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W2042306236 abstract "V-1 is a 12-kDa protein consisting of three consecutive ANK repeats, which are believed to serve as the surface for protein-protein interactions. It is thought to have a role in neural development for its temporal profile of expression during murine cerebellar development, but its precise role remains unknown. Here we applied the proteomic approach to search for protein targets that interact with V-1. The V-1 cDNA attached with a tandem affinity purification tag was expressed in the cultured 293T cells, and the protein complex formed within the cells were captured and characterized by mass spectrometry. We detected two polypeptides specifically associated with V-1, which were identified as the α and β subunits of the capping protein (CP, alternatively called CapZ or β-actinin). CP regulates actin polymerization by capping the barbed end of the actin filament. The V-1·CP complex was detected not only in cultured cells transfected with the V-1 cDNA but also endogenously in cells as well as in murine cerebellar extracts. An analysis of the V-1/CP interaction by surface plasmon resonance spectroscopy showed that V-1 formed a stable complex with the CP heterodimer with a dissociation constant of 1.2 × 10−7m and a molecular stoichiometry of ∼1:1. In addition, V-1 inhibited the CP-regulated actin polymerization in vitro in a dose-dependent manner. Thus, our results suggest that V-1 is a novel component that regulates the dynamics of actin polymerization by interacting with CP and thereby participates in a variety of cellular processes such as actin-driven cell movements and motility during neuronal development. V-1 is a 12-kDa protein consisting of three consecutive ANK repeats, which are believed to serve as the surface for protein-protein interactions. It is thought to have a role in neural development for its temporal profile of expression during murine cerebellar development, but its precise role remains unknown. Here we applied the proteomic approach to search for protein targets that interact with V-1. The V-1 cDNA attached with a tandem affinity purification tag was expressed in the cultured 293T cells, and the protein complex formed within the cells were captured and characterized by mass spectrometry. We detected two polypeptides specifically associated with V-1, which were identified as the α and β subunits of the capping protein (CP, alternatively called CapZ or β-actinin). CP regulates actin polymerization by capping the barbed end of the actin filament. The V-1·CP complex was detected not only in cultured cells transfected with the V-1 cDNA but also endogenously in cells as well as in murine cerebellar extracts. An analysis of the V-1/CP interaction by surface plasmon resonance spectroscopy showed that V-1 formed a stable complex with the CP heterodimer with a dissociation constant of 1.2 × 10−7m and a molecular stoichiometry of ∼1:1. In addition, V-1 inhibited the CP-regulated actin polymerization in vitro in a dose-dependent manner. Thus, our results suggest that V-1 is a novel component that regulates the dynamics of actin polymerization by interacting with CP and thereby participates in a variety of cellular processes such as actin-driven cell movements and motility during neuronal development. The V-1 protein was originally identified in the murine cerebellum as one of the proteins expressed significantly at the initial stage of postnatal development (1Taoka M. Yamakuni T. Song S.Y. Yamakawa Y. Seta K. Okuyama T. Isobe T. Eur. J. Biochem. 1992; 207: 615-620Google Scholar), particularly in the regions where synaptic formation and neuronal migration occur actively during neurogenesis (2Taoka M. Isobe T. Okuyama T. Watanabe M. Kondo H. Yamakawa Y. Ozawa F. Hishinuma F. Kubota M. Minegishi A. Song S.Y. Yamakuni T. J. Biol. Chem. 1994; 269: 9946-9951Google Scholar,3Fujigasaki H. Song S.Y. Kobayashi T. Yamakuni T. Mol. Brain Res. 1996; 40: 203-213Google Scholar). V-1 consists of 117 amino acids containing three contiguous repeats of the ANK motif, alternatively called the cdc10/SWI6 motif (1Taoka M. Yamakuni T. Song S.Y. Yamakawa Y. Seta K. Okuyama T. Isobe T. Eur. J. Biochem. 1992; 207: 615-620Google Scholar, 2Taoka M. Isobe T. Okuyama T. Watanabe M. Kondo H. Yamakawa Y. Ozawa F. Hishinuma F. Kubota M. Minegishi A. Song S.Y. Yamakuni T. J. Biol. Chem. 1994; 269: 9946-9951Google Scholar), which is crucial for a large number of protein-protein interactions (4Sedgwick S.G. Smerdon S.J. Trends Biochem. Sci. 1999; 24: 311-316Google Scholar). Previous studies suggested the potential role of V-1 in the signal transduction pathways leading to catecholamine synthesis or to cardiac hypertrophy. For example, Yamakuni et al. (5Yamakuni T. Yamamoto T. Hoshino M. Song S.Y. Yamamoto H. Kunikata-Sumitomo M. Minegishi A. Kubota M. Ito M. Konishi S. J. Biol. Chem. 1998; 273: 27051-27054Google Scholar, 6Suzuki T. Inagaki H. Yamakuni T. Nagatsu T. Ichinose H. Biochem. Biophys. Res. Commun. 