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c-Src Is Required for Stimulation of Gelsolin-associated Phosphatidylinositol 3-Kinase*

  • Meenakshi Chellaiah
    Affiliations
    Renal Division, Barnes-Jewish Hospital, Washington University School of Medicine, St. Louis, Missouri 63110
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  • Catherine Fitzgerald
    Affiliations
    Renal Division, Barnes-Jewish Hospital, Washington University School of Medicine, St. Louis, Missouri 63110
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  • Ulises Alvarez
    Affiliations
    Renal Division, Barnes-Jewish Hospital, Washington University School of Medicine, St. Louis, Missouri 63110
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  • Keith Hruska
    Correspondence
    To whom correspondence should be addressed: Renal Div., Barnes-Jewish Hospital, North, Washington University School of Medicine, 216 S. Kingshighway, St. Louis, MO 63110. Tel.: 314-454-7771; Fax: 314-454-5126
    Affiliations
    Renal Division, Barnes-Jewish Hospital, Washington University School of Medicine, St. Louis, Missouri 63110
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  • Author Footnotes
    * This work was supported by National Institutes of Health Grants AR41677, DK49728, and DK09976 and funds from the Barnes-Jewish Research Foundation.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:May 08, 1998DOI:https://doi.org/10.1074/jbc.273.19.11908
      We have shown that osteopontin binding to integrin αvβ3 in osteoclasts stimulates gelsolin-associated phosphatidylinositol (PtdIns) 3-hydroxyl kinase (PI 3-kinase), leading to increased levels of gelsolin-bound PtdIns 3,4-P2, PtdIns 4,5-P2, and PtdIns 3,4,5-P3, uncapping of barbed end actin, and actin filament formation. Inhibition of PI 3-kinase activity by wortmannin blocks osteopontin stimulation of actin filament formation, suggesting that activation of gelsolin-associated PI 3-kinase is an important pathway in cytoskeletal regulation. To study the mechanism of gelsolin-associated PI 3-kinase activation, we analyzed anti-gelsolin immunoprecipitates for the association of protein kinases. We demonstrated that c-Src co-immunoprecipitates with gelsolin, and that osteopontin stimulates its activity. Elimination of osteopontin-stimulated Src activity associated with gelsolin through antisense oligodeoxynucleotides blocked the stimulation of PI 3-kinase activity associated with gelsolin and the gelsolin-dependent cytoskeletal reorganization induced by osteopontin, including increased F-actin levels. In addition, treatment of osteoclasts with antisense oligonucleotides to Src reduced bone resorption. Our results demonstrate that osteopontin stimulates gelsolin-associated Src, leading to increased gelsolin-associated PI 3-kinase activity and PtdIns 3,4,5-P3 levels, which facilitate actin filament formation, osteoclast motility, and bone resorption.
      Osteoclasts are multinucleated, giant cells, responsible for bone resorption. Osteoclast adhesion to bone leads to the formation of the osteoclast clear zone: a ring-like adhesion zone circumscribing an area of bone resorption. The cytoplasm of the clear zone contains numerous actin filaments perpendicular to the bone matrix, which are anchored in podosomes (
      • Marchisio P.C.
      • Cirillo D.
      • Naldini L.
      • Primavera M.V.
      • Teti A.
      • Zambonin-Zallone A.
      ,
      • Marchisio P.C.
      • Cirillo D.
      • Teti A.
      • Zambonin-Zallone A.
      • Tarone G.
      ,
      • Zambonin-Zallone A.
      • Teti A.
      • Grano M.
      • Rubinacci A.
      • Abbadini M.
      • Gaboli M.
      • Marchisio C.
      ,
      • Lakkakorpi P.T.
      • Vaananen H.K.
      ,
      • Teti A.
      • Marchisio P.C.
      • Zambonin-Zallone A.
