Phosphorylation of Protein Phosphatase 1 by Cyclin-dependent Protein Kinase 5 during Nerve Growth Factor-induced PC12 Cell Differentiation

The transcription factor Egr-1 activates cyclin-dependent protein kinase 5 (Cdk5) during nerve growth factor (NGF)-induced differentiation of PC12 cells into neurons (Harada, T. Morooka, T., Ogawa, S., and Nishida, E. (2001) Nat. Cell Biol. 3, 453-459). The downstream target of Cdk5 in the Egr-1/Cdk5 pathway is not clear. In this study, we observed that phosphorylation of protein phosphatase 1 (PP1) on Thr320 is reduced in brain extracts from Egr-1-/- mice, indicating that a kinase downstream of Egr-1 phosphorylates PP1. In HEK 293 cells co-transfected with PP1 and Cdk5, Cdk5 phosphorylates PP1. In vitro, Cdk5 purified from bovine brain phosphorylates bacterially expressed recombinant PP1. In NGF-treated PC12 cells, inhibition of Cdk5 by olomoucine or silencing Cdk5 expression by small interfering RNA strategy, suppresses PP1 phosphorylation. Silencing Cdk5 expression by small interfering RNA also blocks NGF-induced neurite outgrowth. Overexpression of PP1 (wild type) promotes NGF-induced differentiation of PC12 cells, whereas that of PP1 (T320A) has no effect. Our data indicate that PP1 is a downstream target of the NGF/Egr-1/Cdk5 pathway during NGF-induced differentiation of PC12 cells and suggest that PP1 phosphorylation promotes neuronal differentiation. The transcription factor Egr-1 activates cyclin-dependent protein kinase 5 (Cdk5) during nerve growth factor (NGF)-induced differentiation of PC12 cells into neurons (Harada, T. Morooka, T., Ogawa, S., and Nishida, E. (2001) Nat. Cell Biol. 3, 453-459). The downstream target of Cdk5 in the Egr-1/Cdk5 pathway is not clear. In this study, we observed that phosphorylation of protein phosphatase 1 (PP1) on Thr320 is reduced in brain extracts from Egr-1-/- mice, indicating that a kinase downstream of Egr-1 phosphorylates PP1. In HEK 293 cells co-transfected with PP1 and Cdk5, Cdk5 phosphorylates PP1. In vitro, Cdk5 purified from bovine brain phosphorylates bacterially expressed recombinant PP1. In NGF-treated PC12 cells, inhibition of Cdk5 by olomoucine or silencing Cdk5 expression by small interfering RNA strategy, suppresses PP1 phosphorylation. Silencing Cdk5 expression by small interfering RNA also blocks NGF-induced neurite outgrowth. Overexpression of PP1 (wild type) promotes NGF-induced differentiation of PC12 cells, whereas that of PP1 (T320A) has no effect. Our data indicate that PP1 is a downstream target of the NGF/Egr-1/Cdk5 pathway during NGF-induced differentiation of PC12 cells and suggest that PP1 phosphorylation promotes neuronal differentiation. Protein phosphatase 1 (PP1) 2The abbreviations used are: PP1, protein phosphatase 1; Cdk, cyclin-dependent protein kinase; NGF, nerve growth factor; siRNA, small interfering RNA; WT, wild type; HA, hemagglutinin; DN, dominant negative; PBS, phosphate-buffered saline; MAP, mitogen-activated protein. is a major Ser/Thr phosphatase in eukaryotic cells participating in a wide variety of cell functions including cell cycle regulation, muscle contraction, glycogen metabolism, cell differentiation, neural function, and signal transduction (for reviews see Refs. 1Cohen P. Annu. Rev. Biochem. 1989; 58: 443-508Crossref Scopus (2161) Google Scholar, 2Oliver C.J. Shenolikar S. Front. Biosci. 1998; 3: 961-972Crossref PubMed Google Scholar, 3Berndt N. Front. Biosci. 1999; 4: 22-42Crossref PubMed Google Scholar). PP1 activity is regulated by separate inhibitory and targeting subunits. Inhibitory subunits suppress PP1 activity, whereas targeting subunits specify substrate specificity and subcellular localization (2Oliver C.J. Shenolikar S. Front. Biosci. 