2002; 293: 962-968Google Scholar, 7Suzuki T. Yamakuni T. Hagiwara M. Ichinose H. J. Biol. Chem. 2002; 277: 40768-40774Google Scholar) demonstrate that the overexpression of V-1 caused a significant increase in the catecholamine level in PC12 cells, presumably through the transcriptional activation of the genes for catecholamine synthesis. In other reports (8Gupta S. Sen S. Biochim. Biophys. Acta. 2002; 1589: 247-260Google Scholar, 9Knuefermann P. Chen P. Misra A. Shi S.P. Abdellatif M. Sivasubramanian N. J. Biol. Chem. 2002; 277: 23888-23897Google Scholar, 10Sivasubramanian N. Adhikary G. Sil P.C. Sen S. J. Biol. Chem. 1996; 271: 2812-2816Google Scholar, 11Sil P. Kandaswamy V. Sen S. Circ. Res. 1998; 82: 1173-1188Google Scholar), V-1 was designated as “myotrophin” and was shown to participate in the cell signaling pathways for the NFkB-mediated activation of protein synthesis in the myocytes. Thus, both of these studies (5Yamakuni T. Yamamoto T. Hoshino M. Song S.Y. Yamamoto H. Kunikata-Sumitomo M. Minegishi A. Kubota M. Ito M. Konishi S. J. Biol. Chem. 1998; 273: 27051-27054Google Scholar, 9Knuefermann P. Chen P. Misra A. Shi S.P. Abdellatif M. Sivasubramanian N. J. Biol. Chem. 2002; 277: 23888-23897Google Scholar) suggested the roles of V-1 in the biological events taking place in the nucleus. However, no biological function has been attributed to V-1 in the cytoplasm in which this molecule predominantly resides within the cells and tissues (5Yamakuni T. Yamamoto T. Hoshino M. Song S.Y. Yamamoto H. Kunikata-Sumitomo M. Minegishi A. Kubota M. Ito M. Konishi S. J. Biol. Chem. 1998; 273: 27051-27054Google Scholar, 9Knuefermann P. Chen P. Misra A. Shi S.P. Abdellatif M. Sivasubramanian N. J. Biol. Chem. 2002; 277: 23888-23897Google Scholar).In this study, we screened for V-1-binding proteins by a novel proteomic approach that combined the tandem affinity purification (TAP) 1The abbreviations used are: TAP, tandem affinity purification; CP, capping protein; GST, glutathioneS-transferase; MS/MS, tandem mass spectrometry; SPR, surface plasmon resonance; CBB, Coomassie Brilliant Blue; 293T, human embryonic kidney 293 T cells 1The abbreviations used are: TAP, tandem affinity purification; CP, capping protein; GST, glutathioneS-transferase; MS/MS, tandem mass spectrometry; SPR, surface plasmon resonance; CBB, Coomassie Brilliant Blue; 293T, human embryonic kidney 293 T cells procedure (12Rigaut G. Shevchenko A. Rutz B. Wilm M. Mann M. Seraphin B. Nat. Biotechnol. 1999; 17: 1030-1032Google Scholar) and mass spectrometry (MS). Following this strategy, we identified capping protein (CP) as a V-1-binding protein. We confirmed the existence of the V-1·CP complex not only in cultured cells transfected with the TAP-tagged V-1 but also endogenously in cells and rat cerebellar extracts. Furthermore, we found that V-1 inhibited the CP-regulated actin polymerization. On the basis of these results, the possible role of V-1 in neuronal development is discussed.DISCUSSIONCP is one of the F-actin-binding proteins that caps the barbed end of actin filaments and nucleates the actin polymerization in a Ca2+-independent (22Maruyama K. Kimura S. Ishii T. Kuroda M. Ohashi K. Muramats S. J. Biochem. 1977; 81: 215-232Google Scholar, 23Caldwell J.E. Heiss S.G. Mermall V. Cooper J.A. Biochemistry. 1989; 28: 8506-8514Google Scholar, 27Maruyama K. Kurokawa H. Oosawa M. Shimaoka S. Yamamoto H. Ito M. J. Biol. Chem. 1990; 265: 8712-8715Google Scholar, 28Casella J.F. Maack D.J. Lin S. J. Biol. Chem. 1986; 261: 10915-10921Google Scholar) and a phosphatidylinositol 4,5-bisphosphate-dependent manner (29Heiss S.G. Cooper J.A. Biochemistry. 1991; 30: 8753-8758Google Scholar, 30Haus U. Hartmann H. Trommler P. Noegel A.A. Schleicher M. Biochem. Biophys. Res. Commun. 1991; 181: 833-839Google Scholar, 31Barkalow K. Witke W. Kwiatkowski D.J. Hartwig J.H. J. Cell Biol. 1996; 134: 389-399Google Scholar, 32DiNubile M.J. Huang S. Biochim. Biophys. Acta. 1997; 1358: 261-278Google Scholar). This activity is thought to be functionally significant, because the actin-based movement of Dictyostelium is proportional to the expression level of CP (33Hug C. Jay P.Y. Reddy I. McNally J.G. Bridgman P.C. Elson E.L. Cooper J.A. Cell. 1995; 81: 591-600Google Scholar) and because CP is essential for the in vitro reconstitution of the cell movement (34Loisel T.P. Boujemaa R. Pantaloni D. Carlier M.F. Nature. 1999; 401: 613-616Google Scholar, 35Pantaloni D. Boujemaa R. Didry D. Gounon P. Carlier M.F. Nat. Cell Biol. 2000; 2: 385-391Google Scholar). In this study, we have shown that the V-1 protein forms a stable stoichiometric complex with CP in vitro as well as in vivo (Figs. 2 and 3) and inhibits the CP-mediated nucleation of actin polymerization. Therefore, we assume that V-1 participates in the regulation of actin dynamics in the cells via the interaction with CP.Our strategy to identify the V-1-interacting molecules was based on the tandem affinity purification of the V-1 complex with a TAP tag followed by protein identification by mass spectrometry. The TAP method was originally developed to analyze interactions among yeast proteins (12Rigaut G. Shevchenko A. Rutz B. Wilm M. Mann M. Seraphin B. Nat. Biotechnol. 1999; 17: 1030-1032Google Scholar). We constructed a mammalian expression vector for the TAP method and applied it to the mammalian 293T cell line. Even though the V-1 protein is a rather minor cellular component and a transient expression system was used for the assay, the method enabled us to isolate a sufficient amount of the V-1·CP complex for characterization by nanospray tandem mass spectrometry (Fig. 1). This affinity-tag technique coupled with mass spectrometry is useful to detect novel protein interactions not only in yeast but also in mammalian cells.The V-1 protein consists of three consecutive ANK repeats with an additional short stretch of sequence (1Taoka M. Yamakuni T. Song S.Y. Yamakawa Y. Seta K. Okuyama T. Isobe T. Eur. J. Biochem. 