Neuronal Cdc2-like Protein Kinase (Cdk5/p25) Is Associated with Protein Phosphatase 1 and Phosphorylates Inhibitor-2

Protein phosphatase 1 (PP1) is complexed with inhibitor 2 (I-2) in the cytosol. In rabbit muscle extract PP1·I-2 is activated upon preincubation with ATP/Mg. This activation is caused by phosphorylation of I-2 on Thr72 by glycogen synthase kinase 3 (GSK3). We have found that PP1·I-2 in bovine brain extract is also activated upon preincubation with ATP/Mg. However, blocking GSK3 action by LiCl inhibited only ∼29% of PP1 activity and indicated that GSK3 is not the sole PP1·I-2 activator in the brain. When bovine brain extract was analyzed by gel filtration PP1·I-2 and neuronal Cdc2-like protein kinase (NCLK), a heterodimer of Cdk5 and the regulatory p25 subunit, co-eluted as a ∼450-kDa size species. The NCLK from the eluted column fractions bound to PP1-specific microcystin-Sepharose and glutathioneS-transferase (GST)-I-2-coated glutathione-agarose beads. Similarly, PP1 from the eluted column fractions was pulled down with GST-Cdk5-coated glutathione-agarose beads. In vitro, NCLK phosphorylated I-2 on Thr72 and activated PP1·I-2 in an ATP/Mg-dependent manner. NCLK bound to PP1 through its Cdk5 subunit and the PP1 binding region was localized to Cdk5 residues 28–41. Our data demonstrate that in brain extract PP1·I-2 and NCLK are associated within a complex of ∼450 kDa and suggest that NCLK is one of the PP1·I-2-activating kinases in the mammalian brain. Protein phosphatase 1 (PP1) is complexed with inhibitor 2 (I-2) in the cytosol. In rabbit muscle extract PP1·I-2 is activated upon preincubation with ATP/Mg. This activation is caused by phosphorylation of I-2 on Thr72 by glycogen synthase kinase 3 (GSK3). We have found that PP1·I-2 in bovine brain extract is also activated upon preincubation with ATP/Mg. However, blocking GSK3 action by LiCl inhibited only ∼29% of PP1 activity and indicated that GSK3 is not the sole PP1·I-2 activator in the brain. When bovine brain extract was analyzed by gel filtration PP1·I-2 and neuronal Cdc2-like protein kinase (NCLK), a heterodimer of Cdk5 and the regulatory p25 subunit, co-eluted as a ∼450-kDa size species. The NCLK from the eluted column fractions bound to PP1-specific microcystin-Sepharose and glutathioneS-transferase (GST)-I-2-coated glutathione-agarose beads. Similarly, PP1 from the eluted column fractions was pulled down with GST-Cdk5-coated glutathione-agarose beads. In vitro, NCLK phosphorylated I-2 on Thr72 and activated PP1·I-2 in an ATP/Mg-dependent manner. NCLK bound to PP1 through its Cdk5 subunit and the PP1 binding region was localized to Cdk5 residues 28–41. Our data demonstrate that in brain extract PP1·I-2 and NCLK are associated within a complex of ∼450 kDa and suggest that NCLK is one of the PP1·I-2-activating kinases in the mammalian brain. protein phosphatase 1 casein kinase fast protein liquid chromatography high pressure liquid chromatography glycogen synthase kinase 3 glutathione S-transferase inhibitor 1 inhibitor 2 4-morpholinepropanesulfonic acid neuronal cdc2-like protein kinase cAMP-dependent protein kinase polymerase chain reaction dithiothreitol mitogen-activated protein kinase polyacrylamide gel electrophoresis PKA inhibitory peptide Protein phosphatase 1 (PP1)1 is a major Ser/Thr phosphatase involved in the regulation of metabolism, cell cycle, cell signaling, muscle contraction, and gene expression (for reviews see Refs. 1Cohen P. Annu. Rev. Biochem. 