Phosphorylation of Protein Phosphatase Inhibitor-1 by Cdk5
2001; Elsevier BV; Volume: 276; Issue: 17 Linguagem: Inglês
10.1074/jbc.m007197200
ISSN1083-351X
AutoresJames Bibb, Akinori Nishi, James P. O’Callaghan, Jernej Ule, Martin J. Lan, Gretchen L. Snyder, Atsuko Horiuchi, Taro Saito, Shin-ichi Hisanaga, Andrew J. Czernik, Angus C. Nairn, Paul Greengard,
Tópico(s)Protein Tyrosine Phosphatases
ResumoProtein phosphatase inhibitor-1 is a prototypical mediator of cross-talk between protein kinases and protein phosphatases. Activation of cAMP-dependent protein kinase results in phosphorylation of inhibitor-1 at Thr-35, converting it into a potent inhibitor of protein phosphatase-1. Here we report that inhibitor-1 is phosphorylated in vitro at Ser-67 by the proline-directed kinases, Cdk1, Cdk5, and mitogen-activated protein kinase. By using phosphorylation state-specific antibodies and selective protein kinase inhibitors, Cdk5 was found to be the only kinase that phosphorylates inhibitor-1 at Ser-67 in intact striatal brain tissue. In vitro and in vivo studies indicated that phospho-Ser-67 inhibitor-1 was dephosphorylated by protein phosphatases-2A and -2B. The state of phosphorylation of inhibitor-1 at Ser-67 was dynamically regulated in striatal tissue by glutamate-dependent regulation ofN-methyl-d-aspartic acid-type channels. Phosphorylation of Ser-67 did not convert inhibitor-1 into an inhibitor of protein phosphatase-1. However, inhibitor-1 phosphorylated at Ser-67 was a less efficient substrate for cAMP-dependent protein kinase. These results demonstrate regulation of a Cdk5-dependent phosphorylation site in inhibitor-1 and suggest a role for this site in modulating the amplitude of signal transduction events that involve cAMP-dependent protein kinase activation. Protein phosphatase inhibitor-1 is a prototypical mediator of cross-talk between protein kinases and protein phosphatases. Activation of cAMP-dependent protein kinase results in phosphorylation of inhibitor-1 at Thr-35, converting it into a potent inhibitor of protein phosphatase-1. Here we report that inhibitor-1 is phosphorylated in vitro at Ser-67 by the proline-directed kinases, Cdk1, Cdk5, and mitogen-activated protein kinase. By using phosphorylation state-specific antibodies and selective protein kinase inhibitors, Cdk5 was found to be the only kinase that phosphorylates inhibitor-1 at Ser-67 in intact striatal brain tissue. In vitro and in vivo studies indicated that phospho-Ser-67 inhibitor-1 was dephosphorylated by protein phosphatases-2A and -2B. The state of phosphorylation of inhibitor-1 at Ser-67 was dynamically regulated in striatal tissue by glutamate-dependent regulation ofN-methyl-d-aspartic acid-type channels. Phosphorylation of Ser-67 did not convert inhibitor-1 into an inhibitor of protein phosphatase-1. However, inhibitor-1 phosphorylated at Ser-67 was a less efficient substrate for cAMP-dependent protein kinase. These results demonstrate regulation of a Cdk5-dependent phosphorylation site in inhibitor-1 and suggest a role for this site in modulating the amplitude of signal transduction events that involve cAMP-dependent protein kinase activation. Control of protein phosphorylation/dephosphorylation occurs through regulation of protein kinase and protein phosphatase activities and is an integral component of intracellular signal transduction. Inhibitor-1 was the first endogenous molecule found to regulate protein phosphatase activity (1Huang F.L. Glinsmann W.H. Eur. J. Biochem. 