1992; 207: 615-620Google Scholar, 2Taoka M. Isobe T. Okuyama T. Watanabe M. Kondo H. Yamakawa Y. Ozawa F. Hishinuma F. Kubota M. Minegishi A. Song S.Y. Yamakuni T. J. Biol. Chem. 1994; 269: 9946-9951Google Scholar, 37Yang Y. Nanduri S. Sen S. Qin J. Structure. 1998; 6: 619-626Google Scholar). The ANK repeat is a structural motif found in many proteins (36Rubin G.M. Yandell M.D. Wortman J.R. Gabor Miklos G.L. Nelson C.R. Hariharan I.K. Fortini M.E. Li P.W. Apweiler R. Fleischmann W. Cherry J.M. Henikoff S. Skupski M.P. Misra S. Ashburner M. Birney E. Boguski M.S. Brody T. Brokstein P. Celniker S.E. Chervitz S.A. Coates D. Cravchik A. Gabrielian A. Galle R.F. Gelbart W.M. George R.A. Goldstein L.S. Gong F. Guan P. Harris N.L. Hay B.A. Hoskins R.A. Li J. Li Z. Hynes R.O. Jones S.J. Kuehl P.M. Lemaitre B. Littleton J.T. Morrison D.K. Mungall C. O'Farrell P.H. Pickeral O.K. Shue C. Vosshall L.B. Zhang J. Zhao Q. Zheng X.H. Lewis S. Science. 2000; 287: 2204-2215Google Scholar) and mediates specific interactions with a diverse array of protein targets (4Sedgwick S.G. Smerdon S.J. Trends Biochem. Sci. 1999; 24: 311-316Google Scholar). In the tertiary structure of V-1 determined by NMR spectroscopy (37Yang Y. Nanduri S. Sen S. Qin J. Structure. 1998; 6: 619-626Google Scholar), the ANK repeats comprise the hairpin-helix-loop-helix modules where the α helices lie along one side providing a structural framework and the hairpins protrude on the other side of the molecule. The hairpins and the surface of the α helices form a groove-like structure, which is believed to be responsible for the contact with the target molecule. This study identified CP as a potential target of V-1. Interestingly, V-1 bound to the functional CP heterodimer consisting of the α and β subunits but did not bind each of the two subunits. This finding is comparable with previous observations that each of the CP subunits was unstable and did not bind actin in vitro and in vivo (26Hug C. Miller T.M. Torres M.A. Casella J.F. Cooper J.A. J. Cell Biol. 1992; 116: 923-931Google Scholar, 38Amatruda J.F. Gattermeir D.J. Karpova T.S. Cooper J.A. J. Cell Biol. 1992; 119: 1151-1162Google Scholar, 39Hart M.C. Cooper J.A. J. Cell Biol. 1999; 147: 1287-1298Google Scholar). Thus, it seems likely that V-1 recognizes the structural interface of the CP heterodimer by its groove-like ANK repeats and covers the F-actin binding surface located in the carboxyl-terminal region of the CPβ subunit (26Hug C. Miller T.M. Torres M.A. Casella J.F. Cooper J.A. J. Cell Biol. 1992; 116: 923-931Google Scholar). However, whether this is the molecular mechanism by which V-1 inhibits the interaction of CP and F-actin awaits further structural investigation.Two distinct proteins, carmil (40Jung G. Remmert K. Wu X. Volosky J.M. Hammer III, J.A. J. Cell Biol. 2001; 153: 1479-1497Google Scholar) and twinfilin (24Palmgren S. Ojala P.J. Wear M.A. Cooper J.A. Lappalainen P. J. Cell Biol. 2001; 155: 251-260Google Scholar), are known to bind CP. Carmil is a scaffold protein containing the CP-binding site, the myosin I-binding site, the short sequence commonly found in several actin monomer-binding proteins, and the acidic stretch that can activate Arp2/3-dependent actin nucleation (40Jung G. Remmert K. Wu X. Volosky J.M. Hammer III, J.A. J. Cell Biol. 2001; 153: 1479-1497Google Scholar). The NH2-terminal region of carmil binds CP, but its activity with CP is unknown. Twinfilin is a ubiquitous actin monomer-binding protein composed of two ADF/cofilin-like domains connected by a short linker region and has a role in the regulation of actin turnover (41Palmgren S. Vartiainen M. Lappalainen P. J. Cell Sci. 2002; 115: 881-886Google Scholar). Twinfilin also forms a stable complex with CP but does not affect its activity. V-1 is neither a scaffold protein nor an actin-binding protein, and it lacks structural homology to either carmil or twinfilin. Thus, V-1 belongs to a novel CP-binding protein category. Recently, Ena/VASP was reported as an anti-capping molecule, which promotes actin filament elongation by associating with the barbed ends of actin and shielding them from CP (42Bear J.E. Svitkina T.M. Krause M. Schafer D.A. Loureiro J.J. Strasser G.A. Maly I.V. Chaga O.Y. Cooper J.A. Borisy G.G. Gertler F.B. Cell. 2002; 109: 509-521Google Scholar). The actin cytoskeleton in the Ena/VASP-deficient cell contained shorter, more highly branched filaments than those in the control cells. V-1 resembles Ena/VASP in the activity of CP-regulated actin polymerization, but whether a similar phenotype can be attributed to the V-1-deficient cell is currently unknown.Previous studies suggested the potential roles of V-1 in nuclear events such as the transcriptional activation of a set of enzymes involved in catecholamine synthesis (5Yamakuni T. Yamamoto T. Hoshino M. Song S.Y. Yamamoto H. Kunikata-Sumitomo M. Minegishi A. Kubota M. Ito M. Konishi S. J. Biol. Chem. 1998; 273: 27051-27054Google Scholar, 6Suzuki T. Inagaki H. Yamakuni T. Nagatsu T. Ichinose H. Biochem. Biophys. Res. Commun. 2002; 293: 962-968Google Scholar, 7Suzuki T. Yamakuni T. Hagiwara M. Ichinose H. J. Biol. Chem. 2002; 277: 40768-40774Google Scholar) or the regulation of de novoprotein synthesis (8Gupta S. Sen S. Biochim. Biophys. Acta. 2002; 1589: 247-260Google Scholar, 9Knuefermann P. Chen P. Misra A. Shi S.P. Abdellatif M. Sivasubramanian N. J. Biol. Chem. 2002; 277: 23888-23897Google Scholar, 10Sivasubramanian N. Adhikary G. Sil P.C. Sen S. J. Biol. Chem. 1996; 271: 2812-2816Google Scholar, 43Schroder H.C. Krasko A. Batel R. Skorokhod A. Pahler S. Kruse M. Muller I.M. Muller W.E. FASEB J. 2000; 14: 2022-2031Google Scholar). This study suggests for the first time that V-1 functions in the molecular events taking place in the cytoplasm in which the major portion of V-1 resides within the cells as revealed by previous immunohistochemical studies (5Yamakuni T. Yamamoto T. Hoshino M. Song S.Y. Yamamoto H. Kunikata-Sumitomo M. Minegishi A. Kubota M. Ito M. Konishi S. J. Biol. Chem. 1998; 273: 27051-27054Google Scholar, 9Knuefermann P. Chen P. Misra A. Shi S.P. Abdellatif M. Sivasubramanian N. J. Biol. Chem. 2002; 277: 23888-23897Google Scholar). V-1 also appeared to be a typical cytoplasmic protein in terms of amino acid sequence with no apparent nuclear localization signal. Our previous studies revealed the characteristic temporal profile of V-1 expression in the developing murine cerebellum at postnatal days 7–12 (2Taoka M. Isobe T. Okuyama T. Watanabe M. Kondo H. Yamakawa Y. Ozawa F. Hishinuma F. Kubota M. Minegishi A. Song S.Y. Yamakuni T. J. Biol. Chem. 1994; 269: 9946-9951Google Scholar). Namely, V-1 expression is particularly significant during the migration of progenitor granule cells from the external to internal granular layer to make synaptic contacts with the target Purkinje cells. Likewise, the dynamics of actin polymerization play a pivotal role in the maturation of granule cells, because the modulation of the barbed ends of actin filaments with cytochalasins changed the behavior of the growth cone (44Zmuda J.F. Rivas R.J. J. Cell Sci. 2000; 113: 2797-2809Google Scholar) and the migration of granule cells (45Rivas R.J. Hatten M.E. J. Neurosci. 1995; 15: 981-989Google Scholar). These observations coupled with the results reported here suggest that V-1 may have a role in the CP-mediated actin-driven cell movements and motility such as granular cell migration and synapse formation. The V-1 protein was originally identified in the murine cerebellum as one of the proteins expressed significantly at the initial stage of postnatal development (1Taoka M. Yamakuni T. Song S.Y. Yamakawa Y. Seta K. Okuyama T. Isobe T. Eur. J. Biochem. 1992; 207: 615-620Google Scholar), particularly in the regions where synaptic formation and neuronal migration occur actively during neurogenesis (2Taoka M. Isobe T. Okuyama T. Watanabe M. Kondo H. Yamakawa Y. Ozawa F. Hishinuma F. Kubota M. Minegishi A. Song S.Y. Yamakuni T. J. Biol. Chem. 1994; 269: 9946-9951Google Scholar,3Fujigasaki H. Song S.Y. Kobayashi T. Yamakuni T. Mol. Brain Res. 1996; 40: 203-213Google Scholar). V-1 consists of 117 amino acids containing three contiguous repeats of the ANK motif, alternatively called the cdc10/SWI6 motif (1Taoka M. Yamakuni T. Song S.Y. Yamakawa Y. Seta K. Okuyama T. Isobe T. Eur. J. Biochem. 1992; 207: 615-620Google Scholar, 2Taoka M. Isobe T. Okuyama T. Watanabe M. Kondo H. Yamakawa Y. Ozawa F. Hishinuma F. Kubota M. Minegishi A. Song S.Y. Yamakuni T. J. Biol. Chem. 1994; 269: 9946-9951Google Scholar), which is crucial for a large number of protein-protein interactions (4Sedgwick S.G. Smerdon S.J. Trends Biochem. Sci. 1999; 24: 311-316Google Scholar). Previous studies suggested the potential role of V-1 in the signal transduction pathways leading to catecholamine synthesis or to cardiac hypertrophy. For example, Yamakuni et al. (5Yamakuni T. Yamamoto T. Hoshino M. Song S.Y. Yamamoto H. Kunikata-Sumitomo M. Minegishi A. Kubota M. Ito M. Konishi S. J. Biol. Chem. 1998; 273: 27051-27054Google Scholar, 6Suzuki T. Inagaki H. Yamakuni T. Nagatsu T. Ichinose H. Biochem. Biophys. Res. Commun. 2002; 293: 962-968Google Scholar, 7Suzuki T. Yamakuni T. Hagiwara M. Ichinose H. J. Biol. Chem. 2002; 277: 40768-40774Google Scholar) demonstrate that the overexpression of V-1 caused a significant increase in the catecholamine level in PC12 cells, presumably through the transcriptional activation of the genes for catecholamine synthesis. In other reports (8Gupta S. Sen S. Biochim. Biophys. Acta. 2002; 1589: 247-260Google Scholar, 9Knuefermann P. Chen P. Misra A. Shi S.P. Abdellatif M. Sivasubramanian N. J. Biol. Chem. 2002; 277: 23888-23897Google Scholar, 10Sivasubramanian N. Adhikary G. Sil P.C. Sen S. J. Biol. Chem. 1996; 271: 2812-2816Google Scholar, 11Sil P. Kandaswamy V. Sen S. Circ. Res. 1998; 82: 1173-1188Google Scholar), V-1 was designated as “myotrophin” and was shown to participate in the cell signaling pathways for the NFkB-mediated activation of protein synthesis in the myocytes. Thus, both of these studies (5Yamakuni T. Yamamoto T. Hoshino M. Song S.Y. Yamamoto H. Kunikata-Sumitomo M. Minegishi A. Kubota M. Ito M. Konishi S. J. Biol. Chem. 1998; 273: 27051-27054Google Scholar, 9Knuefermann P. Chen P. Misra A. Shi S.P. Abdellatif M. Sivasubramanian N. J. Biol. Chem. 2002; 277: 23888-23897Google Scholar) suggested the roles of V-1 in the biological events taking place in the nucleus. However, no biological function has been attributed to V-1 in the cytoplasm in which this molecule predominantly resides within the cells and tissues (5Yamakuni T. Yamamoto T. Hoshino M. Song S.Y. Yamamoto H. Kunikata-Sumitomo M. Minegishi A. Kubota M. Ito M. Konishi S. J. Biol. Chem. 1998; 273: 27051-27054Google Scholar, 9Knuefermann P. Chen P. Misra A. Shi S.P. Abdellatif M. Sivasubramanian N. J. Biol. Chem. 2002; 277: 23888-23897Google Scholar). In this study, we screened for V-1-binding proteins by a novel proteomic approach that combined the tandem affinity purification (TAP) 1The abbreviations used are: TAP, tandem affinity purification; CP, capping protein; GST, glutathioneS-transferase; MS/MS, tandem mass spectrometry; SPR, surface plasmon resonance; CBB, Coomassie Brilliant Blue; 293T, human embryonic kidney 293 T cells 1The abbreviations used are: TAP, tandem affinity purification; CP, capping protein; GST, glutathioneS-transferase; MS/MS, tandem mass spectrometry; SPR, surface plasmon resonance; CBB, Coomassie Brilliant Blue; 293T, human embryonic kidney 293 T cells procedure (12Rigaut G. Shevchenko A. Rutz B. Wilm M. Mann M. Seraphin B. Nat. Biotechnol. 