1989; 58: 453-508Crossref PubMed Scopus (2146) Google Scholar, 2Lee E.Y.C. Zhang L. Zhao S. Wei Q. Zhang J. Qi Z.Q. Belmonte E.R. Frontiers Biosci. 1999; 4: 270-285Crossref PubMed Google Scholar). PP1 is a 37-kDa catalytic subunit bound to two types of regulatory subunits: a targeting subunit and an inhibitory subunit. Targeting subunits confer substrate specificity and localize PP1 to various subcellular compartments. Inhibitory subunits suppress PP1 activity. There are three PP1 inhibitory subunits: inhibitor 1 (I-1), DARPP-32, and inhibitor 2 (I-2) (1Cohen P. Annu. Rev. Biochem. 1989; 58: 453-508Crossref PubMed Scopus (2146) Google Scholar, 2Lee E.Y.C. Zhang L. Zhao S. Wei Q. Zhang J. Qi Z.Q. Belmonte E.R. Frontiers Biosci. 1999; 4: 270-285Crossref PubMed Google Scholar, 3Oliver C.J. Shenolikar S. Frontiers Biosci. 1998; 3: 961-972Crossref PubMed Google Scholar). I-1 and DARPP-32 require phosphorylation for PP1 inhibitory activity, whereas nonphosphorylated I-2 inhibits PP1. These inhibitors are phosphorylated in response to many extracellular stimuli and allow PP1 to respond to various growth factors and hormones (3Oliver C.J. Shenolikar S. Frontiers Biosci. 1998; 3: 961-972Crossref PubMed Google Scholar). In rabbit skeletal muscle extract PP1 is found in both particulate and cytosolic fractions. PP1 in the particulate fraction is active, whereas in the cytosolic fraction it is inactive (1Cohen P. Annu. Rev. Biochem. 1989; 58: 453-508Crossref PubMed Scopus (2146) Google Scholar, 2Lee E.Y.C. Zhang L. Zhao S. Wei Q. Zhang J. Qi Z.Q. Belmonte E.R. Frontiers Biosci. 1999; 4: 270-285Crossref PubMed Google Scholar). The inactive cytosolic enzyme, a PP1·I-2 complex, is activated upon incubation with ATP/Mg and is hence called ATP/Mg-dependent PP1 (1Cohen P. Annu. Rev. Biochem. 1989; 58: 453-508Crossref PubMed Scopus (2146) Google Scholar). An activating factor named Fa is necessary for ATP/Mg-dependent activation of PP1·I-2. Fa has been identified to be glycogen synthase kinase 3 (GSK3) (4Woodgett J.R. Cohen P. Biochim. Biophys. Acta. 1984; 788: 339-347Crossref PubMed Scopus (164) Google Scholar, 5Yang S.D. Vandenheede J.R. Goris J. Merlevede W. J. Biol. Chem. 1980; 255: 11759-11767Abstract Full Text PDF PubMed Google Scholar, 6Hemmings B.A. Yellowless D. Kernohan J.C. Cohen P. Eur. J. Biochem. 1981; 119: 443-451Crossref PubMed Scopus (184) Google Scholar). The ATP/Mg-dependent activation is due to the phosphorylation of I-2 within the PP1·I-2 complex by GSK3. Nonphosphorylated I-2 suppresses PP1 activity within the PP1·I-2 complex. GSK3 phosphorylates I-2 on Thr72 and relieves PP1 from I-2 inhibition (4Woodgett J.R. Cohen P. Biochim. Biophys. Acta. 1984; 788: 339-347Crossref PubMed Scopus (164) Google Scholar, 5Yang S.D. Vandenheede J.R. Goris J. Merlevede W. J. Biol. Chem. 1980; 255: 11759-11767Abstract Full Text PDF PubMed Google Scholar, 6Hemmings B.A. Yellowless D. Kernohan J.C. Cohen P. Eur. J. Biochem. 1981; 119: 443-451Crossref PubMed Scopus (184) Google Scholar, 7Yang S.D. Vandenheede J.R. Merlevede W. J. Biol. Chem. 1981; 256: 10231-10234Abstract Full Text PDF PubMed Google Scholar, 8DePaoli-Roach A.A. J. Biol. Chem. 1984; 259: 12144-12152Abstract Full Text PDF PubMed Google Scholar, 9Park I.K. Roach P. Bondor J. Fox S.P. DePaoli-Roach A.A. J. Biol. Chem. 1994; 269: 944-954Abstract Full Text PDF PubMed Google Scholar). Even though GSK3 is a well-characterized PP1·I-2-activating kinase, several reports suggest that other kinases also phosphorylate I-2 and activate PP1·I-2 (10Chan C.P. McNally S.J. Krebs E.G. Fischer E.H. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 6257-6261Crossref PubMed Scopus (60) Google Scholar, 11Wang Q.M. Guan K.L. Roach P.J. DePaoli-Roach A.A. J. Biol. Chem. 1995; 270: 18352-18358Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 12Puntoni F. Villa-Moruzzi E. Biochem. Biophys. Res. Commun. 1995; 207: 732-739Crossref PubMed Scopus (23) Google Scholar). PP1 is highly expressed in brain (1Cohen P. Annu. Rev. Biochem. 1989; 58: 453-508Crossref PubMed Scopus (2146) Google Scholar). An earlier study found that most of the PP1 in brain extract is inactive and requires incubation with ATP/Mg to become active (13Yang S.D. Fong Y.-L. J. Biol. Chem. 1985; 260: 13464-13470Abstract Full Text PDF PubMed Google Scholar). The is a PP1·I-2 complex, is activated upon incubation with ATP/Mg in the of muscle GSK3. was that brain ATP/Mg-dependent PP1 is in a to its muscle phosphorylation of I-2 (13Yang S.D. Fong Y.-L. J. Biol. Chem. 1985; 260: 13464-13470Abstract Full Text PDF PubMed Google Scholar). of Fa activity was from brain extract (13Yang S.D. Fong Y.-L. J. Biol. Chem. 1985; 260: 13464-13470Abstract Full Text PDF PubMed Google the of activity has it has not been as to kinase PP1·I-2 in the brain. Cdc2-like protein kinase is a heterodimer of protein kinase and a p25 regulatory subunit in J. Qi 1995; PubMed Scopus Google Scholar). a of the protein kinase is expressed in various and cell E. J. PubMed Scopus Google Scholar). However, its kinase activity is only in it is associated with a p25 subunit J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). p25 is a of a protein and is expressed only in E. 1994; PubMed Scopus Google Scholar). NCLK is involved in brain cell cell signaling, and Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, P. E. Full Text Full Text PDF Scopus Google Scholar, A. W. W. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. J. Biol. Chem. Full Text PDF PubMed Google Scholar, L. S. 1999; PubMed Scopus Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). that NCLK is complexed with PP1·I-2 in brain phosphorylates I-2 on and PP1·I-2. Our data suggest that NCLK is one of the kinases that activate PP1·I-2 in the I-2 in was two expression and the polymerase polymerase chain reaction was I-2 as the and and The was for 1 for 1 and for 2 was by a incubation with polymerase The was a and The I-2 from the was by and a of the was a and an as polymerase and The was and of the and a and two and an in the to three and and and and the as for E. J. PubMed Scopus Google as the and the and was with and of the by by I-2 was from as A. P. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google with was in and with When of the was to a of 1 for 3 The was and the was in of and and inhibitor 1 1 1 and The was to and three and was three for a The was for and the was in a for The was and the was a column with and 1 The column was with the and eluted with a of in I-2 for and an column in The column was and eluted with a of in I-2 and for and a column in A. the column with of the I-2 was eluted with a of in A. I-2 and by The was and NCLK J. J. Biol. Chem. Full Text PDF PubMed Google and kinase J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google from bovine brain and rabbit skeletal muscle was a from E. was phosphorylated by kinase J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). PP1 and I-2 from and the Cdk5 subunit of with both and casein kinase 1 and casein kinase 2 have been Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). the region of was was from from as A. W. W. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The of the NCLK peptide substrate has been J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). cAMP-dependent protein kinase substrate and inhibitory peptide from The GSK3 peptide substrate Q.M. C.J. DePaoli-Roach A.A. Roach P. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google was the peptide of the of This peptide was phosphorylated by and the phosphorylated peptide was by The PP1·I-2 complex was from and I-2 as A.A. J. Biol. Chem. 1984; 259: 12144-12152Abstract Full Text PDF PubMed Google in a and The was for and an column with and The column was with the and fractions analyzed by The PP1·I-2 complex was in fractions. and by protein bovine as the of kinase and as J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The of NCLK is on its activity J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). of was on of NCLK and GSK3 peptide by acid was by of microcystin-Sepharose in with of gel filtration fractions from A. The was with The was and the three with of and of The with of and and of the was analyzed by the indicated the of glutathione-agarose with the indicated protein with of the indicated protein and with with and The with of and and of the was analyzed by the indicated ATP/Mg-dependent PP1 activity was as I.K. Roach P. Bondor J. Fox S.P. DePaoli-Roach A.A. J. Biol. Chem. 1994; 269: 944-954Abstract Full Text PDF PubMed Google that the also acid and inhibitor for in a 1 and The was by the of of the to a of the of the The of the various in the 1 and preincubation of acid was to the The was on for and for a The was a and in a liquid to the of and PKA by peptide Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). I-2 phosphorylation by NCLK was in a reaction 1 and was by the of NCLK to the the of the indicated with an of and on a The gel was and and the I-2 in the gel was and in a liquid to the of that was I-2 phosphorylation by was as by in the phosphorylation of of I-2 was by a column and to the data in bovine brain was for 1 in 1 of 1 1 and inhibitor The was for and the was for The was a column with The fraction PP1 activity was an column with The column was with and was eluted with of a in PP1 activity by to and through an gel filtration column column and eluted with 1 1 and PP1 activity, of the fractions was to 1 and and the was a column with The column was with of and eluted with a of in I-2 was phosphorylated for by NCLK in a reaction 1 1 and I-2 was through a in of and for The was by a column with acid as J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google in fractions in of and for The was in 1 1 and a column in The column was and bound peptide was eluted with in the in 1 1 and through an column as one peptide was This peptide was the of and of by a In a study PP1·I-2 was found to be the of PP1 in brain extract (13Yang S.D. Fong Y.-L. J. Biol. Chem. 1985; 260: 13464-13470Abstract Full Text PDF PubMed Google Scholar). brain a bovine brain extract through a column an The ATP/Mg-dependent PP1 activity was analyzed by an gel filtration PP1 activity eluted from the gel filtration column as a of various gel filtration fractions indicated that both PP1 and I-2 in column fractions PP1 activity not The gel filtration indicated that the size of PP1·I-2 in is ∼450 the size of PP1·I-2 is reports E.Y.C. Zhang L. Zhao S. Wei Q. Zhang J. Qi Z.Q. Belmonte E.R. Frontiers Biosci. 1999; 4: 270-285Crossref PubMed Google and that PP1·I-2 is complexed with other protein in the brain. (13Yang S.D. Fong Y.