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

  • Alka Agarwal-Mawal
    Affiliations
    From the Bloomfield Center for Research in Aging, Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, and Department of Neurology and Neurosurgery, McGill University, 3755 Cote Ste-Catherine Road, Montreal, Quebec, H3T 1E2, Canada
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  • Hemant K. Paudel
    Correspondence
    To whom correspondence should be addressed. Tel.: 514-340-8222 (ext 4866); Fax: 514-340-8295; E-mail: [email protected]
    Affiliations
    From the Bloomfield Center for Research in Aging, Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, and Department of Neurology and Neurosurgery, McGill University, 3755 Cote Ste-Catherine Road, Montreal, Quebec, H3T 1E2, Canada
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  • Author Footnotes
    * This work was supported by a grant from Canadian Institute for Health Research.
Open AccessPublished:June 29, 2001DOI:https://doi.org/10.1074/jbc.M010002200
      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.
      PP1
      protein phosphatase 1
      CK
      casein kinase
      FPLC
      fast protein liquid chromatography
      HPLC
      high pressure liquid chromatography
      GSK3
      glycogen synthase kinase 3
      GST
      glutathione S-transferase
      I-1
      inhibitor 1
      I-2
      inhibitor 2
      MOPS
      4-morpholinepropanesulfonic acid
      NCLK
      neuronal cdc2-like protein kinase
      PKA
      cAMP-dependent protein kinase
      PCR
      polymerase chain reaction
      DTT
      dithiothreitol
      MAPK
      mitogen-activated protein kinase
      PAGE
      polyacrylamide gel electrophoresis
      PKI
      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.
      • Cohen P.
      ,
      • Lee E.Y.C.
      • Zhang L.
      • Zhao S.
      • Wei Q.
      • Zhang J.
      • Qi Z.Q.
      • Belmonte E.R.
      ). 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) (
      • Cohen P.
      ,
      • Lee E.Y.C.
      • Zhang L.
      • Zhao S.
      • Wei Q.
      • Zhang J.
      • Qi Z.Q.
      • Belmonte E.R.
      ,
      • Oliver C.J.
      • Shenolikar S.
      ). 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 (
      • Oliver C.J.
      • Shenolikar S.
      ).
      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 (
      • Cohen P.
      ,
      • Lee E.Y.C.
      • Zhang L.
      • Zhao S.
      • Wei Q.
      • Zhang J.
      • Qi Z.Q.
      • Belmonte E.R.
      ). The inactive cytosolic enzyme, a PP1·I-2 complex, is activated upon incubation with ATP/Mg and is hence called ATP/Mg-dependent PP1 (
      • Cohen P.
      ). 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) (
      • Woodgett J.R.
      • Cohen P.
      ,
      • Yang S.D.
      • Vandenheede J.R.
      • Goris J.
      • Merlevede W.
      ,
      • Hemmings B.A.
      • Yellowless D.
      • Kernohan J.C.
      • Cohen P.
      ). 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 (
      • Woodgett J.R.
      • Cohen P.
      ,
      • Yang S.D.
      • Vandenheede J.R.
      • Goris J.
      • Merlevede W.
      ,
      • Hemmings B.A.
      • Yellowless D.
      • Kernohan J.C.
      • Cohen P.
      ,
      • Yang S.D.
      • Vandenheede J.R.
      • Merlevede W.
      ,
      • DePaoli-Roach A.A.
      ,
      • Park I.K.
      • Roach P.
      • Bondor J.
      • Fox S.P.
      • DePaoli-Roach A.A.
      ). 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 (
      • Chan C.P.
      • McNally S.J.
      • Krebs E.G.
      • Fischer E.H.
      ,
      • Wang Q.M.
      • Guan K.L.
      • Roach P.J.
      • DePaoli-Roach A.A.
      ,
      • Puntoni F.
      • Villa-Moruzzi E.
      ).
      PP1 is highly expressed in brain (
      • Cohen P.
      ). An earlier study found that most of the PP1 in brain extract is inactive and requires incubation with ATP/Mg to become active (
      • Yang S.D.
      • Fong Y.-L.
      ). The purified enzyme is a PP1·I-2 complex, which is activated upon incubation with ATP/Mg in the presence of muscle GSK3. It was suggested that brain ATP/Mg-dependent PP1 is regulated in a manner similar to its muscle counterpart, via phosphorylation of I-2 (
      • Yang S.D.
      • Fong Y.-L.
      ). A type of Fa activity was partially purified from porcine brain extract (
      • Yang S.D.
      • Fong Y.-L.
      ), but the identity of this activity has remained unknown. Thus, until now it has not been clear as to which kinase activates PP1·I-2 in the brain.
      Neuronal Cdc2-like protein kinase (NCLK) is a heterodimer of cyclin-dependent protein kinase 5 (Cdk5) and a neuronal-specific p25 regulatory subunit (reviewed in Ref.