      ). Podosomes are small cell processes specific to cells of monocytic origin. Podosomes contain numerous proteins observed in the focal adhesions of other cells. Although podosomes and focal adhesions are related, there are important functional differences. Podosomes are less tightly associated with the substratum and are more highly dynamic, changing in size and location and appearing and disappearing with life spans of 2–12 min (
      • Lakkakorpi P.T.
      • Vaananen H.K.
      ). Osteoclasts are highly motile cells, and podosomes appear to be a preferred cell/matrix attachment mechanism for motility in cells such as macrophages, monocytes, and osteoclasts.
      The adhesion of osteoclasts through podosomes involves interaction of cell surface receptors, integrins, with matrix components. In osteoclasts, integrin αvβ3
      The abbreviations used are: αvβ3, adhesion receptor αvβ3; OP, osteopontin; GRGDS, Gly-Arg-Gly-Asp-Ser cell adhesion sequence; FAK, focal adhesion kinase; PtdIns, phosphatidylinositol; ODN, oligodeoxynucleotide; PI 3-kinase, phosphatidylinositol 3-kinase; PBS, phosphate-buffered saline.
      1The abbreviations used are: αvβ3, adhesion receptor αvβ3; OP, osteopontin; GRGDS, Gly-Arg-Gly-Asp-Ser cell adhesion sequence; FAK, focal adhesion kinase; PtdIns, phosphatidylinositol; ODN, oligodeoxynucleotide; PI 3-kinase, phosphatidylinositol 3-kinase; PBS, phosphate-buffered saline.
      is responsible for adhesion associated with bone resorption (
      • Zambonin-Zallone A.
      • Teti A.
      • Grano M.
      • Rubinacci A.
      • Abbadini M.
      • Gaboli M.
      • Marchisio C.
      ,
      • Davis J.
      • Warwich J.
      • Totty N.
      • Philp R.
      • Helfrich M.
      • Horton M.
      ). The ligands of αvβ3 are many, and they contain the RGD cell adhesion sequence present in several serum and bone matrix proteins. Osteopontin (OP) is an RGD-containing bone matrix protein, which plays a key role in anchoring osteoclasts to bone surfaces. Besides its role as an anchorage protein, OP is produced by osteoclasts in large quantities (
      • Ikeda T.
      • Nomura S.
      • Yamaguchi A.
      • Suda T.
      • Yoshiki S.
      ),2 and its αvβ3integrin receptor, besides location in the podosomes, is found in the osteoclast membrane opposite to the bone matrix (
      • Lakkakorpi P.T.
      • Vaananen H.K.
      ,
      • Horton M.A.
      • Taylor M.L.
      • Arnett T.R.
      • Helfrich M.H.
      ). We have shown that osteopontin binding to basolateral αvβ3 is an autocrine motility factor regulating the shape and depth of osteoclast resorption (
      • Chellaiah M.
      • Kwiatkowski D.
      • Alvarez U.
      • Gillis M.
      • Hruska K.
      ). The intracellular biochemical pathways that integrins regulate, and the cellular functions that they control, have recently been the focus of careful scrutiny. Association of focal adhesion kinase (FAK) with the cell surface at focal adhesions directs interactions with the cytoplasmic domains of integrins and their participation in signal transduction (
      • Ingber D.E.
      ,
      • Kornberg L.J.
      • Earp H.S.
      • Turner C.E.
      • Procop C.
      • Juliano R.L.
      ,
      • Juliano R.L.
      • Haskill S.
      ). Phosphorylation of FAK in response to cell adhesion and other stimuli induce the formation of complexes between FAK and other signaling molecules in vivo, including Src (
      • Cobb B.
      • Schaller M.
      • Leu T.
      • Parsons J.T.
      ,
      • Xing Z.
      • Chen H.C.
      • Noelen J.K.
      • Taylor S.J.
      • Shaloway D.
      • Guan J.L.
      ), Grb2 (
      • Schlaepfer D.D.
      • Hanks S.K.