1976; 1976: 419-426Crossref Scopus (307) Google Scholar). Inhibitor-1 purified from rabbit skeletal muscle is an 18,700-kDa acid- and heat-stable protein composed of 166 amino acids that are highly conserved throughout phylogeny (2Elbrecht A. DiRenzo J. Smith R.G. Shenolikar S. J. Biol. Chem. 1990; 265: 13415-13418Abstract Full Text PDF PubMed Google Scholar, 3Nimmo G.A. Cohen P. Eur. J. Biochem. 1978; 87: 341-351Crossref PubMed Scopus (209) Google Scholar). When phosphorylated at Thr-35 by cAMP-dependent protein kinase (PKA),1 inhibitor-1 selectively and potently inhibits type 1 protein phosphatase (protein phosphatase-1, PP-1) with an IC50 value of ∼1 nm (4Foulkes J.G. Strada S.J. Henderson P.J. Cohen P. Eur. J. Biochem. 1983; 132: 309-313Crossref PubMed Scopus (56) Google Scholar, 5Endo S. Zhou X. Connor J. Wang B. Shenolikar S. Biochemistry. 1996; 35: 522-5228Crossref Scopus (150) Google Scholar, 6Aitken A. Cohen P. FEBS Lett. 1982; 147: 54-58Crossref PubMed Scopus (51) Google Scholar, 7Hemmings Jr., H.C. Greengard P. Tung H.Y.L. Cohen P. Nature. 1984; 310: 503-505Crossref PubMed Scopus (477) Google Scholar). Phospho-Thr-35 inhibitor-1 is dephosphorylated by Ca2+/calmodulin-dependent protein phosphatase 2B (PP-2B, calcineurin) and protein phosphatase 2A (PP-2A), with PP-2B activity predominating in the presence of Ca2+(8Cohen P. Annu. Rev. Biochem. 1989; 58: 453-508Crossref PubMed Scopus (2144) Google Scholar, 9Shenolikar S. Nairn A.C. Adv. Second Messenger Phosphoprotein Res. 1991; 23: 1-121PubMed Google Scholar, 10Shenolikar S. Annu. Rev. Cell Biol. 1994; 10: 55-86Crossref PubMed Scopus (402) Google Scholar, 11Bollen M. Stalmans W. Crit. Rev. Biochem. Mol. Biol. 1992; 27: 227-281Crossref PubMed Scopus (261) Google Scholar). First messengers such as neurotransmitters (e.g.dopamine and acetylcholine) and hormones (e.g. adrenaline) that elevate intracellular cAMP levels promote PKA-dependent phosphorylation of inhibitor-1 at Thr-35 in various tissues. PP-1 inhibition by phospho-Thr-35 inhibitor-1 provides substantial amplification of PKA-dependent signaling cascades and modulates the intensity and duration of a number of physiological responses including regulatory aspects of the cell cycle, gene expression, carbohydrate and lipid metabolism, and synaptic plasticity (12Oliver C.J. Shenolikar S. Front. Biosci. 1998; 3: D961-72Crossref PubMed Google Scholar, 13Connor J.H. Quan H. Oliver C. Shenolikar S. Methods Mol. Biol. 1998; 93: 41-58PubMed Google Scholar, 14Connor J.H. Quan H.N. Ramaswamy N.T. Zhang L. Barik S. Zheng J. Cannon J.F. Lee E.Y.C. Shenolikar S. J. Biol. Chem. 1998; 273: 27716-27724Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 15Mulkey R.M. Endo S. Shenolikar S. Malenka R.C. Nature. 1994; 369: 486-488Crossref PubMed Scopus (898) Google Scholar, 16Hagiwara M. Alberts A. Brindle P. Meinkoth J. Feramisco J. Deng T. Karin M. Shenolikar S. Montminy M. Cell. 1992; 70: 105-113Abstract Full Text PDF PubMed Scopus (404) Google Scholar, 17Alberts A.S. Montminy M. Shenolikar S. Feramisco J.R. Mol. Cell. Biol. 1994; 14: 4398-4407Crossref PubMed Scopus (114) Google Scholar).Inhibitor-1 is widely expressed in mammalian tissue with highest levels occurring in the brain, skeletal muscle, adipose, and kidney tissues (18Hemmings Jr., H.C. Girault J.