      • Lew J.
      • Qi Z.
      • Huang Q.-Q.
      • Paudel H.
      • Matsuura I.
      • Matsushita M.
      • Zhu X.
      • Wang J.H.
      ). Cdk5, a member of the cyclin-dependent protein kinase family, is widely expressed in various tissues and cell lines (
      • Meyerson M.
      • Enders G.H.
      • Wu C.L.
      • Su L.K.
      • Gorka C.
      • Nelson C.
      • Harlow E.
      • Tsai L.-H.
      ). However, its kinase activity is detected only in terminally differentiated neurons where it is associated with a p25 subunit (
      • Lew J.
      • Beaudette K.
      • Litwin C.M.E.
      • Wang J.H.
      ). p25 is a proteolytic fragment of a 35-kDa protein and is expressed only in neurons (
      • Tsai L.-H.
      • Delalle I.
      • Caviness Jr., V.S.
      • Chae T.
      • Harlow E.
      ). NCLK is involved in brain development, neurite outgrowth, cell migration, cell signaling, microtubule dynamics regulation, and Alzheimer's disease pathogenesis (
      • Ohshima T.
      • Ward J.M.
      • Huh C.-G.
      • Longenecker G.
      • Veeranna
      • Pant H.C.
      • Brady R.O.
      • Martin L.J.
      • Kulkarni A.B.
      ,
      • Nlkolic M.
      • Dudek H.
      • Kwon Y.T.
      • Ramos Y.F.M.
      • Tsai L.-H.
      ,
      • Chae T.
      • Kwon Y.T.
      • Bronson R.
      • Dikkes P.
      • Li E.
      • Tsai L.-H.
      ,
      • Sobue K.
      • Agarwal-Mawal A.
      • Li W.
      • Sun W.
      • Miura Y
      • Paudel H.K.
      ,
      • Paudel H.K.
      • Lew J.
      • Ali Z.
      • Wang J.H.
      ,
      • Patrick G.N.
      • Zukerberg L.
      • Nikolic M.
      • de la Monte S.
      • Dikkes P
      • Tsai L.-H.
      ,
      • Paudel H.K.
      ). Herein we show that NCLK is complexed with PP1·I-2 in brain extract, phosphorylates I-2 on Thr72, and activates PP1·I-2. Our data suggest that NCLK is one of the kinases that activate PP1·I-2 in the central nervous system.

      DISCUSSION

      PP1 activity is controlled by three PP1 inhibitory subunits: I-1, DARPP-32, and I-2 (
      • Cohen P.
      ,
      • Lee E.Y.C.
      • Zhang L.
      • Zhao S.
      • Wei Q.
      • Zhang J.
      • Qi Z.Q.
      • Belmonte E.R.
      ,
      • Oliver C.J.
      • Shenolikar S.
      ). I-1 is widely expressed in various tissues, whereas DARPP-32 is found in basal ganglion neurons where it is regulated by dopamine (
      • Cohen P.
      ). PKA phosphorylates I-1 and DARPP-32, and both proteins inhibit PP1 upon PKA phosphorylation (
      • Cohen P.
      ,
      • Lee E.Y.C.
      • Zhang L.
      • Zhao S.
      • Wei Q.
      • Zhang J.
      • Qi Z.Q.
      • Belmonte E.R.
      ,
      • Oliver C.J.
      • Shenolikar S.
      ). Recently, I-1 and DARPP-32 have also been shown to be phosphorylated by NCLK (
      • Huang K.
      • Paudel H.K.
      ,
      • Bibb J.
      • 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.
      ). NCLK phosphorylated I-1 inhibits PP1 in a manner similar to PKA phosphorylated I-1 (
      • Huang K.
      • Paudel H.K.
      ). NCLK phosphorylated DARPP-32, on the other hand, does not inhibit PP1 but is a PKA inhibitor (
      • Bibb J.
      • 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.
      ).
      In a previous study almost all PP1 in brain extract was found to exist as PP1·I-2 and required preincubation with ATP/Mg to become active (
      • Yang S.D.
      • Fong Y.-L.
      ). In this study we partially purified brain PP1·I-2 and found that GSK3 was present in our preparation. Importantly, a complete inhibition of GSK3 activity by LiCl suppressed only ∼29% of ATP/Mg-dependent PP1·I-2 activation in our preparation (Fig. 1 B). These data indicate that GSK3 is not the sole PP1·I-2 activator in the brain.