      • Hunter T.
      • van der Geer P.
      ), Nck (
      • Schwartz M.A.
      • Schaller M.D.
      • Ginsberg M.H.
      ), and PI 3-kinase (
      • Chen H.-C.
      • Guan J.-L.
      ,
      • Guinebault C.
      • Payrastre B.
      • Racaud-Sultan C.
      • Mazarguil H.
      • Breton M.
      • Mauco G.
      • Plantavid M.
      • Chap H.
      ).
      A unique aspect of signaling through αvβ3is its property of responding to soluble ligands (
      • Horton M.A.
      • Taylor M.L.
      • Arnett T.R.
      • Helfrich M.H.
      ,
      • Hruska K.A.
      • Rolnick F.
      • Huskey M.
      • Alvarez U.
      • Cheresh D.
      ,
      • Miyauchi A.
      • Alvarez J.
      • Greenfield R.M.
      • Teti A.
      • Grano M.
      • Colucci S.
      • Zambonin-Zallone A.
      • Ross F.P.
      • Teitelbaum S.L.
      • Cheresh D.
      • Hruska K.A.
      ). We have previously demonstrated the mechanisms of αvβ3 signaling in response to soluble OP (
      • Hruska K.A.
      • Rolnick F.
      • Huskey M.
      • Alvarez U.
      • Cheresh D.
      ,
      • Chellaiah M.
      • Hruska K.A.
      ). Binding of OP- or RGD-containing peptides to αvβ3 stimulated formation of signal generating complexes consisting of FAK, c-Src, and PI 3-kinase associated with αvβ3 (
      • Hruska K.A.
      • Rolnick F.
      • Huskey M.
      • Alvarez U.
      • Cheresh D.
      ,
      • Chellaiah M.
      • Hruska K.A.
      ). OP stimulated PtdIns 3,4-P2 and PtdIns 4,5-P2(PtdIns-P2) and PtdIns 3,4,5-P3(PtdIns-P3) levels in osteoclasts (
      • Hruska K.A.
      • Rolnick F.
      • Huskey M.
      • Alvarez U.
      • Cheresh D.
      ). We further defined one specialized domain of increased PtdIns 3,4-P2 and PtdIns 3,4,5-P3 levels as an actin-capping protein found in the podosome, gelsolin (
      • Chellaiah M.
      • Hruska K.A.
      ). We demonstrated that the increase of PtdIns-P2 and PtdIns-P3 associated with gelsolin, uncapped actin oligomers leading to an increase in F-actin content and actin filament formation (
      • Chellaiah M.
      • Hruska K.A.
      ). PtdIns-P2regulates several actin-binding proteins, including gelsolin (
      • Janmey P.A.
      • Stossel T.P.
      ), profilin (
      • Lassing I.
      • Lindberg U.
      ), α-actinin (
      • Fukami K.
      • Furuhashi K.
      • Inagaki M.
      • Endo T.
      • Hatano S.
      • Takenawa T.
      ), and vinculin (
      • Fukami K.
      • Endo T.
      • Imamura M.
      • Takenawa T.
      ,
      • Johnson R.P.
      • Craig S.W.
      ). Osteopontin affected PtdIns-P2 and -P3 levels associated specifically with gelsolin and not the other proteins (
      • Chellaiah M.
      • Hruska K.A.
      ). A marked increase in the level of PtdIns-P2 with gelsolin leads to the hypothesis that PtdIns-P2 synthesis may be essential for podosome assembly and disassembly. OP treatment also resulted in the increased activity of PI 3-kinase associated with gelsolin. Phosphorylation of PtdIns 4,5-P2 by PI 3-kinase associated with gelsolin leads to the formation of PtdIns 3,4,5-P3 in OP-treated cells. The physiological function of the association of PI 3-kinase with gelsolin is stimulation of actin polymerization during cell motility, and inhibition of PI 3-kinase activity with wortmannin, a specific inhibitor of PI 3-kinase, blocks the increase in F-actin and actin polymerization stimulated by osteopontin (
      • Chellaiah M.
