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Phosphorylation of Protein Phosphatase Inhibitor-1 by Cdk5*

  • James A. Bibb
    Correspondence
    To whom correspondence should be addressed.
    Affiliations
    ‡Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, New York 10021-6399, the
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  • Akinori Nishi
    Affiliations
    ¶Department of Physiology, Kurume University School of Medicine, Kurume, Fukuoka, Japan 830-0011, the

    From the Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, New York 10021-6399, the
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  • James P. O'Callaghan
    Affiliations
    Centers for Disease Control and Prevention, National Institute for Occupational Safety and Health, Morgantown, West Virginia 26505, and the
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  • Jernej Ule
    Affiliations
    ‡Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, New York 10021-6399, the
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  • Martin Lan
    Affiliations
    ‡Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, New York 10021-6399, the
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  • Gretchen L. Snyder
    Affiliations
    ‡Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, New York 10021-6399, the
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  • Atsuko Horiuchi
    Affiliations
    ‡Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, New York 10021-6399, the
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  • Taro Saito
    Affiliations
    **Department of Biological Sciences, Graduate School of Science, Tokyo Metropolitan University, Tokyo, Japan 192-0397
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  • Shin-ichi Hisanaga
    Affiliations
    **Department of Biological Sciences, Graduate School of Science, Tokyo Metropolitan University, Tokyo, Japan 192-0397
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  • Andrew J. Czernik
    Affiliations
    ‡Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, New York 10021-6399, the
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  • Angus C. Nairn
    Affiliations
    ‡Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, New York 10021-6399, the
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  • Paul Greengard
    Affiliations
    ‡Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, New York 10021-6399, the
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  • Author Footnotes
    * This work was supported by a National Research Service award (to J. B.) and by the National Institute of Mental Health and the National Institute of Drug Abuse (to G. L. S., A. C. N., and P. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.This paper is dedicated to the memory of George Kuzmycs (1916–1996) whose contribution to the generation of polyclonal antibodies used in our laboratory has been and will continue to be an invaluable resource.
Open AccessPublished:April 27, 2001DOI:https://doi.org/10.1074/jbc.M007197200
      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 (
      • Huang F.L.
      • Glinsmann W.H.
      ). 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 (
      • Elbrecht A.
      • DiRenzo J.
      • Smith R.G.
      • Shenolikar S.
      ,
      • Nimmo G.A.
      • Cohen P.
      ). 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 (
      • Foulkes J.G.
      • Strada S.J.
      • Henderson P.J.
      • Cohen P.
      ,
      • Endo S.
      • Zhou X.
      • Connor J.
      • Wang B.
      • Shenolikar S.
      ,
      • Aitken A.
      • Cohen P.
      ,
      • Hemmings Jr., H.C.
      • Greengard P.
      • Tung H.Y.L.
      • Cohen P.
      ). 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+(
      • Cohen P.
      ,
      • Shenolikar S.
      • Nairn A.C.
      ,
      • Shenolikar S.
      ,
      • Bollen M.
      • Stalmans W.
      ). 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 (
      • Oliver C.J.