-L. J. Biol. Chem. 1985; 260: 13464-13470Abstract Full Text PDF PubMed Google found that PP1 has activity in brain is complexed with and is activated by preincubation with ATP/Mg not These are with a (13Yang S.D. Fong Y.-L. J. Biol. Chem. 1985; 260: 13464-13470Abstract Full Text PDF PubMed Google and that the PP1·I-2-activating factor is in brain. An that GSK3 is in 1 column fractions not GSK3 is for ATP/Mg-dependent PP1·I-2 gel filtration fractions PP1 activity from A. We an from the fractions with ATP/Mg the in the of the GSK3 inhibitor LiCl Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google to GSK3 action and PP1 activity. PP1 activity was only ∼29% inhibited 1 a kinase that LiCl GSK3 activity in not These data that the brain a kinase other GSK3 that phosphorylates I-2 and PP1. the PP1·I-2-activating kinase 2 inhibitory peptide in the preincubation and the 1 column fractions. PP1 activity was the in in the of not These data indicated that ATP/Mg-dependent PP1 activation in not be due to the of PKA kinases kinase kinase phosphorylates I-2 in Q.M. Guan K.L. Roach P.J. DePaoli-Roach A.A. J. Biol. Chem. 1995; 270: 18352-18358Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). is a PP1·I-2-activating kinase, analyzed 1 column fractions by Our that of and in brain extract to not that and also in 1 fractions not These indicated that and are not for activating PP1·I-2 in I-2 Thr72 is by a I.K. Roach P. Bondor J. Fox S.P. DePaoli-Roach A.A. J. Biol. Chem. 1994; 269: 944-954Abstract Full Text PDF PubMed Google and NCLK an J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). An not as as an NCLK activity that NCLK was in fractions PP1 and a gel filtration to PP1 We the NCLK inhibitor Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, J. L. A. L. L. S. L. Eur. J. Biochem. 1994; PubMed Scopus Google in the preincubation and PP1 and NCLK in the column fractions from A. not only NCLK activity not it also ATP/Mg-dependent PP1 activity by of both and LiCl in the preincubation inhibited of ATP/Mg-dependent PP1 activity 1 blocking NCLK action ATP/Mg-dependent activation of PP1 in These data that ATP/Mg-dependent PP1 activity in the brain also be by the column fractions from 1 with PP1-specific microcystin-Sepharose J. P. J. 1995; PubMed Scopus Google Scholar). The and with PP1 was pulled down with microcystin-Sepharose as NCLK also was pulled down with microcystin-Sepharose 2 We two In the glutathione-agarose with with the column and by an In the glutathione-agarose with with the column and with in and both PP1 and NCLK pulled down with the from the column fractions. PP1 was pulled down with the with not 2 the column fractions from through a An of and indicated that PP1 and NCLK co-eluted from the column not NCLK and PP1 from brain extract not be from other by gel data indicated that NCLK is complexed with PP1·I-2 in the brain extract and is to be a PP1·I-2-activating PP1·I-2 activation I-2 phosphorylation S.D. Vandenheede J.R. Goris J. Merlevede W. J. Biol. Chem. 1980; 255: 11759-11767Abstract Full Text PDF PubMed Google Scholar, 6Hemmings B.A. Yellowless D. Kernohan J.C. Cohen P. Eur. J. Biochem. 