      We found that the ATP/Mg-dependent activation of our partially purified PP1·I-2 preparation was sensitive to the NCLK inhibitor olomoucine (Fig. 1 B). NCLK was pulled down from our preparation with microcystin-Sepharose and GST-I-2-coated glutathione-agarose beads (Fig. 2, A and C). Similarly, PP1 in our preparation bound to glutathione-agarose beads coated with GST-Cdk5 (Fig. 2 E). In vitro, NCLK phosphorylated I-2 on Thr72 and activated PP1·I-2 in an ATP/Mg-dependent manner (Fig. 7). Taken together, these data strongly argue that NCLK is one of the PP1·I-2-activating kinases in the brain. PP1 displays a broad substrate specificity and dephosphorylates targets of many different protein kinases (
      • Cohen P.
      ,
      • Lee E.Y.C.
      • Zhang L.
      • Zhao S.
      • Wei Q.
      • Zhang J.
      • Qi Z.Q.
      • Belmonte E.R.
      ). As discussed above, NCLK phosphorylates I-1 and DARPP-32 (
      • Huang K.
      • Paudel H.K.
      ,
      • Bibb J.
      • 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.
      ). In this study we showed that NCLK also phosphorylates I-2. These observations suggest that NCLK plays a central role in neuronal signaling by phosphorylating PP1 inhibitory subunits I-1, DARPP-32, and I-2.
      It is established that PP1 binds to I-2 (
      • Cohen P.
      ,
      • Lee E.Y.C.
      • Zhang L.
      • Zhao S.
      • Wei Q.
      • Zhang J.
      • Qi Z.Q.
      • Belmonte E.R.
      ,
      • Oliver C.J.
      • Shenolikar S.
      ,
      • DePaoli-Roach A.A.
      ,
      • Park I.K.
      • Roach P.
      • Bondor J.
      • Fox S.P.
      • DePaoli-Roach A.A.
      ). We found that PP1 also binds to NCLK (Fig. 5). These observations suggest that PP1 is the central molecule that holds I-2 and NCLK together within a PP1·I-2·NCLK complex. Our GST pull-down assay demonstrated that, from the gel filtration column fractions containing PP1·I-2·NCLK complex, PP1 and NCLK are pulled down with GST-I-2 (Fig. 2,C and D) and PP1 is pulled down with GST-Cdk5 (Fig. 2 E). These observations raise a question as to how the PP1·I-2·NCLK complex could bind to an exogenous GST-I-2 or GST-Cdk5.
      It has been suggested that, in vivo, free PP1 and I-2 are in a dynamic equilibrium with PP1·I-2 and an excess of I-2 can replace PP1-bound I-2 (
      • Picking W.D.
      • Kudlicki W.
      • Kramer G.
      • Hardesty B.
      • Vandenheede J.R.
      • Merlevede W.
      • Park I.-K.
      • Depaoli-Roach A.
      ,
      • Resink T.J.
      • Hemmings B.A.
      • Tung H.Y.L.
      • Cohen P.
      ). As shown in Fig. 1 A, PP1 in brain extract elutes form a gel filtration column as a component of various species with sizes of ∼40 to ∼450 kDa. Some of these species may represent PP1, PP1·I-2, PP1·NCLK, or PP1·I-2·NCLK. Thus, it is possible that PP1, I-2, and NCLK may also be in a dynamic equilibrium with the PP1·I-2·NCLK complex in the brain. Because I-2 within PP1·I-2 may be displaced by exogenous I-2 (
      • Picking W.D.
      • Kudlicki W.
      • Kramer G.
      • Hardesty B.
      • Vandenheede J.R.
      • Merlevede W.
      • Park I.-K.
      • Depaoli-Roach A.
      ,
      • Resink T.J.
      • Hemmings B.A.
      • Tung H.Y.L.
      • Cohen P.
      ), GST-I-2 could similarly displace I-2 from PP1·I-2·NCLK and form a PP1·GST-I-2·NCLK species. Likewise, GST-Cdk5 may compete with Cdk5 within the PP1·I-2·NCLK complex and displace NCLK to result in the formation of a PP1·I-2·GST-Cdk5 complex.
      I-2 is bound to PP1 in tissue extracts and inhibits PP1 in its nonphosphorylated state. GSK3 phosphorylates I-2 on Thr72within the PP1·I-2 complex. This phosphorylation causes a conformational change in I-2 and also within the PP1·I-2 complex, leading to PP1 activation without complex dissociation (
      • Oliver C.J.
      • Shenolikar S.
      ,
      • DePaoli-Roach A.A.
      ,
      • Park I.K.
      • Roach P.
      • Bondor J.
      • Fox S.P.
      • DePaoli-Roach A.A.
      ,
      • Picking W.D.
      • Kudlicki W.
      • Kramer G.
      • Hardesty B.
      • Vandenheede J.R.
      • Merlevede W.
      • Park I.-K.
      • Depaoli-Roach A.
      ). Activated PP1 rapidly dephosphorylates I-2. This dephosphorylation, however, does not cause immediate loss of PP1 activity and only after some time the complex returns to its inactive conformation (
      • Oliver C.J.
      • Shenolikar S.
      ,
      • DePaoli-Roach A.A.
      ,
      • Park I.K.
      • Roach P.
      • Bondor J.
      • Fox S.P.
      • DePaoli-Roach A.A.
      ,
      • Picking W.D.
      • Kudlicki W.
      • Kramer G.
      • Hardesty B.
      • Vandenheede J.R.
      • Merlevede W.
      • Park I.-K.
      • Depaoli-Roach A.
      ). GSK3 phosphorylation of I-2 within PP1·I-2 has been suggested to be short-lived (
      • Oliver C.J.
      • Shenolikar S.
      ,
      • DePaoli-Roach A.A.
      ,
      • Park I.K.
      • Roach P.
      • Bondor J.
      • Fox S.P.
      • DePaoli-Roach A.A.
      ).
      To further investigate the phosphorylation of I-2 we incubated PP1·I-2 with NCLK in the presence of [γ-32P]ATP/Mg2+. We found that the incubation robustly activated PP1 within PP1·I-2. However, when we analyzed the product of the incubation by SDS-PAGE/autoradiography, we could not detect any significant I-2 phosphorylation (data not shown). These observations suggest that phosphorylation of I-2 by NCLK within the PP1·I-2 complex is also a transient event.
      I-2 is phosphorylated on Ser86, Ser120, and Ser121 in vivo (
      • Holmes C.F.B.
      • Tonks N.K.
      • Major H.
      • Cohen P.
      ), and these sites are phosphorylated by CK2 in vitro (
      • Cohen P.
      ,
      • Holmes C.F.B.
      • Kuret J.
      • Chisholm A.A.K.
      • Cohen P.
      ). CK2 phosphorylation does not activate PP1·I-2 but enhances Thr72phosphorylation by GSK3 (
      • DePaoli-Roach A.A.
      ,
      • Park I.K.
      • Roach P.
      • Bondor J.
      • Fox S.P.
      • DePaoli-Roach A.A.
      ). We found that NCLK also phosphorylates I-2 on Thr72. However, NCLK phosphorylation of I-2 is insensitive to a prior phosphorylation by CK2 (Fig. 4). These data indicate that NCLK and GSK3 display different substrate specificity for I-2 phosphorylation. In neurons CK2/GSK3 and NCLK may activate PP1·I-2 in response to different cellular stimuli. They may also function in different regions of the brain or in different subcellular compartments.
      In this study we showed that a major PP1-binding site is located within a 14-residue-long Cdk5 region (Fig. 6). Within this region there is a34RVRL37 sequence (Fig. 6 A) similar to an RVXF sequence motif found in many PP1-binding proteins (
      • Lee E.Y.C.
      • Zhang L.
      • Zhao S.
      • Wei Q.
      • Zhang J.
      • Qi Z.Q.
      • Belmonte E.R.
      ,
      • Egloff M.-P.
      • Johnson D.F.
      • Moorhead G.
      • Cohen P.T.W.
      • Cohen P.
      • Barford D.
      ,
      • Zhao S.
      • Lee E.Y.C.
      ). This putative PP1-binding site is located in between the ATP binding region and the PSSLAIR helix of Cdk5. This site is identical in Cdk5 from human, bovine, mouse, rat, andXenopus (
      • Meyerson M.
      • Enders G.H.
      • Wu C.L.
      • Su L.K.
      • Gorka C.
      • Nelson C.
      • Harlow E.
      • Tsai L.-H.
      ,
      • Lew J.
      • Winkfein R.J.
      • Paudel H.K.
      • Wang J.H.
      ,
      • Ino H.
      • Ishizuka T.
      • Chiba T.
      • Tatibana M.
      ,
      • Xiong Y.
      • Zhang H.
      • Beach D.H.
      ,
      • Gervasi C.
      • Szaro B.G.
      ) and has a conserved substitution (Asp40 → Glu) in Drosophila (
      • Hellmich M.R.
      • Kennison J.A.
      • Hampton L.L.
      • Battey J.F.
      ). In crystal structure, this sequence is an exposed loop available for interaction with other proteins (
      • De Bondt H.L.
      • Rosenblatt J.
      • Jancarik J.
      • Jones H.D.
      • Morgan D.O.
      • Kim S.-H.
      ,
      • Chou K.-C.
      • Watenpaugh K.D.
      • Heinrikson R.L.
      ). It should also be noted that deletion mutant GST-Cdk5-(42–292) also displays a weak PP1 binding (Fig.6 B), indicating that a low affinity PP1 binding region is located within Cdk5 residues 42–292. More studies will be required to identify this low affinity PP1 binding region.

      Acknowledgments

      We thank Dr. Ernest Y. C. Lee (New York Medical College) for providing purified PP1α.

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