK1 from the cerebral cortex of p35 −/− and +/+ mice to examine whether these two preparations had a differential effect on ERK2 phosphorylation and activity. The animals used for these studies were 3–4 weeks old because MEK1 is expressed at significant levels mainly in the adult brain (37Alessandrini A. Brott B.K. Erikson R.L. Cell Growth Differ. 1997; 8: 505-511PubMed Google Scholar). The MEK1 activity as measured by ERK1/2 phosphorylation was 60–75% higher in the brain extract from the p35 −/− mice as compared with that observed in p35 +/+ mice (Fig.2, B and C). This increase in MEK1 activity correlated with the observed decrease in cdk5 activity in p35 −/− mice (Fig. 2A). The levels of total MEK1 were the same in p35 −/− and +/+ mice as measured by immunoblotting (data not shown). Interestingly, not only did the level of ERK1/2 phosphorylation increase in the p35 −/− mice (Fig.2B), but the amount of phosphorylated ERK1/2 also increased, although the amount of total ERK1/2 remained unchanged (Fig.2D). These data prompted the idea that in vivocdk5/p35 and MEK1 cross-talk might result in regulation of the MAP kinase pathway. To further explore the relationship between cdk5 and MEK1, we compared thein vitro phosphorylation of bacterially expressed MEK1 (inactive), Raf-phosphorylated MEK1 (active), and constitutively active MEK1 (CA-MEK1) by cdk5/p25. Although inactive (unphosphorylated) MEK1 was not phosphorylated by cdk5/p25 (Fig.3A, lane 5), Raf-phosphorylated MEK1 (Fig. 3A, lane 2) and CA-MEK1 (not shown here) were good substrates of cdk5/p25. These data suggested that the Raf-activated MEK1 served as a substrate for cdk5/p25. The effect of cdk5/p25-mediated phosphorylation on MEK1 catalytic activity was then tested using expressed ERK2 as its substrate. In experiments described here the Raf-modified MEK1 with or without cdk5/p35 phosphorylation was used to phosphorylate ERK2. Immunoblot analyses using a phospho-ERK1/2-specific antibody that detects the phosphorylation at the regulatory T and Y residues in the activation loop of ERK2 showed a significant decrease in MEK1 activity (Fig.3B, left panel, lanes 1 and3). Similarly, when the phosphorylation of ERK2 was followed by 32P incorporation, cdk5/p35-mediated phosphorylation of Raf-1, phosphorylated MEK1 showed a decreased phosphorylation of ERK2 (Fig. 3B, right panel, lane 1 versus lane 2). A quantitative measurement of [32P]phosphate incorporation into ERK2 suggested a 75% decrease in MEK1 activity as a result of cdk5/p25 phosphorylation (Fig. 3C, lower panel). Similarly, in another experiment, when an NF-M peptide containing the KSP motif KAKSPVPKSPVEEVKP, a preferred substrate for ERK (30Veeranna Amin N.D. Ahn N.G. Jaffe H. Winters C.A. Grant P. Pant H.C. J. Neurosci. 1998; 18: 4008-4021Crossref PubMed Google Scholar), was incubated in an assay mixture containing ERK2 and CA-MEK1 with and without cdk5/p25, the phosphorylation of the peptide was reduced by ∼40% in the presence of cdk5/p25 (Fig. 3C,upper panel). These experiments supported the idea that the activation of ERK2 by CA-MEK1 is inhibited by cdk5/p35-mediated phosphorylation of CA-MEK1. NGF stimulates the Ras-Raf-MEK-ERK (MAP kinase) pathway in PC12 cells, which results in neuronal differentiation (33Cowley S. Paterson H. Kemp P. Marshall C.J. Cell. 1994; 77: 841-852Abstract Full Text PDF PubMed Scopus (1854) Google Scholar). Also, cdk5 is active in PC12 cells because p35 is endogenously expressed in these cells (38Yan G.Z. Ziff E.B. J. Neurosci. 1995; 15: 6200-6212Crossref PubMed Google Scholar). To examine the effect of cdk5 on the MAP kinase pathway, PC12 cells were treated with roscovitine, a specific cdk5 inhibitor shown to inhibit endogenous cdk5 activity in cultured cells (17Bibb 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. Nature. 1999; 402: 669-671Crossref PubMed Scopus (491) Google Scholar, 39Patrick G.N. Zukerberg L. Nikolic M. de la Monte S. Dikkes P. Tsai L.H. Nature. 1999; 402: 615-622Crossref PubMed Scopus (1323) Google Scholar). Subsequent treatment of these cells for 25 min with NGF stimulated the MAP kinase pathway as indicated by enhanced phosphorylation of ERK1/2 (Fig.4A, lane 2). Interestingly, when the cells were stimulated with NGF in the presence of roscovitine, the increase in ERK phosphorylation was about 3-fold higher (Fig. 4A, lane 3). The effect of cdk5 on the kinetics of MAP kinase activation was also tested (Fig.4B). PC12 cells were treated with NGF for different times in the presence or absence of roscovitine. In the absence of roscovitine, NGF stimulated the MEK1-dependent MAP kinase pathway in a manner reported by several groups. The MEK activity (as judged by phosphorylated ERK1/2 levels) was near maximal at 20 min after NGF treatment. In the presence of roscovitine, however, there was a slight increase in phospho-ERK1/2 levels after 15 min of NGF treatment followed by a significant increase in ph" @default.