1981; 119: 443-451Crossref PubMed Scopus (184) Google Scholar, 7Yang S.D. Vandenheede J.R. Merlevede W. J. Biol. Chem. 1981; 256: 10231-10234Abstract Full Text PDF PubMed Google Scholar, 8DePaoli-Roach A.A. J. Biol. Chem. 1984; 259: 12144-12152Abstract Full Text PDF PubMed Google Scholar, 9Park I.K. Roach P. Bondor J. Fox S.P. DePaoli-Roach A.A. J. Biol. Chem. 1994; 269: 944-954Abstract Full Text PDF PubMed Google NCLK phosphorylates We I-2 with NCLK in the of and the analyzed by I-2 was phosphorylated in a and NCLK of of NCLK in study was from brain to see NCLK by other to various kinases that kinase not and Similarly, kinase PKA GSK3 activity not and the phosphorylation of I-2 by NCLK was to 2 NCLK inhibitor in the and I-2 inhibited I-2 phosphorylation by NCLK in a and data that NCLK phosphorylates PP1·I-2 activation requires phosphorylation of I-2 on Thr72 S.D. Vandenheede J.R. Goris J. Merlevede W. J. Biol. Chem. 1980; 255: 11759-11767Abstract Full Text PDF PubMed Google Scholar, 6Hemmings B.A. Yellowless D. Kernohan J.C. Cohen P. Eur. J. Biochem. 1981; 119: 443-451Crossref PubMed Scopus (184) Google Scholar, 7Yang S.D. Vandenheede J.R. Merlevede W. J. Biol. Chem. 1981; 256: 10231-10234Abstract Full Text PDF PubMed Google Scholar, 8DePaoli-Roach A.A. J. Biol. Chem. 1984; 259: 12144-12152Abstract Full Text PDF PubMed Google Scholar, 9Park I.K. Roach P. Bondor J. Fox S.P. DePaoli-Roach A.A. J. Biol. Chem. 1994; 269: 944-954Abstract Full Text PDF PubMed Google Scholar). NCLK phosphorylates I-2 I-2 was and the was an one eluted from the column not fractions and with The was through a column and an one peptide was peptide was by a and identified as the and of peptide The by not be that was This was by the of the on data and the acid of I-2 S.J. Cohen 1994; PubMed Scopus Google that from I-2 residues and that Thr72 is the phosphorylation NCLK phosphorylates Thr72 of of acid was by a The identified is a that not be in a The acid was by a The identified is a that not be GSK3 phosphorylates I-2 on However, GSK3 phosphorylates I-2 nonphosphorylated I-2 A.A. J. Biol. Chem. 1984; 259: 12144-12152Abstract Full Text PDF PubMed Google Scholar, 9Park I.K. Roach P. Bondor J. Fox S.P. DePaoli-Roach A.A. J. Biol. Chem. 1994; 269: 944-954Abstract Full Text PDF PubMed Google Scholar). found that NCLK also phosphorylates I-2 on phosphorylation of I-2 by NCLK and GSK3. NCLK and GSK3 and of of We phosphorylated I-2 and I-2 by NCLK GSK3 and the NCLK of of I-2 and of of GSK3 as of of I-2 and of of NCLK phosphorylated I-2 and I-2 in a manner. on the other phosphorylated I-2 the nonphosphorylated These data that GSK3 and NCLK substrate specificity for I-2 We found that NCLK is complexed with PP1 in brain extract NCLK to glutathione-agarose with with and with PP1 was found to be associated with not with These data that NCLK to PP1 through its Cdk5 subunit. the PP1 binding region within Cdk5 three Cdk5 residues residues and residues with with and with in and PP1 bound to of that bound PP1 and that of Cdk5 residues bound PP1 and the of Cdk5 residues These indicated that a major PP1 binding region is within the Cdk5 residues to demonstrate that NCLK the PP1·I-2 complex from PP1 and PP1·I-2 was with ATP/Mg in the of and PP1 activity was in preincubation of PP1·I-2 with ATP/Mg not PP1 activity, as PP1·I-2 preincubation with ATP/Mg in the of NCLK PP1 activity by PP1 activity is by three PP1 inhibitory subunits: DARPP-32, and I-2 (1Cohen P. Annu. Rev. Biochem. 1989; 58: 453-508Crossref PubMed Scopus (2146) Google Scholar, 2Lee E.Y.C. Zhang L. Zhao S. Wei Q. Zhang J. Qi Z.Q. Belmonte E.R. Frontiers Biosci. 1999; 4: 270-285Crossref PubMed Google Scholar, 3Oliver C.J. Shenolikar S. Frontiers Biosci. 1998; 3: 961-972Crossref PubMed Google Scholar). I-1 is expressed in various whereas DARPP-32 is found in it is by (1Cohen P. Annu. Rev. Biochem. 1989; 58: 453-508Crossref PubMed Scopus (2146) Google Scholar). PKA phosphorylates I-1 and DARPP-32, and both PP1 upon PKA phosphorylation (1Cohen P. Annu. Rev. Biochem. 