1999; 17: 1030-1032Google Scholar) and mass spectrometry (MS). Following this strategy, we identified capping protein (CP) as a V-1-binding protein. We confirmed the existence of the V-1·CP complex not only in cultured cells transfected with the TAP-tagged V-1 but also endogenously in cells and rat cerebellar extracts. Furthermore, we found that V-1 inhibited the CP-regulated actin polymerization. On the basis of these results, the possible role of V-1 in neuronal development is discussed. DISCUSSIONCP is one of the F-actin-binding proteins that caps the barbed end of actin filaments and nucleates the actin polymerization in a Ca2+-independent (22Maruyama K. Kimura S. Ishii T. Kuroda M. Ohashi K. Muramats S. J. Biochem. 1977; 81: 215-232Google Scholar, 23Caldwell J.E. Heiss S.G. Mermall V. Cooper J.A. Biochemistry. 1989; 28: 8506-8514Google Scholar, 27Maruyama K. Kurokawa H. Oosawa M. Shimaoka S. Yamamoto H. Ito M. J. Biol. Chem. 1990; 265: 8712-8715Google Scholar, 28Casella J.F. Maack D.J. Lin S. J. Biol. Chem. 1986; 261: 10915-10921Google Scholar) and a phosphatidylinositol 4,5-bisphosphate-dependent manner (29Heiss S.G. Cooper J.A. Biochemistry. 1991; 30: 8753-8758Google Scholar, 30Haus U. Hartmann H. Trommler P. Noegel A.A. Schleicher M. Biochem. Biophys. Res. Commun. 1991; 181: 833-839Google Scholar, 31Barkalow K. Witke W. Kwiatkowski D.J. Hartwig J.H. J. Cell Biol. 1996; 134: 389-399Google Scholar, 32DiNubile M.J. Huang S. Biochim. Biophys. Acta. 1997; 1358: 261-278Google Scholar). This activity is thought to be functionally significant, because the actin-based movement of Dictyostelium is proportional to the expression level of CP (33Hug C. Jay P.Y. Reddy I. McNally J.G. Bridgman P.C. Elson E.L. Cooper J.A. Cell. 1995; 81: 591-600Google Scholar) and because CP is essential for the in vitro reconstitution of the cell movement (34Loisel T.P. Boujemaa R. Pantaloni D. Carlier M.F. Nature. 1999; 401: 613-616Google Scholar, 35Pantaloni D. Boujemaa R. Didry D. Gounon P. Carlier M.F. Nat. Cell Biol. 2000; 2: 385-391Google Scholar). In this study, we have shown that the V-1 protein forms a stable stoichiometric complex with CP in vitro as well as in vivo (Figs. 2 and 3) and inhibits the CP-mediated nucleation of actin polymerization. Therefore, we assume that V-1 participates in the regulation of actin dynamics in the cells via the interaction with CP.Our strategy to identify the V-1-interacting molecules was based on the tandem affinity purification of the V-1 complex with a TAP tag followed by protein identification by mass spectrometry. The TAP method was originally developed to analyze interactions among yeast proteins (12Rigaut G. Shevchenko A. Rutz B. Wilm M. Mann M. Seraphin B. Nat. Biotechnol. 1999; 17: 1030-1032Google Scholar). We constructed a mammalian expression vector for the TAP method and applied it to the mammalian 293T cell line. Even though the V-1 protein is a rather minor cellular component and a transient expression system was used for the assay, the method enabled us to isolate a sufficient amount of the V-1·CP complex for characterization by nanospray tandem mass spectrometry (Fig. 1). This affinity-tag technique coupled with mass spectrometry is useful to detect novel protein interactions not only in yeast but also in mammalian cells.The V-1 protein consists of three consecutive ANK repeats with an additional short stretch of sequence (1Taoka M. Yamakuni T. Song S.Y. Yamakawa Y. Seta K. Okuyama T. Isobe T. Eur. J. Biochem. 1992; 207: 615-620Google Scholar, 2Taoka M. Isobe T. Okuyama T. Watanabe M. Kondo H. Yamakawa Y. Ozawa F. Hishinuma F. Kubota M. Minegishi A. Song S.Y. Yamakuni T. J. Biol. Chem. 1994; 269: 9946-9951Google Scholar, 37Yang Y. Nanduri S. Sen S. Qin J. Structure. 1998; 6: 619-626Google Scholar). The ANK repeat is a structural motif found in many proteins (36Rubin G.M. Yandell M.D. Wortman J.R. Gabor Miklos G.L. Nelson C.R. Hariharan I.K. Fortini M.E. Li P.W. Apweiler R. Fleischmann W. Cherry J.M. Henikoff S. Skupski M.P. Misra S. Ashburner M. Birney E. Boguski M.S. Brody T. Brokstein P. Celniker S.E. Chervitz S.A. Coates D. Cravchik A. Gabrielian A. Galle R.F. Gelbart W.M. George R.A. Goldstein L.S. Gong F. Guan P. Harris N.L. Hay B.A. Hoskins R.A. Li J. Li Z. Hynes R.O. Jones S.J. Kuehl P.M. Lemaitre B. Littleton J.T. Morrison D.K. Mungall C. O'Farrell P.H. Pickeral O.K. Shue C. Vosshall L.B. Zhang J. Zhao Q. Zheng X.H. Lewis S. Science. 2000; 287: 2204-2215Google Scholar) and mediates specific interactions with a diverse array of protein targets (4Sedgwick S.G. Smerdon S.J. Trends Biochem. Sci. 1999; 24: 311-316Google Scholar). In the tertiary structure of V-1 determined by NMR spectroscopy (37Yang Y. Nanduri S. Sen S. Qin J. Structure. 1998; 6: 619-626Google Scholar), the ANK repeats comprise the hairpin-helix-loop-helix modules where the α helices lie along one side providing a structural framework and the hairpins protrude on the other side of the molecule. The hairpins and the surface of the α helices form a groove-like structure, which is believed to be responsible for the contact with the target molecule. This study identified CP as a potential target of V-1. Interestingly, V-1 bound to the functional CP heterodimer consisting of the α and β subunits but did not bind each of the two subunits. This finding is comparable with previous observations that each of the CP subunits was unstable and did not bind actin in vitro and in vivo (26Hug C. Miller T.M. Torres M.A. Casella J.F. Cooper J.A. J. Cell Biol. 1992; 116: 923-931Google Scholar, 38Amatruda J.F. Gattermeir D.J. Karpova T.S. Cooper J.A. J. Cell Biol. 1992; 119: 1151-1162Google Scholar, 39Hart M.C. Cooper J.A. J. Cell Biol. 1999; 147: 1287-1298Google Scholar). Thus, it seems likely that V-1 recognizes the structural interface of the CP heterodimer by its groove-like ANK repeats and covers the F-actin binding surface located in the carboxyl-terminal region of the CPβ subunit (26Hug C. Miller T.M. Torres M.A. Casella J.F. Cooper J.A. J. Cell Biol. 1992; 116: 923-931Google Scholar). However, whether this is the molecular mechanism by which V-1 inhibits the interaction of CP and F-actin awaits further structural investigation.Two distinct proteins, carmil (40Jung G. Remmert K. Wu X. Volosky J.M. Hammer III, J.A. J. Cell Biol. 2001; 153: 1479-1497Google Scholar) and twinfilin (24Palmgren S. Ojala P.J. Wear M.A. Cooper J.A. Lappalainen P. J. Cell Biol. 2001; 155: 251-260Google Scholar), are known to bind CP. Carmil is a scaffold protein containing the CP-binding site, the myosin I-binding site, the short sequence commonly found in several actin monomer-binding proteins, and the acidic stretch that can activate Arp2/3-dependent actin nucleation (40Jung G. Remmert K. Wu X. Volosky J.M. Hammer III, J.A. J. Cell Biol. 2001; 153: 1479-1497Google Scholar). The NH2-terminal region of carmil binds CP, but its activity with CP is unknown. Twinfilin is a ubiquitous actin monomer-binding protein composed of two ADF/cofilin-like domains connected by a short linker region and has a role in the regulation of actin turnover (41Palmgren S. Vartiainen M. Lappalainen P. J. Cell Sci. 2002; 115: 881-886Google Scholar). Twinfilin also forms a stable complex with CP but does not affect its activity. V-1 is neither a scaffold protein nor an actin-binding protein, and it lacks structural homology to either carmil or twinfilin. Thus, V-1 belongs to a novel CP-binding protein category. Recently, Ena/VASP was reported as an anti-capping molecule, which promotes actin filament elongation by associating with the barbed ends of actin and shielding them from CP (42Bear J.E. Svitkina T.M. Krause M. Schafer D.A. Loureiro J.J. Strasser G.A. Maly I.V. Chaga O.Y. Cooper J.A. Borisy G.G. Gertler F.B. Cell. 2002; 109: 509-521Google Scholar). The actin cytoskeleton in the Ena/VASP-deficient cell contained shorter, more highly branched filaments than those in the control cells. V-1 resembles Ena/VASP in the activity of CP-regulated actin polymerization, but whether a similar phenotype can be attributed to the V-1-deficient cell is currently unknown.Previous studies suggested the potential roles of V-1 in nuclear events such as the transcriptional activation of a set of enzymes involved in catecholamine synthesis (5Yamakuni T. Yamamoto T. Hoshino M. Song S.Y. Yamamoto H. Kunikata-Sumitomo M. Minegishi A. Kubota M. Ito M. Konishi S. J. Biol. Chem. 1998; 273: 27051-27054Google Scholar, 6Suzuki T. Inagaki H. Yamakuni T. Nagatsu T. Ichinose H. Biochem. Biophys. Res. Commun. 2002; 293: 962-968Google Scholar, 7Suzuki T. Yamakuni T. Hagiwara M. Ichinose H. J. Biol. Chem. 2002; 277: 40768-40774Google Scholar) or the regulation of de novoprotein synthesis (8Gupta S. Sen S. Biochim. Biophys. Acta. 2002; 1589: 247-260Google Scholar, 9Knuefermann P. Chen P. Misra A. Shi S.P. Abdellatif M. Sivasubramanian N. J. Biol. Chem. 2002; 277: 23888-23897Google Scholar, 10Sivasubramanian N. Adhikary G. Sil P.C. Sen S. J. Biol. Chem. 1996; 271: 2812-2816Google Scholar, 43Schroder H.C. Krasko A. Batel R. Skorokhod A. Pahler S. Kruse M. Muller I.M. Muller W.E. FASEB J. 2000; 14: 2022-2031Google Scholar). This study suggests for the first time that V-1 functions in the molecular events taking place in the cytoplasm in which the major portion of V-1 resides within the cells as revealed by previous immunohistochemical studies (5Yamakuni T. Yamamoto T. Hoshino M. Song S.Y. Yamamoto H. Kunikata-Sumitomo M. Minegishi A. Kubota M. Ito M. Konishi S. J. Biol. Chem. 1998; 273: 27051-27054Google Scholar, 9Knuefermann P. Chen P. Misra A. Shi S.P. Abdellatif M. Sivasubramanian N. J. Biol. Chem. 2002; 277: 23888-23897Google Scholar). V-1 also appeared to be a typical cytoplasmic protein in terms of amino acid sequence with no apparent nuclear localization signal. Our previous studies revealed the characteristic temporal profile of V-1 expression in the developing murine cerebellum at postnatal days 7–12 (2Taoka M. Isobe T. Okuyama T. Watanabe M. Kondo H. Yamakawa Y. Ozawa F. Hishinuma F. Kubota M. Minegishi A. Song S.Y. Yamakuni T. J. Biol. Chem. 1994; 269: 9946-9951Google Scholar). Namely, V-1 expression is particularly significant during the migration of progenitor granule cells from the external to internal granular layer to make synaptic contacts with the target Purkinje cells. Likewise, the dynamics of actin polymerization play a pivotal role in the maturation of granule cells, because the modulation of the barbed ends of actin filaments with cytochalasins changed the behavior of the growth cone (44Zmuda J.F. Rivas R.J. J. Cell Sci. 2000; 113: 2797-2809Google Scholar) and the migration of granule cells (45Rivas R.J. Hatten M.E. J. Neurosci. 1995; 15: 981-989Google Scholar). These observations coupled with the results reported here suggest that V-1 may have a role in the CP-mediated actin-driven cell movements and motility such as granular cell migration and synapse formation. CP is one of the F-actin-binding proteins that caps the barbed end of actin filaments and nucleates the actin polymerization in a Ca2+-independent (22Maruyama K. Kimura S. Ishii T. Kuroda M. Ohashi K. Muramats S. J. Biochem. 1977; 81: 215-232Google Scholar, 23Caldwell J.E. Heiss S.G. Mermall V. Cooper J.A. Biochemistry. 1989; 28: 8506-8514Google Scholar, 27Maruyama K. Kurokawa H. Oosawa M. Shimaoka S. Yamamoto H. Ito M. J. Biol. Chem. 1990; 265: 8712-8715Google Scholar, 28Casella J.F. Maack D.J. Lin S. J. Biol. Chem. 1986; 261: 10915-10921Google Scholar) and a phosphatidylinositol 4,5-bisphosphate-dependent manner (29Heiss S.G. Cooper J.A. Biochemistry. 1991; 30: 8753-8758Google Scholar, 30Haus U. Hartmann H. Trommler P. Noegel A.A. Schleicher M. Biochem. Biophys. Res. Commun. 1991; 181: 833-839Google Scholar, 31Barkalow K. Witke W. Kwiatkowski D.J. Hartwig J.H. J. Cell Biol. 1996; 134: 389-399Google Scholar, 32DiNubile M.J. Huang S. Biochim. Biophys. Acta. 1997; 1358: 261-278Google Scholar). This activity is thought to be functionally significant, because the actin-based movement of Dictyostelium is proportional to the expression level of CP (33Hug C. Jay P.Y. Reddy I. McNally J.G. Bridgman P.C. Elson E.L. Cooper J.A. Cell. 1995; 81: 591-600Google Scholar) and because CP is essential for the in vitro reconstitution of the cell movement (34Loisel T.P. Boujemaa R. Pantaloni D. Carlier M.F. Nature. 1999; 401: 613-616Google Scholar, 35Pantaloni D. Boujemaa R. Didry D. Gounon P. Carlier M.F. Nat. Cell Biol. 2000; 2: 385-391Google Scholar). In this study, we have shown that the V-1 protein forms a stable stoichiometric complex with CP in vitro as well as in vivo (Figs. 2 and 3) and inhibits the CP-mediated nucleation of actin polymerization. Therefore, we assume that V-1 participates in the regulation of actin dynamics in the cells via the interaction with CP. Our strategy to identify the V-1-interacting molecules was based on the tandem affinity purification of the V-1 complex with a TAP tag followed by protein identification by mass spectrometry. The TAP method was originally developed to analyze interactions among yeast proteins (12Rigaut G. Shevchenko A. Rutz B. Wilm M. Mann M. Seraphin B. Nat. Biotechnol. 1999; 17: 1030-1032Google Scholar). We constructed a mammalian expression vector for the TAP method and applied it to the mammalian 293T cell line. Even though the V-1 protein is a rather minor cellular component and a transient expression system was used for the assay, the method enabled us to isolate a sufficient amount of the V-1·CP complex for characterization by nanospray tandem mass spectrometry (Fig. 1). This affinity-tag technique coupled with mass spectrometry is useful to detect novel protein interactions not only in yeast but also in mammalian cells. The V-1 protein consists of three consecutive ANK repeats with an additional short stretch of sequence (1Taoka M. Yamakuni T. Song S.Y. Yamakawa Y. Seta K. Okuyama T. Isobe T. Eur. J. Biochem. 1992; 207: 615-620Google Scholar, 2Taoka M. Isobe T. Okuyama T. Watanabe M. Kondo H. Yamakawa Y. Ozawa F. Hishinuma F. Kubota M. Minegishi A. Song S.Y. Yamakuni T. J. Biol. Chem. 1994; 269: 9946-9951Google Scholar, 37Yang Y. Nanduri S. Sen S. Qin J. Structure. 1998; 6: 619-626Google Scholar). The ANK repeat is a structural motif found in many proteins (36Rubin G.M. Yandell M.D. Wortman J.R. Gabor Miklos G.L. Nelson C.R. Hariharan I.K. Fortini M.E. Li P.W. Apweiler R. Fleischmann W. Cherry J.M. Henikoff S. Skupski M.P. Misra S. Ashburner M. Birney E. Boguski M.S. Brody T. Brokstein P. Celniker S.E. Chervitz S.A. Coates D. Cravchik A. Gabrielian A. Galle R.F. Gelbart W.M. George R.A. Goldstein L.S. Gong F. Guan P. Harris N.L. Hay B.A. Hoskins R.A. Li J. Li Z. Hynes R.O. Jones S.J. Kuehl P.M. Lemaitre B. Littleton J.T. Morrison D.K. Mungall C. O'Farrell P.H. Pickeral O.K. Shue C. Vosshall L.B. Zhang J. Zhao Q. Zheng X.H. Lewis S. Science. 2000; 287: 2204-2215Google Scholar) and mediates specific interactions with a diverse array of protein targets (4Sedgwick S.G. Smerdon S.J. Trends Biochem. Sci. 1999; 24: 311-316Google Scholar). In the tertiary structure of V-1 determined by NMR spectroscopy (37Yang Y. Nanduri S. Sen S. Qin J. Structure. 1998; 6: 619-626Google Scholar), the ANK repeats comprise the hairpin-helix-loop-helix modules where the α helices lie along one side providing a structural framework and the hairpins protrude on the other side of the molecule. The hairpins and the surface of the α helices form a groove-like structure, which is believed to be responsible for the contact with the target molecule. This study identified CP as a potential target of V-1. Interestingly, V-1 bound to the functional CP heterodimer consisting of the α and β subunits but did not bind each of the two subunits. This finding is comparable with previous observations that each of the CP subunits was unstable and did not bind actin in vitro and in vivo (26Hug C. Miller T.M. Torres M.A. Casella J.F. Cooper J.A. J. Cell Biol. 1992; 116: 923-931Google Scholar, 38Amatruda J.F. Gattermeir D.J. Karpova T.S. Cooper J.A. J. Cell Biol. 1992; 119: 1151-1162Google Scholar, 39Hart M.C. Cooper J.A. J. Cell Biol. 1999; 147: 1287-1298Google Scholar). Thus, it seems likely that V-1 recognizes the structural interface of the CP heterodimer by its groove-like ANK repeats and covers the F-actin binding surface located in the carboxyl-terminal region of the CPβ subunit (26Hug C. Miller T.M. Torres M.A. Casella J.F. Cooper J.A. J. Cell Biol. 1992; 116: 923-931Google Scholar). However, whether this is the molecular mechanism by which V-1 inhibits the interaction of CP and F-actin awaits further structural investigation. Two distinct proteins, carmil (40Jung G. Remmert K. Wu X. Volosky J.M. Hammer III, J.A. J. Cell Biol. 2001; 153: 1479-1497Google Scholar) and twinfilin (24Palmgren S. Ojala P.J. Wear M.A. Cooper J.A. Lappalainen P. J. Cell Biol. 2001; 155: 251-260Google Scholar), are known to bind CP. Carmil is a scaffold protein containing the CP-binding site, the myosin I-binding site, the short sequence commonly found in several actin monomer-binding proteins, and the acidic stretch that can activate Arp2/3-dependent actin nucleation (40Jung G. Remmert K. Wu X. Volosky J.M. Hammer III, J.A. J. Cell Biol. 2001; 153: 1479-1497Google Scholar). The NH2-terminal region of carmil binds CP, but its activity with CP is unknown. Twinfilin is a ubiquitous actin monomer-binding protein composed of two ADF/cofilin-like domains connected by a short linker region and has a role in the regulation of actin turnover (41Palmgren S. Vartiainen M. Lappalainen P. J. Cell Sci. 2002; 115: 881-886Google Scholar). Twinfilin also forms a stable complex with CP but does not affect its activity. V-1 is neither a scaffold protein nor an actin-binding protein, and it lacks structural homology to either carmil or twinfilin. Thus, V-1 belongs to a novel CP-binding protein category. Recently, Ena/VASP was reported as an anti-capping molecule, which promotes actin filament elongation by associating with the barbed ends of actin and shielding them from CP (42Bear J.E. Svitkina T.M. Krause M. Schafer D.A. Loureiro J.J. Strasser G.A. Maly I.V. Chaga O.Y. Cooper J.A. Borisy G.G. Gertler F.B. Cell. 2002; 109: 509-521Google Scholar). The actin cytoskeleton in the Ena/VASP-deficient cell contained shorter, more highly branched filaments than those in the control cells. V-1 resembles Ena/VASP in the activity of CP-regulated actin polymerization, but whether a similar phenotype can be attributed to the V-1-deficient cell is currently unknown. Previous studies suggested the potential roles of V-1 in nuclear events such as the transcriptional activation of a set of enzymes involved in catecholamine synthesis (5Yamakuni T. Yamamoto T. Hoshino M. Song S.Y. Yamamoto H. Kunikata-Sumitomo M. Minegishi A. Kubota M. Ito M. Konishi S. J. Biol. Chem. 1998; 273: 27051-27054Google Scholar, 6Suzuki T. Inagaki H. Yamakuni T. Nagatsu T. Ichinose H. Biochem. Biophys. Res. Commun. 2002; 293: 962-968Google Scholar, 7Suzuki T. Yamakuni T. Hagiwara M. Ichinose H. J. Biol. Chem. 2002; 277: 40768-40774Google Scholar) or the regulation of de novoprotein synthesis (8Gupta S. Sen S. Biochim. Biophys. Acta. 2002; 1589: 247-260Google Scholar, 9Knuefermann P. Chen P. Misra A. Shi S.P. Abdellatif M. Sivasubramanian N. J. Biol. Chem. 2002; 277: 23888-23897Google Scholar, 10Sivasubramanian N. Adhikary G. Sil P.C. Sen S. J. Biol. Chem. 1996; 271: 2812-2816Google Scholar, 43Schroder H.C. Krasko A. Batel R. Skorokhod A. Pahler S. Kruse M. Muller I.M. Muller W.E. FASEB J. 2000; 14: 2022-2031Google Scholar). This study suggests for the first time that V-1 functions in the molecular events taking place in the cytoplasm in which the major portion of V-1 resides within the cells as revealed by previous immunohistochemical studies (5Yamakuni T. Yamamoto T. Hoshino M. Song S.Y. Yamamoto H. Kunikata-Sumitomo M. Minegishi A. Kubota M. Ito M. Konishi S. J. Biol. Chem. 1998; 273: 27051-27054Google Scholar, 9Knuefermann P. Chen P. Misra A. Shi S.P. Abdellatif M. Sivasubramanian N. J. Biol. Chem. 2002; 277: 23888-23897Google Scholar). V-1 also appeared to be a typical cytoplasmic protein in terms of amino acid sequence with no apparent nuclear localization signal. Our previous studies revealed the characteristic temporal profile of V-1 expression in the developing murine cerebellum at postnatal days 7–12 (2Taoka M. Isobe T. Okuyama T. Watanabe M. Kondo H. Yamakawa Y. Ozawa F. Hishinuma F. Kubota M. Minegishi A. Song S.Y. Yamakuni T. J. Biol. Chem. 1994; 269: 9946-9951Google Scholar). Namely, V-1 expression is particularly significant during the migration of progenitor granule cells from the external to internal granular layer to make synaptic contacts with the target Purkinje cells. Likewise, the dynamics of actin polymerization play a pivotal role in the maturation of granule cells, because the modulation of the barbed ends of actin filaments with cytochalasins changed the behavior of the growth cone (44Zmuda J.F. Rivas R.J. J. Cell Sci. 2000; 113: 2797-2809Google Scholar) and the migration of granule cells (45Rivas R.J. Hatten M.E. J. Neurosci. 1995; 15: 981-989Google Scholar). These observations coupled with the results reported here suggest that V-1 may have a role in the CP-mediated actin-driven cell movements and motility such as granular cell migration and synapse formation." @default.
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W2042306236 title "V-1, a Protein Expressed Transiently during Murine Cerebellar Development, Regulates Actin Polymerization via Interaction with Capping Protein" @default.
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