      • Hruska K.A.
      ).
      Examination of the signaling pathways leading to integrin-mediated activation of PI 3-kinase suggests that members of the Src family of nonreceptor kinases may play a role (
      • Pleiman C.M.
      • Hertz W.M.
      • Cambier J.C.
      ,
      • Escobedo J.A.
      • Navankassatusas K.
      • Kavanaugh W.M.
      • Milfay D.
      • Fried V.
      • Williams L.T.
      ,
      • Otsu M.
      • Hiles I.
      • Gout I.
      • Fry M.J.
      • Ruizlarrea F.
      • Panayotou G.
      • Thompson A.
      • Dhand R.
      • Hsuan J.
      • Totty N.
      • Smith A.D.
      • Morgan S.J.
      • Ciurtneidge S.A.
      • Parker P.J.
      • Waterfield M.A.
      ,
      • Liu X.
      • Marengere L.E.M.
      • Koch C.A.
      • Pawson T.
      ). PI 3-kinase activity is associated with c-Src, and the associated kinase activity increases quickly upon stimulation with thrombin in platelets (
      • Gutkind J.S.
      • Lacal P.M.
      • Robbins K.C.
      ). Several lines of evidence reveal that PI 3-kinase is activated by c-Src (
      • Fukui Y.
      • Kornbluth S.
      • Jong S.M.
      • Wang L.H.
      • Hanafusa H.
      ,
      • Fukui Y.
      • Hanafusa H.
      ). c-Src itself plays an important role in the osteoclast. Deletion of the gene for c-Src in mice results in impaired osteoclast polarization, failure of bone resorption, and osteopetrosis (
      • Horne W.C.
      • Neff L.
      • Chatterjee D.
      • Lomri A.
      • Levy J.B.
      • Baron R.
      ,
      • Lowe C.
      • Yoneda T.
      • Boyce B.F.
      • Chen H.
      • Mundy G.R.
      • Soriano P.
      ), Osteoclasts from c-Src deficient mice lack ruffled borders and have impaired bone resorptive activity in vitro (
      • Boyce B.F.
      • Yoneda T.
      • Lowe C.
      • Soriano P.
      • Mundy G.R.
      ). Wortmannin inhibited PI 3-kinase activity in osteoclasts both in vivo and in vitro and also ruffled border formation and bone resorption (
      • Nakamura I.
      • Takahashi N.
      • Sasaki T.
      • Tanaka S.
      • Udagawa N.
      • Murakami H.
      • Kimura K.
      • Kubuyama Y.
      • Kurokawa T.
      • Suda T.
      • Fukui Y.
      ). These results suggest that both Src and PI3-kinase activity are important in osteoclastic bone resorption.
      To examine the role of c-Src in the pathway to activation of gelsolin-associated PI 3-kinase from OP/αvβ3 integrin-mediated signaling, we have utilized an antisense strategy to disrupt the function of Src in the avian osteoclast system. Our results demonstrate that c-Src activity associated with the actin-binding protein gelsolin was increased upon treatment with OP. Furthermore, this increase in Src activity was necessary for the activation of gelsolin-associated PI 3-kinase, and for the subsequent increase in F-actin content and actin filament formation stimulated by liganding of αvβ3. Thus, c-Src is upstream of gelsolin-associated PI 3-kinase, and it activates PI 3-kinase.
      Since the c-Src antisense oligodeoxynucleotides-treated osteoclasts failed to form an organized podosome-containing clear zone, and were deficient in bone resorption, our data address an important second issue regarding the Src knockout phenotype. Our results, at leastin vitro, demonstrate that the osteoclast defect of the Src−/− mouse is not species-specific. They suggest that the avian osteoclasts also possess cellular sites where Src function cannot be substituted for by another member of the Src superfamily.