• W2000335680 created "2016-06-24" @default.
• W2000335680 creator A5005402708 @default.
• W2000335680 creator A5026450827 @default.
• W2000335680 creator A5031655398 @default.
• W2000335680 creator A5036538943 @default.
• W2000335680 creator A5041282587 @default.
• W2000335680 creator A5045280240 @default.
• W2000335680 creator A5066949514 @default.
• W2000335680 creator A5078572190 @default.
• W2000335680 creator A5090823444 @default.
• W2000335680 date "2002-01-01" @default.
• W2000335680 modified "2023-09-30" @default.
• W2000335680 title "Phosphorylation of MEK1 by cdk5/p35 Down-regulates the Mitogen-activated Protein Kinase Pathway" @default.
• W2000335680 cites W1490481380 @default.
• W2000335680 cites W1497321897 @default.
• W2000335680 cites W1563116834 @default.
• W2000335680 cites W1568595238 @default.
• W2000335680 cites W1594266482 @default.
• W2000335680 cites W1637051517 @default.
• W2000335680 cites W1681161665 @default.
• W2000335680 cites W1766779621 @default.
• W2000335680 cites W1851307228 @default.
• W2000335680 cites W1899552864 @default.
• W2000335680 cites W1976385326 @default.
• W2000335680 cites W1977403160 @default.
• W2000335680 cites W1989480480 @default.
• W2000335680 cites W1993954144 @default.
• W2000335680 cites W1996410021 @default.
• W2000335680 cites W2001521752 @default.
• W2000335680 cites W2002860017 @default.
• W2000335680 cites W2009204794 @default.
• W2000335680 cites W2021958350 @default.
• W2000335680 cites W2036623899 @default.
• W2000335680 cites W2038901745 @default.
• W2000335680 cites W2042629507 @default.
• W2000335680 cites W2048595114 @default.
• W2000335680 cites W2048949076 @default.
• W2000335680 cites W2052086914 @default.
• W2000335680 cites W2054187948 @default.
• W2000335680 cites W2054937093 @default.
• W2000335680 cites W2060153749 @default.
• W2000335680 cites W2068088732 @default.
• W2000335680 cites W2072310191 @default.
• W2000335680 cites W2076823024 @default.
• W2000335680 cites W2081119681 @default.
• W2000335680 cites W2083451944 @default.
• W2000335680 cites W2085136839 @default.
• W2000335680 cites W2085931606 @default.
• W2000335680 cites W2098719212 @default.
• W2000335680 cites W2098921291 @default.
• W2000335680 cites W2099082103 @default.
• W2000335680 cites W2128500531 @default.
• W2000335680 cites W2131571794 @default.
• W2000335680 cites W2132503351 @default.
• W2000335680 cites W2143658086 @default.
• W2000335680 cites W2148519196 @default.
• W2000335680 cites W2162322742 @default.
• W2000335680 cites W2169927110 @default.
• W2000335680 cites W2171367003 @default.
• W2000335680 doi "https://doi.org/10.1074/jbc.m109324200" @default.
• W2000335680 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/11684694" @default.
• W2000335680 hasPublicationYear "2002" @default.
• W2000335680 type Work @default.
• W2000335680 sameAs 2000335680 @default.
• W2000335680 citedByCount "147" @default.
• W2000335680 countsByYear W20003356802012 @default.
• W2000335680 countsByYear W20003356802013 @default.
• W2000335680 countsByYear W20003356802014 @default.
• W2000335680 countsByYear W20003356802015 @default.
• W2000335680 countsByYear W20003356802016 @default.
• W2000335680 countsByYear W20003356802017 @default.
• W2000335680 countsByYear W20003356802018 @default.
• W2000335680 countsByYear W20003356802019 @default.
• W2000335680 countsByYear W20003356802020 @default.
• W2000335680 countsByYear W20003356802021 @default.
• W2000335680 countsByYear W20003356802022 @default.
• W2000335680 crossrefType "journal-article" @default.
• W2000335680 hasAuthorship W2000335680A5005402708 @default.
• W2000335680 hasAuthorship W2000335680A5026450827 @default.
• W2000335680 hasAuthorship W2000335680A5031655398 @default.
• W2000335680 hasAuthorship W2000335680A5036538943 @default.
• W2000335680 hasAuthorship W2000335680A5041282587 @default.
• W2000335680 hasAuthorship W2000335680A5045280240 @default.
• W2000335680 hasAuthorship W2000335680A5066949514 @default.
• W2000335680 hasAuthorship W2000335680A5078572190 @default.
• W2000335680 hasAuthorship W2000335680A5090823444 @default.
• W2000335680 hasBestOaLocation W20003356801 @default.
• W2000335680 hasConcept C11960822 @default.
• W2000335680 hasConcept C121738310 @default.
• W2000335680 hasConcept C124160383 @default.
• W2000335680 hasConcept C132149769 @default.
• W2000335680 hasConcept C137361374 @default.
• W2000335680 hasConcept C159479382 @default.
• W2000335680 hasConcept C184235292 @default.
• W2000335680 hasConcept C185592680 @default.
• W2000335680 hasConcept C82495950 @default.
• W2000335680 hasConcept C86803240 @default.

• next