      • Shenolikar S.
      ,
      • Connor J.H.
      • Quan H.
      • Oliver C.
      • Shenolikar S.
      ,
      • Connor J.H.
      • Quan H.N.
      • Ramaswamy N.T.
      • Zhang L.
      • Barik S.
      • Zheng J.
      • Cannon J.F.
      • Lee E.Y.C.
      • Shenolikar S.
      ,
      • Mulkey R.M.
      • Endo S.
      • Shenolikar S.
      • Malenka R.C.
      ,
      • Hagiwara M.
      • Alberts A.
      • Brindle P.
      • Meinkoth J.
      • Feramisco J.
      • Deng T.
      • Karin M.
      • Shenolikar S.
      • Montminy M.
      ,
      • Alberts A.S.
      • Montminy M.
      • Shenolikar S.
      • Feramisco J.R.
      ).
      Inhibitor-1 is widely expressed in mammalian tissue with highest levels occurring in the brain, skeletal muscle, adipose, and kidney tissues (
      • Hemmings Jr., H.C.
      • Girault J.-A.
      • Nairn A.C.
      • Bertuzzi G.
      • Greengard P.
      ,
      • MacDougall L.K.
      • Cambell D.G.
      • Hubbard M.J.
      • Cohen P.
      ,
      • Lowenstein P.R.
      • Shering A.F.
      • MacDougall L.K.
      • Cohen P.
      ,
      • Gustafson E.L.
      • Girault J.-A.
      • Hemmings Jr., H.C.
      • Nairn A.C.
      • Greengard P.
      ,
      • Barbas H.
      • Gustafson E.L.
      • Greengard P.
      ,
      • Alder R.
      • Barbas H.
      ,
      • Sakagami H.
      • Ebina K.
      • Kondo H.
      ,
      • Allen P.
      • Hvalby Ø.
      • Jensen V.
      • Ramsay M.
      • Chaudhry F.A.
      • Storm- Mathisen J.
      • Morris R.
      • Andersen P.
      • Greengard P.
      ,
      • Mikkelsen J.D.
      • Gustafson E.L.
      ). 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 (
      • Gustafson E.L.
      • Girault J.-A.
      • Hemmings Jr., H.C.
      • Nairn A.C.
      • Greengard P.
      ). 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 (
      • Mulkey R.M.
      • Endo S.
      • Shenolikar S.
      • Malenka R.C.
      ,
      • Blitzer R.D.
      • Connor J.H.
      • Brown G.P.
      • Wong T.
      • Shenolikar S.
      • Iyengar R.
      • Landau E.M.
      ). Mice lacking inhibitor-1 display deficits in long term potentiation induction (
      • Allen P.
      • Hvalby Ø.
      • Jensen V.
      • Ramsay M.
      • Chaudhry F.A.
      • Storm- Mathisen J.
      • Morris R.
      • Andersen P.
      • Greengard P.
      ).
      In the dopaminoceptive medium spiny neurons of the striatum, inhibitor-1 is co-expressed with a homologous PP-1 inhibitor, DARPP-32 (
      • Nairn A.C.
      • Hemmings Jr., H.C.
      • Walaas S.I.
      • Greengard P.
      ). Inhibitor-1 is ∼10 times less abundant than DARPP-32 in the striatum (
      • Gustafson E.L.
      • Girault J.-A.
      • Hemmings Jr., H.C.
      • Nairn A.C.
      • Greengard P.
      ,
      • Hemmings Jr., H.C.
      • Nairn A.C.
      • Aswad D.W.
      • Greengard P.
      ,
      • Ouimet C.C.
      • Langley-Gullion K.C.
      • Greengard P.
      ), 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 (
      • Williams K.R.
      • Hemmings Jr., H.C.
      • LoPresti M.B.
      • Konigsberg W.H.
      • Greengard P.
      ) 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 (
      • Hemmings Jr., H.C.
      • Greengard P.
      • Tung H.Y.L.
      • Cohen P.
      ). 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 (
      • Greengard P.
      • Allen P.B.
      • Nairn A.C.
      ,
      • Desdouits F.
      • Sciliano J.C.
      • Greengard P.
      • Girault J.-A.
      ,
      • Girault J.-A.
      • Hemmings Jr., H.C.
      • Williams K.R.
      • Nairn A.C.
      • Greengard P.
      ). 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 (
      • Bibb J.A.
      • Snyder G.L.
      • Nishi A.
      • Yan Z.
      • Meijer L.
      • Fienberg A.A.
      • Tsai L.-H.
      • Kwon Y.T.
      • Girault J.-A.
      • Czernik A.J.
      • Huganir R.L.
      • Hemmings Jr., H.C.
      • Nairn A.C.
      • Greengard P.
      ). 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.

      DISCUSSION

      We 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 (
      • Hemmings Jr., H.C.
      • Nairn A.C.
      • Aswad D.W.
      • Greengard P.
      ,
      • Ouimet C.C.
      • Langley-Gullion K.C.
      • Greengard P.
      ,
      • Foulkes J.G.
      • Cohen P.
      ,
      • Nimmo G.A.
      • Cohen P.
      ). 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 (
      • Nikolic M.
      • Dudek H.
      • Kwon Y.T.
      • Ramos Y.F.
      • Tsai L.H.
      ), development of the nervous system (
      • Ohshima T.
      • Ward J.M.
      • Huh C.-G.
      • Longenecker G.
      • Veeranna Pant H.C.
      • Brady R.O.
      • Martin L.J.
      • Kulkarni A.B.
      ), and regulation of dopamine signaling through phosphorylation of DARPP-32 in the striatum (
      • Bibb J.A.
      • Snyder G.L.
      • Nishi A.
      • Yan Z.
      • Meijer L.
      • Fienberg A.A.
      • Tsai L.-H.
      • Kwon Y.T.
      • Girault J.-A.
      • Czernik A.J.
      • Huganir R.L.
      • Hemmings Jr., H.C.
      • Nairn A.C.
      • Greengard P.
      ). Cdk5 may also play a role in muscle development (
      • Lazaro J.B.
      • Kitzmann M.
      • Poul M.A.
      • Vandromme M.
      • Lamb N.J.
      • Fernandez A.
      ). 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.
      J. 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 (
      • King M.M.
      • Huang C.Y.
      • Chock P.B.
      • Nairn A.C.
      • Hemmings Jr., H.C.
      • Chan K.-F.J.
      • Greengard P.
      ), and PP-2B is highly concentrated in striatal neurons (
      • Goto S.
      • Matsukado Y.
      • Mihara Y.
      • Inoue N.
      • Miyamoto E.
      ). 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 (
      • Zhang 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.
      ). 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 (
      • Huang F.L.
      • Glinsmann W.H.
      ,
      • Aitken A.
      • Bilham T.
      • Cohen P.
      ,
      • Nimmo G.A.
      • Cohen P.
      ,
      • Cohen P.
      • Rylatt D.B.
      • Nimmo G.A.
      ). 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 (
      • Aitken A.
      • Bilham T.
      • Cohen P.
      ). 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 (
      • Endo S.
      • Zhou X.
      • Connor J.
      • Wang B.
      • Shenolikar S.
      ,
      • Aitken A.
      • Cohen P.
      ). All these findings are in contradiction to a recent report by Huang and Paudel (
      • Huang K.-X.
      • Paudel H.K.
      ) 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 (
      • Endo S.
      • Zhou X.
      • Connor J.
      • Wang B.
      • Shenolikar S.
      ,
      • Huang H.B.
      • Horiuchi A.
      • Watanabe T.
      • Shih S.-R.
      • Tsay H.-J.
      • Li H.-C.
      • Greengard P.
      • Nairn A.C.
      ,
      • Goldberg J.
      • Huang H.B.
      • Kwon Y.G.
      • Greengard P.
      • Nairn A.C.
      • Kuriyan J.
      ). 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 (
      • Endo S.
      • Zhou X.
      • Connor J.
      • Wang B.
      • Shenolikar S.
      ,
      • Huang H.-B.
      • Horiuchi A.
      • Goldberg J.
      • Greengard P.
      • Nairn A.C.
      ,
      • Huang H.B.
      • Horiuchi A.
      • Watanabe T.
      • Shih S.-R.
      • Tsay H.-J.
      • Li H.-C.
      • Greengard P.
      • Nairn A.C.
      ,
      • Goldberg J.
      • Huang H.B.
      • Kwon Y.G.
      • Greengard P.
      • Nairn A.C.
      • Kuriyan J.
      ). Phosphorylation of DARPP-32 at other sites does not directly affect PP-1 inhibitory activity (
      • Desdouits F.
      • Sciliano J.C.
      • Greengard P.
      • Girault J.-A.
      ,
      • Girault J.-A.
      • Hemmings Jr., H.C.
      • Williams K.R.
      • Nairn A.C.
      • Greengard P.
      ).
      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 (
      • Eto M.
      • Karginov A.
      • Brautigan D.L.
      ,
      • Zhang J.
      • Zhang L.
      • Zhao S.
      • Lee E.Y.
      ,
      • Allen P.B.
      • Ouimet C.C.
      • Greengard P.
      ,
      • Jagiello I.
      • Beullens M.
      • Stalmans W.
      • Bollen M.
      ). 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.

      Acknowledgments

      We thank Shirish Shenolikar, Duke University Medical Center, for the pUC120 inhibitor-1 subclone; Alan R. Saltiel and David T. Dudely, Parke-Davis Pharmaceutical Research Division, for the GST-ERK1 and GST-MEK bacterial expression vectors and PD 98059; J. G. Seidman, Harvard Medical School for the PP-2Bα/− mice; Laurent Meijer, Center National de la Recherché Scientifique for roscovitine and sea star Cdk1; and Steven L. Pelech, University of British Columbia for sea star Cdk1 and activated MAPK. We acknowledge the technical assistance of Jean Whitesell at Cocalico Biologicals, Inc., and Joseph Fernandez of the Rockefeller University Protein/DNA Technology Center.

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