      DISCUSSION

      In this report, we demonstrate that Src is associated with gelsolin and its stimulation is required for OP stimulation of gelsolin-associated PI 3-kinase. Src immunoprecipitates with gelsolin, and OP stimulates the kinase activity of Src associated with gelsolin in a Triton-soluble fraction of cell lysates. The cellular function of gelsolin-associated Src was demonstrated by the absence of podosomes and cytoskeletal reorganization stimulated by osteopontin when Src was missing in the gelsolin-associated complex.
      Activation of nonreceptor protein tyrosine kinases play a central role in assembly of focal adhesions and the propagation of signals triggered by integrin receptors (
      • Burridge K.
      • Fath K.
      • Kelly T.
      • Nuckolls G.
      • Turner C.
      ,
      • Schaller M.
      • Parsons J.T.
      ). Src-dependent phosphorylation of FAK and the focal adhesion-associated proteins contributes to their assembly and activates signaling events (
      • Richardson A.
      • Parsons T.
      ). Activation of cell surface integrins has been shown to rapidly increase the tyrosine phosphorylation of FAK and the focal adhesion-associated proteins, paxillin and tensin (
      • Schwartz M.A.
      • Schaller M.D.
      • Ginsberg M.H.
      ,
      • Guan J.L.
      • Trevithick J.
      • Hynes R.O.
      ,
      • Bockholt S.M.
      • Burridge K.
      ). Phosphorylation of FAK on tyrosine 397 creates a binding site for the SH2 domain of Src (and Fyn) and results in the formation of a FAK/Src complex, which is an early event in assembly of focal adhesion complexes and the activation of integrin signaling pathways (
      • Schwartz M.A.
      • Schaller M.D.
      • Ginsberg M.H.
      ,
      • Schaller M.
      • Parsons J.T.
      ,
      • Schlaepfer D.D.
      • Hunter T.
      ). In agreement with this concept, our data suggest that Src is required for podosome assembly, since they were absent in the Src antisense-treated cells.
      We have previously shown that treatment of osteoclasts with OP stimulated formation of signal-generating complexes consisting of αvβ3, FAK, PI 3-kinase, and Src (
      • Hruska K.A.
      • Rolnick F.
      • Huskey M.
      • Alvarez U.
      • Cheresh D.
      ). OP stimulates synthesis of phosphoinositides such as PtdIns-P2, PtdIns 3,4-P2, and PtdIns-P3 through the activation of the respective phospholipid kinases in osteoclasts (
      • Hruska K.A.
      • Rolnick F.
      • Huskey M.
      • Alvarez U.
      • Cheresh D.
      ). The increase in PtdIns-P2 and PtdIns-P3 levels associated with Triton-soluble gelsolin leads to actin uncapping, stimulation of F-actin formation, and actin filament formation in osteoclasts. The increase in PtdIns-P3 levels associated with gelsolin is due to translocation of PI 3-kinase to gelsolin in a Triton-soluble cell fraction (
      • Chellaiah M.
      • Hruska K.A.
      ), and the increase in PI 4,5-P2 may be due to activation of PI 4-phosphate 5-kinase. Chong et al.(
      • Chong L.D.
      • Traynor-Kaplan A.
      • Bokoch G.M.
      • Schwartz M.A.
      ) have suggested that liganding of integrins by matrix activates PI 4-phosphate 5-kinase. The increase in the levels of PtdIns 4,5-P2 associated with gelsolin in OP-treated cells are decreased by pretreatment with antibody to integrin αvβ3 (LM609) (
      • Chellaiah M.
      • Hruska K.A.
      ) or herbimycin A. The findings of gelsolin-associated Src stimulated by osteopontin suggest that Src might activate gelsolin-associated PI 3-kinase. This implication was substantiated by our findings that antisense ODNs to Src blocked OP-induced PI 3-kinase activation. Translocation and activation of gelsolin-associated PI 3-kinase may have occurred through binding of the p85 subunit of PI 3-kinase to gelsolin-associated Src (
      • Liu X.