1989; 58: 453-508Crossref PubMed Scopus (2146) Google Scholar, 2Lee E.Y.C. Zhang L. Zhao S. Wei Q. Zhang J. Qi Z.Q. Belmonte E.R. Frontiers Biosci. 1999; 4: 270-285Crossref PubMed Google Scholar, 3Oliver C.J. Shenolikar S. Frontiers Biosci. 1998; 3: 961-972Crossref PubMed Google Scholar). I-1 and DARPP-32 have also been to be phosphorylated by NCLK Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google J. A. L. A.A. P. 1999; PubMed Scopus Google Scholar). NCLK phosphorylated I-1 inhibits PP1 in a to PKA phosphorylated I-1 Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). NCLK phosphorylated DARPP-32, on the other not PP1 is a PKA inhibitor J. A. L. A.A. P. 1999; PubMed Scopus Google Scholar). In a study PP1 in brain extract was found to as PP1·I-2 and preincubation with ATP/Mg to become active (13Yang S.D. Fong Y.-L. J. Biol. Chem. 1985; 260: 13464-13470Abstract Full Text PDF PubMed Google Scholar). In study brain PP1·I-2 and found that GSK3 was in a inhibition of GSK3 activity by LiCl only ∼29% of ATP/Mg-dependent PP1·I-2 activation in 1 These data that GSK3 is not the sole PP1·I-2 activator in the brain. We found that the ATP/Mg-dependent activation of PP1·I-2 was to the NCLK inhibitor 1 NCLK was pulled down from with microcystin-Sepharose and glutathione-agarose and Similarly, PP1 in bound to glutathione-agarose with 2 In vitro, NCLK phosphorylated I-2 on Thr72 and activated PP1·I-2 in an ATP/Mg-dependent data that NCLK is one of the PP1·I-2-activating kinases in the brain. PP1 a substrate specificity and of many protein kinases (1Cohen P. Annu. Rev. Biochem. 1989; 58: 453-508Crossref PubMed Scopus (2146) Google Scholar, 2Lee E.Y.C. Zhang L. Zhao S. Wei Q. Zhang J. Qi Z.Q. Belmonte E.R. Frontiers Biosci. 1999; 4: 270-285Crossref PubMed Google Scholar). NCLK phosphorylates I-1 and DARPP-32 Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, J. A. L. A.A. P. 1999; PubMed Scopus Google Scholar). In study that NCLK also phosphorylates These suggest that NCLK a in neuronal by PP1 inhibitory subunits DARPP-32, and is that PP1 to I-2 (1Cohen P. Annu. Rev. Biochem. 1989; 58: 453-508Crossref PubMed Scopus (2146) Google Scholar, 2Lee E.Y.C. Zhang L. Zhao S. Wei Q. Zhang J. Qi Z.Q. Belmonte E.R. Frontiers Biosci. 1999; 4: 270-285Crossref PubMed Google Scholar, 3Oliver C.J. Shenolikar S. Frontiers Biosci. 1998; 3: 961-972Crossref PubMed Google Scholar, 8DePaoli-Roach A.A. J. Biol. Chem. 1984; 259: 12144-12152Abstract Full Text PDF PubMed Google Scholar, 9Park I.K. Roach P. Bondor J. Fox S.P. DePaoli-Roach A.A. J. Biol. Chem. 1994; 269: 944-954Abstract Full Text PDF PubMed Google Scholar). We found that PP1 also to NCLK These suggest that PP1 is the that I-2 and NCLK within a complex. Our from the gel filtration column fractions complex, PP1 and NCLK are pulled down with and and PP1 is pulled down with 2 These a as to the complex to an has been in PP1 and I-2 are in a with PP1·I-2 and an of I-2 I-2 W. Vandenheede J.R. Merlevede W. A. PubMed Scopus Google Scholar, B.A. Cohen P. Eur. J. Biochem. PubMed Scopus Google Scholar). in 1 PP1 in brain extract a gel filtration column as a of various with of to ∼450 of it is that and NCLK also be in a with the complex in the brain. I-2 within PP1·I-2 be by I-2 W. Vandenheede J.R. Merlevede W. A. PubMed Scopus Google Scholar, B.A. Cohen P. Eur. J. Biochem. PubMed Scopus Google I-2 from and a species. with Cdk5 within the complex and NCLK to in the of a complex. I-2 is bound to PP1 in and inhibits PP1 in its nonphosphorylated GSK3 phosphorylates I-2 on the PP1·I-2 complex. This phosphorylation a in I-2 and also within the PP1·I-2 complex, to PP1 activation complex (3Oliver C.J. Shenolikar S. Frontiers Biosci. 