-A. Nairn A.C. Bertuzzi G. Greengard P. J. Neurochem. 1992; 59: 1053-1061Crossref PubMed Scopus (55) Google Scholar, 19MacDougall L.K. Cambell D.G. Hubbard M.J. Cohen P. Biochim. Biophys. Acta. 1989; 1010: 218-226Crossref PubMed Scopus (44) Google Scholar, 20Lowenstein P.R. Shering A.F. MacDougall L.K. Cohen P. Brain Res. 1995; 575: 80-92Crossref Scopus (11) Google Scholar, 21Gustafson E.L. Girault J.-A. Hemmings Jr., H.C. Nairn A.C. Greengard P. J. Comp. Neurol. 1991; 310: 170-188Crossref PubMed Scopus (43) Google Scholar, 22Barbas H. Gustafson E.L. Greengard P. J. Comp. Neurol. 1993; 334: 1-18Crossref PubMed Scopus (27) Google Scholar, 23Alder R. Barbas H. Neuroreports. 1995; 6: 2368-2372Crossref PubMed Scopus (10) Google Scholar, 24Sakagami H. Ebina K. Kondo H. Mol. Brain Res. 1994; 25: 7-18Crossref PubMed Scopus (35) Google Scholar, 25Allen P. Hvalby Ø. Jensen V. Ramsay M. Chaudhry F.A. Storm- Mathisen J. Morris R. Andersen P. Greengard P. J. Neurosci. 2000; 20: 3537-3543Crossref PubMed Google Scholar, 26Mikkelsen J.D. Gustafson E.L. Brain Res. 1993; 623: 147-154Crossref PubMed Scopus (4) Google Scholar). Within the brain, the highest levels of inhibitor-1 immunoreactivity are associated with the dentate gyrus of the hippocampus and the neostriatum and substantia nigra of the basal ganglia (21Gustafson E.L. Girault J.-A. Hemmings Jr., H.C. Nairn A.C. Greengard P. J. Comp. Neurol. 1991; 310: 170-188Crossref PubMed Scopus (43) Google Scholar). Control of PP-1 by inhibitor-1 in the hippocampus is thought to be an important component of the mechanisms underlying learning and memory, including long term potentiation and long term depression (15Mulkey R.M. Endo S. Shenolikar S. Malenka R.C. Nature. 1994; 369: 486-488Crossref PubMed Scopus (898) Google Scholar, 27Blitzer R.D. Connor J.H. Brown G.P. Wong T. Shenolikar S. Iyengar R. Landau E.M. Science. 1998; 280: 1940-1943Crossref PubMed Scopus (356) Google Scholar). Mice lacking inhibitor-1 display deficits in long term potentiation induction (25Allen P. Hvalby Ø. Jensen V. Ramsay M. Chaudhry F.A. Storm- Mathisen J. Morris R. Andersen P. Greengard P. J. Neurosci. 2000; 20: 3537-3543Crossref PubMed Google Scholar).In the dopaminoceptive medium spiny neurons of the striatum, inhibitor-1 is co-expressed with a homologous PP-1 inhibitor, DARPP-32 (28Nairn A.C. Hemmings Jr., H.C. Walaas S.I. Greengard P. J. Neurochem. 1998; 50: 257-262Crossref Scopus (38) Google Scholar). Inhibitor-1 is ∼10 times less abundant than DARPP-32 in the striatum (21Gustafson E.L. Girault J.-A. Hemmings Jr., H.C. Nairn A.C. Greengard P. J. Comp. Neurol. 1991; 310: 170-188Crossref PubMed Scopus (43) Google Scholar, 29Hemmings Jr., H.C. Nairn A.C. Aswad D.W. Greengard P. J. Neurosci. 1984; 4: 99-110Crossref PubMed Google Scholar, 30Ouimet C.C. Langley-Gullion K.C. Greengard P. Brain Res. 1998; 808: 8-12Crossref PubMed Scopus (141) Google Scholar), which constitutes about 0.25% of total striatal protein and is estimated to occur at a concentration of 50 μm. The NH2-terminal residues 9–50 of inhibitor-1 and DARPP-32 display 60% identity (31Williams K.R. Hemmings Jr., H.C. LoPresti M.B. Konigsberg W.H. Greengard P. J. Biol. Chem. 1986; 261: 1890-1903Abstract Full Text PDF PubMed Google Scholar) and phosphorylation of the homologous residue on DARPP-32 (Thr-34) converts it into an inhibitor of PP-1 with an IC50 value identical to that of inhibitor-1 (7Hemmings Jr., H.C. Greengard P. Tung H.Y.L. Cohen P. Nature. 