1998; 3: 961-972Crossref PubMed Google Scholar). PP1 activity is also regulated by phosphorylation (3Berndt N. Front. Biosci. 1999; 4: 22-42Crossref PubMed Google Scholar, 4Dhadwala M. Da Cruz e Silva E.F. Hall F.L. Williams R.T. Carbonaro-Hall D.A. Nairn A.C. Greengard P. Berndt N. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6408-6412Crossref PubMed Scopus (229) Google Scholar, 5Kown Y.-G. Lee S.-Y. Choi Y. Greengard P. Nairn A.C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2168-2173Crossref PubMed Scopus (181) Google Scholar, 6Liu C.W.Y. Wang R.-H. Dhadwala M. Schonthal A.H. Villa-Moruzzi E. Berndt N. J. Biol. Chem. 1999; 274: 29470-29475Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 7Yamano H. Ishii K. Yanagida M. EMBO J. 1994; 13: 5310-5318Crossref PubMed Scopus (100) Google Scholar, 8Berndt N.M. Dhadwala M. Liu C.W.Y. Curr. Biol. 1997; 7: 375-386Abstract Full Text Full Text PDF PubMed Google Scholar, 9Puntoni F. Villa-Moruzzi E. Arch. Biochem. Biophys. 1997; 340: 177-184Crossref PubMed Scopus (31) Google Scholar). In dividing mammalian cells, PP1 is phosphorylated by cyclin-dependent protein kinases Cdk1 and Cdk2 on Thr320 (3Berndt N. Front. Biosci. 1999; 4: 22-42Crossref PubMed Google Scholar, 4Dhadwala M. Da Cruz e Silva E.F. Hall F.L. Williams R.T. Carbonaro-Hall D.A. Nairn A.C. Greengard P. Berndt N. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6408-6412Crossref PubMed Scopus (229) Google Scholar, 5Kown Y.-G. Lee S.-Y. Choi Y. Greengard P. Nairn A.C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2168-2173Crossref PubMed Scopus (181) Google Scholar, 6Liu C.W.Y. Wang R.-H. Dhadwala M. Schonthal A.H. Villa-Moruzzi E. Berndt N. J. Biol. Chem. 1999; 274: 29470-29475Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). When Thr320 is phosphorylated, PP1 activity is inhibited (4Dhadwala M. Da Cruz e Silva E.F. Hall F.L. Williams R.T. Carbonaro-Hall D.A. Nairn A.C. Greengard P. Berndt N. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6408-6412Crossref PubMed Scopus (229) Google Scholar, 5Kown Y.-G. Lee S.-Y. Choi Y. Greengard P. Nairn A.C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2168-2173Crossref PubMed Scopus (181) Google Scholar, 6Liu C.W.Y. Wang R.-H. Dhadwala M. Schonthal A.H. Villa-Moruzzi E. Berndt N. J. Biol. Chem. 1999; 274: 29470-29475Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). In yeast PP1 homologue dis2 is phosphorylated on Thr316 (corresponding to Thr320 of mammalian PP1), and the overexpression of a dis2 (T316A) mutant causes cell cycle arrest (7Yamano H. Ishii K. Yanagida M. EMBO J. 1994; 13: 5310-5318Crossref PubMed Scopus (100) Google Scholar). Introduction of a PP1-T320A mutant into synchronized mammalian cells at the late G1 phase prevented cells from entering the S phase (8Berndt N.M. Dhadwala M. Liu C.W.Y. Curr. Biol. 1997; 7: 375-386Abstract Full Text Full Text PDF PubMed Google Scholar). It was concluded that PP1 Thr320 phosphorylation is required for S phase initiation (3Berndt N. Front. Biosci. 1999; 4: 22-42Crossref PubMed Google Scholar, 8Berndt N.M. Dhadwala M. Liu C.W.Y. Curr. Biol. 1997; 7: 375-386Abstract Full Text Full Text PDF PubMed Google Scholar). The role of PP1 phosphorylation in other cell activities is unclear. Cyclin-dependent protein kinase 5 (Cdk5) is a heterodimer of a catalytic Cdk5 and a regulatory p25 subunit (10Lew J. Qi Z. Huang Q.-Q. Paudel H. Matsura I. Matsushita M. Zhu X. Wang J.H. Neurobiol. Aging. 1995; 16: 263-268Crossref PubMed Scopus Google Scholar, Nat. Rev. Biol. PubMed Scopus Google Scholar, P. P. J. Biochem. PubMed Scopus Google Scholar, I. T. E. 1994; PubMed Scopus Google Scholar). The p25 subunit is a of and and p25 the catalytic activity of Cdk5 subunit I. T. E. 1994; PubMed Scopus Google Scholar). Cdk5 is in dividing cells in cells J. 1995; PubMed Google Scholar, T. T. S. E. Nat. Cell Biol. 3: PubMed Scopus Google and is expressed in neurons (10Lew J. Qi Z. Huang Q.-Q. Paudel H. Matsura I. Matsushita M. Zhu X. Wang J.H. Neurobiol. Aging. 1995; 16: 263-268Crossref PubMed Scopus Google Scholar, Nat. Rev. Biol. PubMed Scopus Google Scholar, P. P. J. Biochem. PubMed Scopus Google Scholar, I. T. E. 1994; PubMed Scopus Google Scholar). Cdk5 is in brain neuronal differentiation, and cell and When PC12 cells with nerve growth factor into The transcription factor Egr-1 growth is by and is for the differentiation J. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar). activates Cdk5 Egr-1 T. T. S. E. Nat. Cell Biol. 3: PubMed Scopus Google Scholar, F. Wang Wang X. PubMed Scopus Google and inhibition of Cdk5 activity blocks NGF-induced neurite T. T. S. E. Nat. Cell Biol. 3: PubMed Scopus Google Scholar). indicate that Cdk5 is a of the pathway and that in Cdk5 activity is required for differentiation of PC12 cells to In this study, we observed that PP1 phosphorylation is reduced in the brain extracts of Egr-1-/- mice, that a kinase that downstream of Egr-1 phosphorylates PP1 in the to Cdk5 phosphorylates PP1. we that PP1 is phosphorylated by Cdk5 in vitro, in mammalian cells and in PC12 also that in PC12 cells, PP1 phosphorylation by Cdk5 activity, blocks neurite outgrowth. Overexpression of PP1 not PP1 (T320A) promotes differentiation. Our data indicate that Cdk5 phosphorylates PP1 in and suggest that PP1 phosphorylation has a role in neuronal differentiation. in wild or Egr-1-/- a The Egr-1 J. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google a from of of the to the of the of and the of the for The by by and and and was from the and no was the was from Egr-1-/- and no was the The at of by and brain was in of 1 1 and 1 of and a The at for a The used to Protein and was purified from of bovine brain K. A. Y. Paudel J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, A. Paudel J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). PP1 PP1 was purified from E. M. Berndt N. Biol. 1998; Google Scholar). and Cdk5 from that and p25 and from Cruz and T. Paudel PubMed Scopus Google Scholar, T. S. Paudel PubMed Scopus Google Scholar). for PP1 phosphorylated on Thr320 was from Cell was from was into the mammalian expression by was and and in the mutant PP1 (T320A) was by the The and and of the by in was a from that and T. S. Paudel PubMed Scopus Google Scholar). Cell and cells in with bovine and HEK 293 cells T. Paudel PubMed Scopus Google Scholar, T. S. Paudel PubMed Scopus Google Scholar). of the cells the cells PC12 cells on was and cell differentiation was A. Paudel 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). the the cells in 1 1 and and and of of and a from Cell PC12 cells with Cell the was The cells at the and cells on at with in for with and by with bovine and for The cells with in bovine and for at The cells with for 1 at and and a was on the a T. M. M. J. E. M. J. Cell Biol. 1997; PubMed Scopus Google Scholar, I. J. I. Curr. Biol. 1994; 4: Full Text Full Text PDF PubMed Scopus Google Scholar). that or the cell to and cells that or neurite outgrowth. that or with the or the of the cell to S. H. P. C.J. 1994; Full Text PDF PubMed Scopus Google Scholar). cells in In of PP1 by Cdk5 was at in a of PP1, 1 1 and of The was by of Cdk5 into the the the the and with of and by or Cdk5 activity in brain was K. A. Y. Paudel J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google a substrate of Cdk5 J. Wang J.H. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The protein of brain was to by with The of the and The was by the of of brain to of the at the was by of The at for and for 5 a at The was and for the of into the substrate a K. A. Y. Paudel J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Cdk5 activity is expressed the of to the protein in the brain PP1 in Egr-1-/- Egr-1 PP1 expression in we from and Egr-1-/- was by in PP1 is expressed in and Egr-1-/- of of PP1 protein in and and Egr-1-/- and data indicate that Egr-1 not expression of PP1 in PP1 is phosphorylated on Thr320 in (4Dhadwala M. Da Cruz e Silva E.F. Hall F.L. Williams R.T. Carbonaro-Hall D.A. Nairn A.C. Greengard P. Berndt N. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6408-6412Crossref PubMed Scopus (229) Google Scholar, 5Kown Y.-G. Lee S.-Y. Choi Y. Greengard P. Nairn A.C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2168-2173Crossref PubMed Scopus (181) Google Scholar, 6Liu C.W.Y. Wang R.-H. Dhadwala M. Schonthal A.H. Villa-Moruzzi E. Berndt N. J. Biol. Chem. 1999; 274: 29470-29475Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). observed that PP1 in brain is to that with PP1 phosphorylated on Thr320 that PP1 is phosphorylated in the Egr-1 PP1 we brain of and Egr-1-/- by observed that PP1 is phosphorylated on Thr320 in of Egr-1-/- that the of PP1 in is and that in Egr-1-/- and indicate that PP1 phosphorylation is in Egr-1-/- Egr-1 is a transcription factor J. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, J. PubMed Scopus Google and PP1 phosphorylation by the expression of a kinase that phosphorylates PP1. Thr320 of PP1 is a phosphorylation by cyclin-dependent Cdk1 and Cdk2 (3Berndt N. Front. Biosci. 1999; 4: 22-42Crossref PubMed Google Scholar, 4Dhadwala M. Da Cruz e Silva E.F. Hall F.L. Williams R.T. Carbonaro-Hall D.A. Nairn A.C. Greengard P. Berndt N. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6408-6412Crossref PubMed Scopus (229) Google Scholar, 5Kown Y.-G. Lee S.-Y. Choi Y. Greengard P. Nairn A.C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2168-2173Crossref PubMed Scopus (181) Google Scholar, 6Liu C.W.Y. Wang R.-H. Dhadwala M. Schonthal A.H. Villa-Moruzzi E. Berndt N. J. Biol. Chem. 1999; 274: 29470-29475Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Cdk1 and Cdk2 not expressed in whereas Cdk5 a substrate specificity with Cdk1 and Cdk2 J. Wang J.H. J. Biol. Chem. Full Text PDF PubMed Google and is the major cyclin-dependent kinase in the neurons of mammalian brain (10Lew J. Qi Z. Huang Q.-Q. Paudel H. Matsura I. Matsushita M. Zhu X. Wang J.H. Neurobiol. Aging. 1995; 16: 263-268Crossref PubMed Scopus Google Scholar, Nat. Rev. Biol. PubMed Scopus Google Scholar, P. P. J. Biochem. PubMed Scopus Google Scholar). that Egr-1 activates Cdk5 in neurons by the expression of subunit of Cdk5 T. T. S. E. Nat. Cell Biol. 3: PubMed Scopus Google Scholar, F. Wang Wang X. PubMed Scopus Google Scholar). Egr-1 activates brain Cdk5, Egr-1-/- to reduced and Cdk5 this we the of Cdk5, and p25 by and Cdk5 activity in and Egr-1-/- brain is to we the of on the of p25 and The Cdk5 was the of the of and p25 was in in Egr-1-/- brain extracts and The Cdk5 activity in Egr-1-/- brain extracts was that of and Cdk5 activity reduced in Egr-1-/- The of reduced PP1 phosphorylation to reduced Cdk5 activity in Egr-1-/- brain that Cdk5 a kinase that phosphorylates PP1 in the Cdk5 Cdk5 phosphorylates PP1, we the specificity of PP1 is phosphorylated on Thr320 in cells by Cdk1 and we or mutant (T320A) in HEK 293 cells and cell by and (T320A) phosphorylated by and to data that is for PP1 phosphorylated on co-transfected with and in HEK 293 of (T320A) with and with Cdk5 with to to no kinase activity co-transfected with in cells I. T. E. 1994; PubMed Scopus Google kinase activity of Cdk5 co-transfected with in HEK 293 cells not cells and by for the expression of the and PP1 phosphorylation. The of phosphorylated PP1 was on the of PP1 and phosphorylated PP1. in in cells co-transfected with and was phosphorylated and and see In cells co-transfected with and phosphorylation was and and see data indicate that PP1 is phosphorylated by Cdk5 and by Cdk5 and we HEK 293 cells, co-transfected with and with the Cdk5 olomoucine A. Paudel J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. M. A. S. J. Biochem. 