1998; 3: 961-972Crossref PubMed Google Scholar, 8DePaoli-Roach A.A. J. Biol. Chem. 1984; 259: 12144-12152Abstract Full Text PDF PubMed Google Scholar, 9Park I.K. Roach P. Bondor J. Fox S.P. DePaoli-Roach A.A. J. Biol. Chem. 1994; 269: 944-954Abstract Full Text PDF PubMed Google Scholar, W. Vandenheede J.R. Merlevede W. A. PubMed Scopus Google Scholar). PP1 This not of PP1 activity and only the complex to its inactive (3Oliver C.J. Shenolikar S. Frontiers Biosci. 1998; 3: 961-972Crossref PubMed Google Scholar, 8DePaoli-Roach A.A. J. Biol. Chem. 1984; 259: 12144-12152Abstract Full Text PDF PubMed Google Scholar, 9Park I.K. Roach P. Bondor J. Fox S.P. DePaoli-Roach A.A. J. Biol. Chem. 1994; 269: 944-954Abstract Full Text PDF PubMed Google W. Vandenheede J.R. Merlevede W. A. PubMed Scopus Google Scholar). GSK3 phosphorylation of I-2 within PP1·I-2 has been to be (3Oliver C.J. Shenolikar S. Frontiers Biosci. 1998; 3: 961-972Crossref PubMed Google Scholar, 8DePaoli-Roach A.A. J. Biol. Chem. 1984; 259: 12144-12152Abstract Full Text PDF PubMed Google Scholar, 9Park I.K. Roach P. Bondor J. Fox S.P. DePaoli-Roach A.A. J. Biol. Chem. 1994; 269: 944-954Abstract Full Text PDF PubMed Google Scholar). the phosphorylation of I-2 PP1·I-2 with NCLK in the of We found that the incubation activated PP1 within PP1·I-2. However, analyzed the of the incubation by not I-2 phosphorylation not These suggest that phosphorylation of I-2 by NCLK within the PP1·I-2 complex is also a I-2 is phosphorylated on and in Cohen P. Biochim. Biophys. Acta. PubMed Scopus Google and are phosphorylated by in (1Cohen P. Annu. Rev. Biochem. 1989; 58: 453-508Crossref PubMed Scopus (2146) Google Scholar, J. Cohen P. Biochim. Biophys. Acta. PubMed Scopus Google Scholar). phosphorylation not activate PP1·I-2 by GSK3 A.A. J. Biol. Chem. 1984; 259: 12144-12152Abstract Full Text PDF PubMed Google Scholar, 9Park I.K. Roach P. Bondor J. Fox S.P. DePaoli-Roach A.A. J. Biol. Chem. 1994; 269: 944-954Abstract Full Text PDF PubMed Google Scholar). We found that NCLK also phosphorylates I-2 on However, NCLK phosphorylation of I-2 is to a phosphorylation by These data that NCLK and GSK3 substrate specificity for I-2 In and NCLK activate PP1·I-2 in response to also in of the brain in subcellular compartments. In study that a major is within a Cdk5 region region is to an found in many E.Y.C. Zhang L. Zhao S. Wei Q. Zhang J. Qi Z.Q. Belmonte E.R. Frontiers Biosci. 1999; 4: 270-285Crossref PubMed Google Scholar, Cohen Cohen P. D. J. PubMed Scopus Google Scholar, S. E.Y.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). This is in the binding region and the of This is in Cdk5 from E. J. PubMed Scopus Google Scholar, J. J. Biol. Chem. Full Text PDF PubMed Google Scholar, Res. 1994; PubMed Scopus Google Scholar, Zhang Full Text PDF PubMed Scopus Google Scholar, Res. Res. 1995; PubMed Scopus Google and has a in 1994; PubMed Scopus Google Scholar). In is an for with other J. J. PubMed Scopus Google Scholar, Biochem. Biophys. Res. Commun. 1999; 259: PubMed Scopus Google Scholar). also be that also a PP1 binding that a PP1 binding region is within Cdk5 residues be to PP1 binding We for

Fetched live from OpenAlex and de-inverted. Abstracts are not stored in this database: the inverted indexes are 8.6 GB of the frame’s 9.3 GB of text, and the host has 13 GB free.