      • Marengere L.E.M.
      • Koch C.A.
      • Pawson T.
      ), resulting in p85 phosphorylation and subsequent formation and activation of p85/p110 heterodimers (the active form of PI 3-kinase).
      The PI 3-kinases are a family of phospholipid kinases that phosphorylate PtdIns in the 3-hydroxyl position of the inositol ring. The prototypical PI 3-kinase is a heterodimer comprised of a regulatory subunit, p85, and a catalytic subunit, p110. The catalytic subunit is activated by association with p85 and association is stimulated by tyrosine phosphorylation of the p85 SH2 domain. p85 is a known substrate of Src. In recent years, several isoforms of p85 have been discovered, and isoforms of p110 have also been found. In addition, catalytic subunits of PI 3-kinase that do not require association with the regulatory subunit have been discovered. The forms of PI 3-kinase in the osteoclast have not been determined. However, the PI 3-kinase associated with gelsolin is a prototypical form (
      • Chellaiah M.
      • Hruska K.A.
      ), and gelsolin-associated p85 phosphorylation is stimulated by osteopontin/αvβ3 (
      • Chellaiah M.
      • Hruska K.A.
      ).
      Several lines of evidence suggest that c-Src plays a role in regulating actin rearrangement in normal cells. Analysis of murine fibroblasts over expressing c-Src (K+) and dominant negative variants of Src (K−) have demonstrated pronounced actin rearrangement in K+ cells, which was absent in K− cells (
      • Chang J.H.
      • Gill S.
      • Settleman J.
      • Parsons S.J.
      ). These results suggest a role for c-Src in actin rearrangement. Moreover, it has been shown that oncogenic function of v-Src requires its translocation to the cytoskeleton at sites of focal adhesion (
      • Fincham V.J.
      • Unlu M.
      • Brunton V.G.
      • Pitts J.D.
      • Wyke J.A.
      • Frame M.C.
      ). High levels of c-Src expression are part of the osteoclast phenotype, and other members of the Src family, which account for redundancy in other cells, do not compensate for the absence of Src in the Src−/− mice (
      • Horne W.C.
      • Neff L.
      • Chatterjee D.
      • Lomri A.
      • Levy J.B.
      • Baron R.
      ). Therefore, we sought to determine whether Src activation is essential for changes in the actin cytoskeleton during stimulation of bone resorption by OP in avian osteoclasts. Failure of osteopontin-stimulated actin stress fiber formation by Src antisense ODN-treated cells demonstrates that activation of gelsolin-associated Src is an important regulatory mechanism in osteoclast function. Src has been shown to be required for the formation of ruffled borders and bone resorption, but Src is not required for osteoclast development (
      • Suda T.
      • Takahashi N.
      • Martin T.J.
      ,
      • Hall T.J.
      • Schaeublin M.
      • Missbach M.
      ,
      • Hawkins P.T.
      • Jackson T.R.
      • Stephens L.R.
      ). Inhibition of ruffled border formation and the in vitro pit forming activity of osteoclast by wortmannin confirms the role of PI 3-kinase in pathways controlling osteoclastic bone resorption (
      • Nakamura I.
      • Takahashi N.
      • Sasaki T.
      • Tanaka S.
      • Udagawa N.
      • Murakami H.
      • Kimura K.
      • Kubuyama Y.
      • Kurokawa T.
      • Suda T.
      • Fukui Y.
      ,
      • DeCorte V.
      • Gettemans J.
      • Vandekerckhove J.
      ). Thus, the antisense Src knock-out experiments may explain not only the role of gelsolin-associated PI 3-kinase in cellular responses, but also how it is activated and the pathway coupled to this response.