1984; 310: 503-505Crossref PubMed Scopus (477) Google Scholar). The activity of DARPP-32 is regulated through phosphorylation at other sites. Phosphorylation of Ser-102 by casein kinase 2 and Ser-137 by casein kinase 1 potentiates PKA phosphorylation and attenuates PP-2B dephosphorylation of Thr-34, respectively (32Greengard P. Allen P.B. Nairn A.C. Neuron. 1999; 23: 435-447Abstract Full Text Full Text PDF PubMed Scopus (638) Google Scholar, 33Desdouits F. Sciliano J.C. Greengard P. Girault J.-A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2682-2685Crossref PubMed Scopus (81) Google Scholar, 34Girault J.-A. Hemmings Jr., H.C. Williams K.R. Nairn A.C. Greengard P. J. Biol. Chem. 1989; 264: 21748-21759Abstract Full Text PDF PubMed Google Scholar). Phosphorylation of DARPP-32 by cyclin-dependent kinase 5 (Cdk5) at Thr-75 prevents DARPP-32 from serving as a PKA substrate and converts DARPP-32 into a competitive inhibitor of PKA (35Bibb 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 (487) Google Scholar). Following residue 50, very little primary sequence homology exists between inhibitor-1 and DARPP-32, and inhibitor-1 does not contain phosphorylation sites homologous to those found in DARPP-32 other than Thr-35. Therefore, it seemed possible that the activity of inhibitor-1 is differentially regulated by phosphorylation of other putative sites.In this report we show that inhibitor-1 is phosphorylated on Ser-67 by MAP kinase and by two members of the cyclin-dependent protein kinase family in vitro but serves as a substrate only for Cdk5 in striatal neurons. We demonstrate that this site of phosphorylation is predominantly dephosphorylated by PP-2A and PP-2B. Kinetic analyses indicate that phosphorylation at Ser-67 reduces the ability of inhibitor-1 to serve as a substrate for PKA but has no effect on PP-1 inhibition.DISCUSSIONWe report here that protein phosphatase inhibitor-1 is efficiently phosphorylated at Ser-67 by three different proline-directed kinasesin vitro, Cdk1, Cdk5, and MAP kinase. The apparentKm values for these phosphorylation reactions were similar to the concentration of inhibitor-1 estimated to occur in striatal medium spiny neurons (29Hemmings Jr., H.C. Nairn A.C. Aswad D.W. Greengard P. J. Neurosci. 1984; 4: 99-110Crossref PubMed Google Scholar, 30Ouimet C.C. Langley-Gullion K.C. Greengard P. Brain Res. 1998; 808: 8-12Crossref PubMed Scopus (141) Google Scholar, 57Foulkes J.G. Cohen P. Eur. J. Biochem. 1979; 97: 251-256Crossref PubMed Scopus (94) Google Scholar, 58Nimmo G.A. Cohen P. Eur. J. Biochem. 1978; 87: 353-365Crossref PubMed Scopus (88) Google Scholar). Moreover, immunoblot analysis indicated that Cdk5 and MAP kinase, but not Cdk1, are present in adult striatum. Treatment of striatal slices with selective protein kinase inhibitors indicated that Cdk5 phosphorylates Ser-67 inhibitor-1 in the striatum but that MAP kinase does not. These studies using striatal slices were consistent with our biochemical results and indicate that Cdk5 is the predominant kinase responsible for phosphorylation of inhibitor-1 at Ser-67 in intact striatal tissue. Cdk5 functions in neurite outgrowth (59Nikolic M. Dudek H. Kwon Y.T. Ramos Y.F. Tsai L.H. Genes Dev. 1996; 10: 816-825Crossref PubMed Scopus (529) Google Scholar), development of the nervous system (60Ohshima 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 (802) Google Scholar), and regulation of dopamine signaling through phosphorylation of DARPP-32 in the striatum (35Bibb 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 (487) Google Scholar). Cdk5 may also play a role in muscle development (61Lazaro J.B. Kitzmann M. Poul M.A. Vandromme M. Lamb N.J. Fernandez A. J. Cell Sci. 1997; 110: 1251-1260Crossref PubMed Google Scholar). A substantial level of basal phosphorylation of inhibitor-1 at Ser-67 was also detected in adult rat hippocampus, and in skeletal muscle and kidney tissue. 3J. Bibb, unpublished results. These observations suggest that Cdk1 and/or MAP kinase could also be responsible for the phosphorylation of Ser-67 in peripheral tissue.Phospho-Ser-67 inhibitor-1 was found to be a substrate for protein phosphatases PP-2A and PP-2B in vitro. In other in vitro protein phosphatase assays using purified protein phosphatase catalytic subunits and standard substrates as controls, PP-2B was found to be more efficient than PP-2A and PP-2C at dephosphorylating phospho-Ser-67 inhibitor-1. PP-1 could not dephosphorylate phospho-Ser-67 inhibitor-1 at all (data not shown). Both phospho-Thr-35 of inhibitor-1 and phospho-Thr-34 of DARPP-32 have been shown to be very efficient substrates for PP-2B (62King M.M. Huang C.Y. Chock P.B. Nairn A.C. Hemmings Jr., H.C. Chan K.-F.J. Greengard P. J. Biol. Chem. 1984; 259: 8080-8083Abstract Full Text PDF PubMed Google Scholar), and PP-2B is highly concentrated in striatal neurons (63Goto S. Matsukado Y. Mihara Y. Inoue N. Miyamoto E. Brain Res. 1986; 397: 161-172Crossref PubMed Scopus (149) Google Scholar). Treatment of slices with NMDA, which activates PP-2B by increasing intracellular Ca2+, caused an almost complete loss of phospho-Ser-67 levels. Conversely, cyclosporin A, a PP-2B inhibitor, increased phospho-Ser-67 levels. Basal phospho-Ser-67 inhibitor-1 levels were increased in striatal slices from PP-2Bα−/−mice. The residual effects of NMDA on Ser-67 in PP-2Bα−/− may be attributed to the activity of the β-isoform of PP-2B (56Zhang B.W. Zimmer G. Chen J. Ladd D. Li E. Alt F.W. Wiederrecht G. Cryan J. O'Neill E.A. Seidman C.E. Abbas A.K. Seidman J.G. J. Exp. Med. 1996; 183: 413-420Crossref PubMed Scopus (138) Google Scholar). Thus, a variety of data suggest that both PP-2A and PP-2B may dephosphorylate phospho-Ser-67 inhibitor-1 under basal conditions, but PP-2B may function as the predominant phosphatase in the presence of elevated Ca2+ levels.Phosphorylation of inhibitor-1 at Ser-67 by Cdk5 had no effect on PP-1 inhibitory activity. These results are also in complete agreement with numerous previous reports that used inhibitor-1 purified from rabbit muscle. It has been demonstrated that inhibitor-1, as well as its homolog, DARPP-32, only become potent inhibitors of PP-1 after phosphorylation by PKA and that the Thr-35 nonphosphorylated form of inhibitor-1 is devoid of PP-1 inhibitory activity (1Huang F.L. Glinsmann W.H. Eur. J. Biochem. 1976; 1976: 419-426Crossref Scopus (307) Google Scholar, 50Aitken A. Bilham T. Cohen P. Eur. J. Biochem. 1982; 126: 235-246Crossref PubMed Scopus (118) Google Scholar, 58Nimmo G.A. Cohen P. Eur. J. Biochem. 1978; 87: 353-365Crossref PubMed Scopus (88) Google Scholar, 64Cohen P. Rylatt D.B. Nimmo G.A. FEBS Lett. 1977; 76: 182-186Crossref PubMed Scopus (69) Google Scholar). In early studies, inhibitor-1 isolated from rabbit skeletal muscle was found, by direct amino acid sequence analysis, to be phosphorylated at Ser-67 with a stoichiometry of 0.5–0.7 mol/mol (50Aitken A. Bilham T. Cohen P. Eur. J. Biochem. 1982; 126: 235-246Crossref PubMed Scopus (118) Google Scholar). That preparation was found to inhibit PP-1 with a Ki of 1.6 nm only when phosphorylated at Thr-35 by PKA. Without phosphorylation by PKA, the endogenous protein could not inhibit PP-1 at detectable levels even at a concentration 1,000-fold above theKi. Peptide fragments lacking the sequence surrounding phospho-Ser-67 were fully active when phosphorylated by PKA, and a phosphopeptide containing residues 61–71 was inactive (5Endo S. Zhou X. Connor J. Wang B. Shenolikar S. Biochemistry. 1996; 35: 522-5228Crossref Scopus (150) Google Scholar,6Aitken A. Cohen P. FEBS Lett. 1982; 147: 54-58Crossref PubMed Scopus (51) Google Scholar). All these findings are in contradiction to a recent report by Huang and Paudel (65Huang K.-X. Paudel H.K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5824-5829Crossref PubMed Scopus (46) Google Scholar) that suggested that phosphorylation of inhibitor-1 at Ser-67 converted it into a potent inhibitor of recombinant PP-1 purified from bacteria.The present studies are also completely consistent with extensive studies that have established that PP-1 is inhibited by a common conserved region at the NH2 terminus of inhibitor-1 and DARPP-32 (5Endo S. Zhou X. Connor J. Wang B. Shenolikar S. Biochemistry. 1996; 35: 522-5228Crossref Scopus (150) Google Scholar, 66Huang H.B. Horiuchi A. Watanabe T. Shih S.-R. Tsay H.-J. Li H.-C. Greengard P. Nairn A.C. J. Biol. Chem. 1999; 274: 7870-7878Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 67Goldberg J. Huang H.B. Kwon Y.G. Greengard P. Nairn A.C. Kuriyan J. Nature. 1995; 376: 745-753Crossref PubMed Scopus (740) Google Scholar). In addition to the region surrounding the phosphorylated Thr-35 residue (Thr-34 in DARPP-32), which interacts with the active site of PP-1, a short docking motif (RKIXF, residues 8–12) in the two proteins is required for potent inhibition and interacts with a defined region that is removed from the active site on PP-1 (5Endo S. Zhou X. Connor J. Wang B. Shenolikar S. Biochemistry. 1996; 35: 522-5228Crossref Scopus (150) Google Scholar, 54Huang H.-B. Horiuchi A. Goldberg J. Greengard P. Nairn A.C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3530-3535Crossref PubMed Scopus (76) Google Scholar, 66Huang H.B. Horiuchi A. Watanabe T. Shih S.-R. Tsay H.-J. Li H.-C. Greengard P. Nairn A.C. J. Biol. Chem. 1999; 274: 7870-7878Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 67Goldberg J. Huang H.B. Kwon Y.G. Greengard P. Nairn A.C. Kuriyan J. Nature. 1995; 376: 745-753Crossref PubMed Scopus (740) Google Scholar). Phosphorylation of DARPP-32 at other sites does not directly affect PP-1 inhibitory activity (33Desdouits F. Sciliano J.C. Greengard P. Girault J.-A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2682-2685Crossref PubMed Scopus (81) Google Scholar, 34Girault J.-A. Hemmings Jr., H.C. Williams K.R. Nairn A.C. Greengard P. J. Biol. Chem. 1989; 264: 21748-21759Abstract Full Text PDF PubMed Google Scholar).Our results indicated that phosphorylation of inhibitor-1 at Ser-67 slightly altered its efficiency as a substrate for PKA when phosphorylated on Ser-67. The stoichiometry of phosphorylation in striatal tissue under basal conditions was determined to be 0.34 mol/mol, allowing an estimate of the concentration of phospho-Ser-67 in the striatum of about 1.7 μm. By increasing theKm for PKA phosphorylation from 1.7 to 9.5 μm, phospho-Ser-67 may serve as a fine control mechanism for regulating the degree to which the effects of PKA activation are amplified. Recently, additional PP-1 inhibitor proteins have been identified, some of which demonstrate selective inhibitory activity only when PP-1 occurs in association with other regulatory factors (68Eto M. Karginov A. Brautigan D.L. Biochemistry. 1999; 38: 16952-16957Crossref PubMed Scopus (89) Google Scholar, 69Zhang J. Zhang L. Zhao S. Lee E.Y. Biochemistry. 1998; 37: 16728-16734Crossref PubMed Scopus (53) Google Scholar, 70Allen P.B. Ouimet C.C. Greengard P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9956-9961Crossref PubMed Scopus (381) Google Scholar, 71Jagiello I. Beullens M. Stalmans W. Bollen M. J. Biol. Chem. 1995; 270: 17257-17263Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Similarly, phospho-Ser-67 inhibitor-1 may serve a regulatory function only in the context of a protein complex. Phospho-Ser-67 inhibitor-1 could also facilitate subcellular localization or be involved in the regulation of some unknown function of inhibitor-1. It will also be interesting to see if phosphorylation of inhibitor-1 at Ser-67 is involved in the mechanisms underlying synaptic plasticity in the brain and carbohydrate and lipid metabolism in peripheral tissues. Control of protein phosphorylation/dephosphorylation occurs through regulation of protein kinase and protein phosphatase activities and is an integral component of intracellular signal transduction. Inhibitor-1 was the first endogenous molecule found to regulate protein phosphatase activity (1Huang F.L. Glinsmann W.H. Eur. J. Biochem. 1976; 1976: 419-426Crossref Scopus (307) Google Scholar). Inhibitor-1 purified from rabbit skeletal muscle is an 18,700-kDa acid- and heat-stable protein composed of 166 amino acids that are highly conserved throughout phylogeny (2Elbrecht A. DiRenzo J. Smith R.G. Shenolikar S. J. Biol. Chem. 1990; 265: 13415-13418Abstract Full Text PDF PubMed Google Scholar, 3Nimmo G.A. Cohen P. Eur. J. Biochem. 1978; 87: 341-351Crossref PubMed Scopus (209) Google Scholar). When phosphorylated at Thr-35 by cAMP-dependent protein kinase (PKA),1 inhibitor-1 selectively and potently inhibits type 1 protein phosphatase (protein phosphatase-1, PP-1) with an IC50 value of ∼1 nm (4Foulkes J.G. Strada S.J. Henderson P.J. Cohen P. Eur. J. Biochem. 1983; 132: 309-313Crossref PubMed Scopus (56) Google Scholar, 5Endo S. Zhou X. Connor J. Wang B. Shenolikar S. Biochemistry. 1996; 35: 522-5228Crossref Scopus (150) Google Scholar, 6Aitken A. Cohen P. FEBS Lett. 1982; 147: 54-58Crossref PubMed Scopus (51) Google Scholar, 7Hemmings Jr., H.C. Greengard P. Tung H.Y.L. Cohen P. Nature. 1984; 310: 503-505Crossref PubMed Scopus (477) Google Scholar). Phospho-Thr-35 inhibitor-1 is dephosphorylated by Ca2+/calmodulin-dependent protein phosphatase 2B (PP-2B, calcineurin) and protein phosphatase 2A (PP-2A), with PP-2B activity predominating in the presence of Ca2+(8Cohen P. Annu. Rev. Biochem. 1989; 58: 453-508Crossref PubMed Scopus (2144) Google Scholar, 9Shenolikar S. Nairn A.C. Adv. Second Messenger Phosphoprotein Res. 1991; 23: 1-121PubMed Google Scholar, 10Shenolikar S. Annu. Rev. Cell Biol. 1994; 10: 55-86Crossref PubMed Scopus (402) Google Scholar, 11Bollen M. Stalmans W. Crit. Rev. Biochem. Mol. Biol. 1992; 27: 227-281Crossref PubMed Scopus (261) Google Scholar). First messengers such as neurotransmitters (e.g.dopamine and acetylcholine) and hormones (e.g. adrenaline) that elevate intracellular cAMP levels promote PKA-dependent phosphorylation of inhibitor-1 at Thr-35 in various tissues. PP-1 inhibition by phospho-Thr-35 inhibitor-1 provides substantial amplification of PKA-dependent signaling cascades and modulates the intensity and duration of a number of physiological responses including regulatory aspects of the cell cycle, gene expression, carbohydrate and lipid metabolism, and synaptic plasticity (12Oliver C.J. Shenolikar S. Front. Biosci. 1998; 3: D961-72Crossref PubMed Google Scholar, 13Connor J.H. Quan H. Oliver C. Shenolikar S. Methods Mol. Biol. 1998; 93: 41-58PubMed Google Scholar, 14Connor J.H. Quan H.N. Ramaswamy N.T. Zhang L. Barik S. Zheng J. Cannon J.F. Lee E.Y.C. Shenolikar S. J. Biol. Chem. 1998; 273: 27716-27724Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 15Mulkey R.M. Endo S. Shenolikar S. Malenka R.C. Nature. 1994; 369: 486-488Crossref PubMed Scopus (898) Google Scholar, 16Hagiwara M. Alberts A. Brindle P. Meinkoth J. Feramisco J. Deng T. Karin M. Shenolikar S. Montminy M. Cell. 1992; 70: 105-113Abstract Full Text PDF PubMed Scopus (404) Google Scholar, 17Alberts A.S. Montminy M. Shenolikar S. Feramisco J.R. Mol. Cell. Biol. 1994; 14: 4398-4407Crossref PubMed Scopus (114) Google Scholar). Inhibitor-1 is widely expressed in mammalian tissue with highest levels occurring in the brain, skeletal muscle, adipose, and kidney tissues (18Hemmings Jr., H.C. Girault J.-A. Nairn A.C. Bertuzzi G. Greengard P. J. Neurochem. 1992; 59: 1053-1061Crossref PubMed Scopus (55) Google Scholar, 19MacDougall L.K. Cambell D.G. Hubbard M.J. Cohen P. Biochim. Biophys. Acta. 1989; 1010: 218-226Crossref PubMed Scopus (44) Google Scholar, 20Lowenstein P.R. Shering A.F. MacDougall L.K. Cohen P. Brain Res. 1995; 575: 80-92Crossref Scopus (11) Google Scholar, 21Gustafson E.L. Girault J.-A. Hemmings Jr., H.C. Nairn A.C. Greengard P. J. Comp. Neurol. 1991; 310: 170-188Crossref PubMed Scopus (43) Google Scholar, 22Barbas H. Gustafson E.L. Greengard P. J. Comp. Neurol. 1993; 334: 1-18Crossref PubMed Scopus (27) Google Scholar, 23Alder R. Barbas H. Neuroreports. 1995; 6: 2368-2372Crossref PubMed Scopus (10) Google Scholar, 24Sakagami H. Ebina K. Kondo H. Mol. Brain Res. 1994; 25: 7-18Crossref PubMed Scopus (35) Google Scholar, 25Allen P. Hvalby Ø. Jensen V. Ramsay M. Chaudhry F.A. Storm- Mathisen J. Morris R. Andersen P. Greengard P. J. Neurosci. 2000; 20: 3537-3543Crossref PubMed Google Scholar, 26Mikkelsen J.D. Gustafson E.L. Brain Res. 1993; 623: 147-154Crossref PubMed Scopus (4) Google Scholar). Within the brain, the highest levels of inhibitor-1 immunoreactivity are associated with the dentate gyrus of the hippocampus and the neostriatum and substantia nigra of the basal ganglia (21Gustafson E.L. Girault J.-A. Hemmings Jr., H.C. Nairn A.C. Greengard P. J. Comp. Neurol. 1991; 310: 170-188Crossref PubMed Scopus (43) Google Scholar). 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