1994; PubMed Scopus Google or cells for PP1 phosphorylation by PP1 was phosphorylated in PP1 phosphorylation with olomoucine When the of olomoucine PP1 phosphorylation was at on we that Cdk5 phosphorylates PP1 on Thr320 in HEK 293 cells with and The p25 regulatory subunit of Cdk5 is by the of protein I. T. E. 1994; PubMed Scopus Google Scholar). p25 activates Cdk5, is the of Cdk5 Nat. Rev. Biol. PubMed Scopus Google Scholar, P. P. J. Biochem. PubMed Scopus Google Scholar). phosphorylates PP1, we and in HEK 293 cells and for expression of the and phosphorylation The of phosphorylated in cells with and was in cells with not that phosphorylates PP1. In Cdk5 phosphorylates PP1 we in kinase expressed recombinant PP1 was with purified Cdk5 in the of for for PP1 phosphorylation by was observed in Cdk5 with and PP1 not phosphorylated with In PP1, Cdk5, and PP1 phosphorylation with that Cdk5 phosphorylates PP1 in The Cdk5 used in this was purified from bovine brain the that a kinase in Cdk5 phosphorylated PP1, we in kinase for in the of of the Cdk5 olomoucine with olomoucine of PP1 phosphorylation When the of olomoucine was to PP1 phosphorylation was indicate that Cdk5 phosphorylates PP1 in PP1 with the Cdk5 and PP1, we co-transfected with or (T320A) in HEK 293 cells and was to or was or in with data with A. Paudel J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google and that PP1 with also with (T320A) indicating that of Thr320 to not with the of PP1 to PP1 is phosphorylated on Thr320 in HEK 293 cells 3, data also indicate that Thr320 phosphorylation not PP1 from to PP1 during cells with into neurons P. PubMed Scopus Google Scholar). In cells, activates in activates Cdk5 by the expression of T. T. S. E. Nat. Cell Biol. 3: PubMed Scopus Google Scholar). Cdk5 activity is required for PC12 cell differentiation T. T. S. E. Nat. Cell Biol. 3: PubMed Scopus Google Scholar). It is that Cdk5 phosphorylates PP1, and this phosphorylation for neuronal differentiation. this we PC12 cells with for and the with for 1 of PC12 cells to of PC12 cells with and growth of NGF-treated PC12 cells for the not in PP1 is expressed and is phosphorylated in PC12 cells to that the of PP1 and and phosphorylated PP1 and not with in data indicate that the of PP1 and phosphorylated PP1 not during NGF-induced PC12 cell differentiation. Cdk5 in PP1 in NGF-induced PC12 with a J. 1995; PubMed Google that Cdk1 and Cdk2 expressed in PC12 cells to for on of not data suggest that PP1 phosphorylated by Cdk1 Cdk2 at and 1 that kinases to for PP1 phosphorylation with of differentiation. Cdk5 is PC12 cells to for and with Cdk5 cells and for PP1 phosphorylation. in olomoucine inhibited PP1 phosphorylation in a and the of PP1 and data indicate that a kinase to olomoucine phosphorylates PP1 in PC12 in to Cdk5, also Cdk1 and Cdk2 J. M. A. S. J. Biochem. 1994; PubMed Scopus Google Scholar). to Cdk5 phosphorylates PP1, we Cdk5 expression by PC12 cells with or cells to for and by The of Cdk5, PP1, and phosphorylated PP1 and Cdk5 expression was not by in and 1 cells the of Cdk5 was in cells with and Cdk5 expression by and PP1 expression was at and indicating that of Cdk5 expression not PP1 PP1 phosphorylation on the other was in and cells 1 and in on PP1 phosphorylation was by and by the of phosphorylated PP1 was that of silencing Cdk5 expression PP1 phosphorylation in PC12 cells with for on we concluded that Cdk5 phosphorylates PP1 during NGF-induced differentiation of PC12 of Silencing Cdk5 on PC12 Cell the role of PP1 we PP1 phosphorylation by Cdk5 expression and neurite outgrowth. PC12 cells with and and the cells to The of phosphorylated PP1 in cells with 1 and of not The of cells neurite in cells is at 1 and to at in cells with the of phosphorylated PP1 is reduced not and the of cells neurite not and at 1 and from 1 to cells a of phosphorylated PP1 and the of cells neurite outgrowth. with PP1 phosphorylation at 1 and a of neurite to that observed in cell with cells with of phosphorylated PP1 and not the in the of cells neurite outgrowth. data that of Cdk5 expression by blocks PP1 phosphorylation and neurite outgrowth. of Overexpression of PP1 and PP1 (T320A) on NGF-induced PC12 in to PP1 also phosphorylates a of Nat. Rev. Biol. PubMed Scopus Google Scholar). Cdk5 expression by also suppress phosphorylation of other the of cells with to to differentiation, to phosphorylation of Cdk5 other PP1. to the role of PP1 we PC12 cells with and cells with to In cells, Cdk5 to and the of phosphorylated PP1. In (T320A) and cells, the of phosphorylated PP1 to at the of cells to differentiation by neurite outgrowth. The cells or or the of the cell S. H. P. C.J. 1994; Full Text PDF PubMed Scopus Google Scholar). neurons that mammalian brain and or that from of the cell we also the of in and and of and (T320A) cells In cells, this to of and (T320A) cells and cells, of cells neurite cells, and and and or neurite (T320A) cells, and or and to or In and of cells and and to differentiation or overexpression of (T320A) not PC12 differentiation. Overexpression of neurite neurite and the of indicate that overexpression of PP1 promotes neuronal differentiation. Cdk1 and Cdk2 and PP1 in dividing cells, and this phosphorylation role in cell cycle (3Berndt N. Front. Biosci. 1999; 4: 22-42Crossref PubMed Google Scholar, 4Dhadwala M. Da Cruz e Silva E.F. Hall F.L. Williams R.T. Carbonaro-Hall D.A. Nairn A.C. Greengard P. Berndt N. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6408-6412Crossref PubMed Scopus (229) Google Scholar, 5Kown Y.-G. Lee S.-Y. Choi Y. Greengard P. Nairn A.C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2168-2173Crossref PubMed Scopus (181) Google Scholar, 6Liu C.W.Y. Wang R.-H. Dhadwala M. Schonthal A.H. Villa-Moruzzi E. Berndt N. J. Biol. Chem. 1999; 274: 29470-29475Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 7Yamano H. Ishii K. Yanagida M. EMBO J. 1994; 13: 5310-5318Crossref PubMed Scopus (100) Google Scholar, 8Berndt N.M. Dhadwala M. Liu C.W.Y. Curr. Biol. 1997; 7: 375-386Abstract Full Text Full Text PDF PubMed Google Scholar, 9Puntoni F. Villa-Moruzzi E. Arch. Biochem. Biophys. 1997; 340: 177-184Crossref PubMed Scopus (31) Google Scholar). PP1 phosphorylation by Cdk5 in the brain and in NGF-treated PC12 cells, we used that with PP1 phosphorylated on the specificity of the by PP1 (T320A) Our that PP1 is phosphorylated with Cdk5 in HEK 293 cells In NGF-treated PC12 cells, silencing Cdk5 expression or Cdk5 activity reduced PP1 phosphorylation in purified Cdk5 phosphorylated bacterially expressed recombinant PP1 data indicate that Cdk5 phosphorylates PP1 in and in It is that in HEK 293 cells, the phosphorylation of PP1 is to olomoucine data suggest that PP1 is also phosphorylated by a other and Cdk5 in H. H. S. U. EMBO J. PubMed Scopus Google that in neurons PP1 was Cdk5 was indicating that Cdk5 suppresses PP1 activity in the also that Cdk5 not PP1 in and that Cdk5 PP1 by a that not PP1 phosphorylation. in phosphorylation data H. H. S. U. EMBO J. PubMed Scopus Google not in phosphorylation we to the of the and with in phosphorylation of PP1 (4Dhadwala M. Da Cruz e Silva E.F. Hall F.L. Williams R.T. Carbonaro-Hall D.A. Nairn A.C. Greengard P. Berndt N. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6408-6412Crossref PubMed Scopus (229) Google Scholar, 6Liu C.W.Y. Wang R.-H. Dhadwala M. Schonthal A.H. Villa-Moruzzi E. Berndt N. J. Biol. Chem. 1999; 274: 29470-29475Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). PP1 (4Dhadwala M. Da Cruz e Silva E.F. Hall F.L. Williams R.T. Carbonaro-Hall D.A. Nairn A.C. Greengard P. Berndt N. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6408-6412Crossref PubMed Scopus (229) Google and phosphorylation in the of of PP1 PP1 phosphorylation the of the kinase purified phosphorylates PP1, purified is PP1 is not phosphorylated (4Dhadwala M. Da Cruz e Silva E.F. Hall F.L. Williams R.T. Carbonaro-Hall D.A. Nairn A.C. Greengard P. Berndt N. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6408-6412Crossref PubMed Scopus (229) Google Scholar). It is that H. H. S. U. EMBO J. PubMed Scopus Google not of PP1 used a Cdk5 that was not purified in phosphorylation PC12 cells and used for neuronal differentiation P. PubMed Scopus Google Scholar). In cells, of to cell to the kinase phosphorylates with factor Egr-1 J. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar). Egr-1 was a transcription factor of PC12 cells J. PubMed Scopus Google Scholar). The pathway is of the major by and to neurite of Cdk5 activity blocks neurite T. T. S. E. Nat. Cell Biol. 3: PubMed Scopus Google the of Cdk5 in this pathway not In the study, we observed that PP1 is phosphorylated in PC12 cells to When Cdk5 expression is PP1 phosphorylation is In and in HEK 293 cells, Cdk5 phosphorylates PP1 and Our data that PP1 is phosphorylated by Cdk5 in PC12 cells and suggest that PP1 is downstream of Cdk5 in the In dividing PC12 cells, Cdk5 is and is cells with J. 1995; PubMed Google Scholar, T. T. S. E. Nat. Cell Biol. 3: PubMed Scopus Google Scholar). PP1 phosphorylation to with Cdk5 in PP1 is phosphorylated in dividing cells and phosphorylated at the cells to and the differentiation that PP1 phosphorylation is at a during the of dividing PC12 cells to neuronal PC12 from the cell cycle is a for differentiation, and has that and differentiation for differentiation PubMed Scopus Google Scholar). Cdk1 and in dividing cells, in the neuronal differentiation J. 1995; PubMed Google Scholar). Cdk5 on the other is in dividing cells in cells J. 1995; PubMed Google Scholar, T. T. S. E. Nat. Cell Biol. 3: PubMed Scopus Google Scholar). It is that PC12 cells to NGF, PP1 is phosphorylated by Cdk1 of PP1 and phosphorylated by Cdk1 and Cdk2 and and by a of phosphorylated PP1 is and this to to neuronal differentiation. PC12 cells with Cdk5 expression and PP1 phosphorylation differentiation by a reduced of cells with neurite with cells with of Cdk5 and phosphorylated PP1 that PP1 phosphorylation required for neurite outgrowth. Cdk5 phosphorylates a of to the of PP1 we PP1 or PP1 (T320A) into PC12 PP1 neurite the of and neurite to the PP1 (T320A) on the other not of the differentiation in this the the PP1 and PP1 (T320A) is that is phosphorylated, data suggest that PP1 phosphorylation promotes neuronal differentiation. in the of and phosphorylated in and of PP1 S. PubMed Scopus Google Scholar). It is that PP1 phosphorylation PP1 and required for differentiation M. PubMed Scopus Google Scholar). Cdk5 the subunit for activity Nat. Rev. Biol. PubMed Scopus Google Scholar). In PC12 cells, Egr-1 the expression of and activates Cdk5 T. T. S. E. Nat. Cell Biol. 3: PubMed Scopus Google Scholar). of a of NGF-induced expression T. T. S. E. Nat. 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