      That activation of gelsolin-associated PI 3-kinase is dependent upon Src activation is evident from the in vitro Src knock-out experiments. Although osteoclast gelsolin appears to be central to localization of tyrosine kinases involved in regulation of the actin cytoskeleton, the exact mechanism of gelsolin/Src interaction is unknown. The activation of gelsolin-associated PI 3-kinase by Src does not necessarily mean that it is directly associated with gelsolin. An intriguing question is by what mechanisms are Src and PI 3-kinase associated with gelsolin. There is now compelling evidence that PI 3-kinase lipid products directly associate with the SH2 domain of kinases (
      • Rameh L.E.
      • Chen C.S.
      • Cantley L.C.
      ). This observation suggests that the stimulation of Src association with gelsolin may serve to organize the gelsolin/PI 3-kinase complex. We have previously demonstrated that OP-stimulated translocation of PI 3-kinase from Triton-insoluble to Triton-soluble gelsolin (
      • Chellaiah M.
      • Hruska K.A.
      ). OP stimulated the levels of gelsolin-associated PtdIns 4,5-P2 which is a known substrate for PI 3-kinase. The increase in the synthesis of PtdIns 3,4,5-P3 in Triton-soluble gelsolin by OP may function in the recruitment of signaling molecules. Further studies need to be directed at determining how these interactions occur and are regulated in vivo.

      Acknowledgments

      We thank Dr. Brian Bennett for assistance with confocal microscopy and data processing, Dr. John Connolly for helpful comments on the manuscript, and Kathy Jones and Helen Odle for secretarial assistance.

      References

        • Marchisio P.C.
        • Cirillo D.
        • Naldini L.
        • Primavera M.V.
        • Teti A.
        • Zambonin-Zallone A.
        J. Cell Biol. 1984; 99: 1696-1705
        • Marchisio P.C.
        • Cirillo D.
        • Teti A.
        • Zambonin-Zallone A.
        • Tarone G.
        Exp. Cell Res. 1987; 169: 202-214
        • Zambonin-Zallone A.
        • Teti A.
        • Grano M.
        • Rubinacci A.
        • Abbadini M.
        • Gaboli M.
        • Marchisio C.
        Exp. Cell Res. 1989; 182: 645-652
        • Lakkakorpi P.T.
        • Vaananen H.K.
        J. Bone Miner. Res. 1991; 6: 817-826
        • Teti A.
        • Marchisio P.C.
        • Zambonin-Zallone A.
        Am. J. Physiol. 1991; 261: C1-C7
        • Davis J.
        • Warwich J.
        • Totty N.
        • Philp R.
        • Helfrich M.
        • Horton M.
        J. Cell Biol. 1989; 109: 1817-1826
        • Ikeda T.
        • Nomura S.
        • Yamaguchi A.
        • Suda T.
        • Yoshiki S.
        J. Histochem. Cytochem. 1992; 40: 1079-1088
        • Horton M.A.
        • Taylor M.L.
        • Arnett T.R.
        • Helfrich M.H.
        Exp. Cell Res. 1991; 195: 368-375
        • Chellaiah M.
        • Kwiatkowski D.
        • Alvarez U.
        • Gillis M.
        • Hruska K.
        J. Bone Miner. Res. 1997; 12: S137
        • Ingber D.E.
        Curr. Opin. Cell Biol. 1991; 3: 841-848
        • Kornberg L.J.
        • Earp H.S.
        • Turner C.E.
        • Procop C.
        • Juliano R.L.
        Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8392-8396
        • Juliano R.L.
        • Haskill S.
        J. Cell Biol. 1993; 120: 577-585
        • Cobb B.
        • Schaller M.
        • Leu T.
        • Parsons J.T.
        Mol. Cell. Biol. 1994; 14: 147-155
        • Xing Z.
        • Chen H.C.
        • Noelen J.K.
        • Taylor S.J.
        • Shaloway D.
        • Guan J.L.
        Mol. Cell. