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Amphiphysin 1 Binds the Cyclin-dependent Kinase (cdk) 5 Regulatory Subunit p35 and Is Phosphorylated by cdk5 and cdc2*

  • Scott R. Floyd
    Affiliations
    Howard Hughes Medical Institute and Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510
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  • Elena B. Porro
    Affiliations
    Howard Hughes Medical Institute and Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115
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  • Vladimir I. Slepnev
    Affiliations
    Howard Hughes Medical Institute and Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510
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  • Gian-Carlo Ochoa
    Affiliations
    Howard Hughes Medical Institute and Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510
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  • Li-Huei Tsai
    Affiliations
    Howard Hughes Medical Institute and Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115
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  • Pietro De Camilli
    Correspondence
    To whom correspondence should be addressed:
    Affiliations
    Howard Hughes Medical Institute and Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510
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  • Author Footnotes
    * This work was supported in part by National Institutes of Health Grants NS36251 and CA46128 and United States Army Medical Research and Development Command Grant DAMD17-97-7068 (to P. D. C.) and National Institutes of Health Grants NS37007 (to L.-H. T.).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.
Open AccessPublished:March 01, 2001DOI:https://doi.org/10.1074/jbc.M008932200
      Amphiphysin 1 is a phosphoprotein expressed at high levels in neurons, where it participates in synaptic vesicle endocytosis and neurite outgrowth. It is a substrate for cyclin-dependent kinase (cdk) 5, a member of the cyclin-dependent protein kinase family, which has been functionally linked to neuronal migration and neurite outgrowth via its action on the actin cytoskeleton. The yeast homologue of amphiphysin, Rvs167, functions in endocytosis and actin dynamics, is phosphorylated by the cdk5 homologue Pho85, and binds the Pho85 regulatory subunit Pcl2. We show here that amphiphysin 1 interacts with the cdk5-activating subunit p35 and that this interaction is mediated by the conserved NH2-terminal region of amphiphysin. Amphiphysin 1 colocalizes with p35 in the growth cones of neurons and at actin-rich peripheral lamellipodia in transfected fibroblasts. Amphiphysin is phosphorylated by cdk5 in a region including serines 272, 276, and 285. Amphiphysin 1 is also phosphorylated by the cdc2/cyclin B kinase complex in the same region and undergoes mitotic phosphorylation in dividing cells. These data indicate that phosphorylation by members of the cyclin-dependent kinase family is a conserved property of amphiphysin and suggest that this phosphorylation may play an important physiological role both in mitosis and in differentiated cells.
      cdk
      cyclin-dependent kinase
      GST
      glutathioneS-transferase
      PAGE
      polyacrylamide gel electrophoresis
      HPLC
      high pressure liquid chromatography
      CHO
      Chinese hamster ovary
      Amphiphysin 1 and 2 are SH3 domain-containing proteins concentrated in nerve terminals of mature neurons (
      • Lichte B.
      • Veh R.W.
      • Meyer H.E.
      • Kilimann M.W.
      ,
      • De Camilli P.
      • Thomas A.
      • Cofiell R.
      • Folli F.
      • Lichte B.
      • Piccolo G.
      • Meinck H.M.
      • Austoni M.
      • Fassetta G.
      • Bottazzo G.
      • Bates D.
      • Cartlidge N.
      • Solimena M.
      • Kilimann M.W.
      ). They belong to a protein family conserved from yeast to humans, whose members play pleiotropic roles in endocytosis, actin function, and regulation of growth control. Studies of the yeast amphiphysin homologue Rvs167 have shown that this protein is phosphorylated by the Pho85 kinase and binds Pcl2, an activator of this kinase (
      • Lee J.
      • Colwill K.
      • Aneliunas V.
      • Tennyson C.
      • Moore L.
      • Ho Y.
      • Andrews B.
      ). The Pho85 kinase is a member of the cyclin-dependent kinase family and functions in cellular growth control as well as endocytosis and actin function. The homologue of the Pho85 kinase in mammalian cells is cdk51 (
      • Huang D.
      • Patrick G.
      • Moffat J.
      • Tsai L.H.
      • Andrews B.
      ,
      • Nishizawa M.
      • Kanaya Y.
      • Toh E.A.
      ). Its two activators, p35 and p39 (
      • Tsai L.H.
      • Delalle I.
      • Caviness Jr., V.S.
      • Chae T.
      • Harlow E.
      ,
      • Lew J.
      • Huang Q.Q.
      • Qi Z.
      • Winkfein R.J.
      • Aebersold R.
      • Hunt T.
      • Wang J.H.
      ,
      • Tang D.
      • Yeung J.
      • Lee K.Y.
      • Matsushita M.
      • Matsui H.
      • Tomizawa K.
      • Hatase O.
      • Wang J.H.
      ), which represent functional homologues of Pcl2, are expressed only in neurons and developing muscle cells (
      • Tsai L.H.
      • Delalle I.
      • Caviness Jr., V.S.
      • Chae T.
      • Harlow E.
      ,
      • Lew J.
      • Huang Q.Q.
      • Qi Z.
      • Winkfein R.J.
      • Aebersold R.
      • Hunt T.
      • Wang J.H.
      ,
      • Philpott A.
      • Porro E.B.
      • Kirschner M.W.
      • Tsai L.H.
      ). Consistent with these homologies, amphiphysin 1 is a substrate for cdk5 and is a component of a high molecular weight complex in brain that also contains cdk5/p35 (
      • Rosales J.L.
      • Nodwell M.J.
      • Johnston R.N.
      • Lee K.Y.
      ). In addition to its role in neurons, amphiphysin is likely to function outside the nervous system because it is expressed in other cell types (
      • Floyd S.R.
      • Butler M.H.
      • Cremona O.
      • David C.
      • Freyberg Z.
      • Zhang X.
      • Solimena M.
      • Tokunaga A.
      • Ishizu H.
      • Tsutsui K.
      • De Camilli P.
      ,
      • Wigge P.
      • Kohler K.
      • Vallis Y.
      • Doyle C.A.
      • Owen D.
      • Hunt S.P.
      • McMahon H.T.
      ). In this study we have further characterized the relationship between amphiphysin 1 and the cdk5/p35 kinase complex. We also show that amphiphysin 1 is phosphorylated by cdc2, another cyclin-dependent kinase family member, and undergoes mitotic phosphorylation.

      DISCUSSION

      In this study we report evidence for a functional link between amphiphysin 1 and the cdk5 kinase complex. We have demonstrated thein vivo occurrence of an interaction between amphiphysin and the cdk5 regulatory subunit p35 by immunoprecipitation and immunofluorescence. This interaction is analogous to the Pcl2-Rvs167 interaction in yeast. We have also shown that p35 binds to the evolutionary conserved NH2-terminal region of amphiphysin 1, in agreement with the reported binding of Pcl2 to the NH2-terminal moiety of Rvs167 (
      • Lee J.
      • Colwill K.
      • Aneliunas V.
      • Tennyson C.
      • Moore L.
      • Ho Y.
      • Andrews B.
      ). Thus, our findings point to a further similarity between the mammalian p35/cdk5/amphiphysin 1 and the yeast Pcl2/Pho85/Rvs167 protein networks. We also mapped the main cdk5 phosphorylation site to a small fragment containing three serine residues in the central region of amphiphysin 1, just upstream of the binding sites for clathrin and the clathrin adaptor AP-2, although phosphorylation of this site did not appear to affect AP-2 or clathrin binding.
      S. R. Floyd, E. B. Porro, V. I. Slepnev, G.-C. Ochoa, L.-H. Tsai, and P. De Camilli, unpublished observations.
      Because Rvs167, like several other members of the amphiphysin family, does not contain recognizable clathrin or AP-2 binding sites (
      • Bauer F.
      • Urdaci M.
      • Aigle M.
      • Crouzet M.
      ,
      • Sakamuro D.
      • Elliott K.J.
      • Wechsler-Reya R.
      • Prendergast G.C.
      ,
      • Butler M.H.
      • David C.
      • Ochoa G.-C.
      • Freyberg Z.
      • Daniell L.
      • Grabs D.
      • Cremona O.
      • De Camilli P.
      ), it is not unexpected that cdk5 phosphorylation may not represent a conserved mechanism to regulate these interactions.
      Studies of p35/cdk5 have revealed a key role of this complex in the regulation of actin function at the leading edge of neuronal processes with important functional implications for neuronal migration and growth cone navigation (
      • Nikolic M.
      • Dudek H.
      • Kwon Y.T.
      • Ramos Y.F.
      • Tsai L.H.
      ,
      • Chae T.
      • Kwon Y.T.
      • Bronson R.
      • Dikkes P.
      • Li E.
      • Tsai L.H.
      ,
      • Nikolic M.
      • Chou M.M.
      • Lu W.
      • Mayer B.J.
      • Tsai L.H.
      ). Likewise, independent studies have suggested a role of amphi- physin and its binding partners in growth cone dynamics (
      • Mundigl O.
      • Ochoa G.C.
      • David C.
      • Slepnev V.I.
      • Kabanov A.
      • De Camilli P.
      ). Amphiphysin has also been implicated in endocytosis, a process for which a key role of actin is emerging (
      • Wendland B.
      • Emr S.D.
      • Riezman H.
      ,
      • Qualmann B.
      • Kessels M.M.
      • Kelly R.B.
      ). Although a function of cdk5 phosphorylation in endocytosis has not been reported, in yeast, Pho85, Plc2, and Rvs167 mutations have strikingly similar effects on both actin function and endocytosis (
      • Lee J.
      • Colwill K.
      • Aneliunas V.
      • Tennyson C.
      • Moore L.
      • Ho Y.
      • Andrews B.
      ). We suggest therefore that amphiphysin, p35, and cdk5 may be interrelated in their physiological functions in vivo. Our demonstration that amphiphysin 1 and p35 colocalize with each other and with actin in lamellipodia of transfected cells supports this possibility. We note that an interaction between amphiphysin 2 and the cAbl kinase has been reported (
      • Kadlec L.
      • Pendergast A.M.
      ). cAbl has recently been shown to phosphorylate and activate cdk5 and to bind cdk5 through the bridging protein cables (
      • Zukerberg L.R.
      • Patrick G.N.
      • Nikolic M.
      • Humbert S.
      • Wu C.L.
      • Lanier L.M.
      • Gertler F.B.
      • Vidal M.
      • Van Etten R.A.
      • Tsai L.H.
      ). In addition, cAbl is known to have regulatory actions on the actin cytoskeleton and participate in neuronal development (
      • Lanier L.M.
      • Gertler F.B.
      ,
      • Koleske A.J.
      • Gifford A.M.
      • Scott M.L.
      • Nee M.
      • Bronson R.T.
      • Miczek K.A.
      • Baltimore D.
      ).
      Finally, we have shown that amphiphysin 1 undergoes mitotic phosphorylation. The same region that is a target for cdk5 phosphorylation can also be phosphorylated by the mitotic cdc2/cyclin B1 complex. Thus, phosphorylation of amphiphysin 1 in this region may have a similar conserved function in both neurons and dividing cells. One of the critical events that correlates with mitosis is the dramatic rearrangement of the peripheral cytoplasm that results in the partial dissociation of cells from the substratum. Cyclin-dependent kinase phosphorylation of amphiphysin may help to produce local changes in the actin cytoskeleton that are crucial for the dynamic properties of dividing cells, neuronal growth cones, the leading edge of migrating cells, and the function of the mature synapse.

      Acknowledgments

      We thank Drs. Graham Warren and Yanzhuang Wang for the generous provision of reagents and advice on cdc2 phosphorylation. We thank Warren T. Kim for aid with neuronal cultures and immunofluorescence.

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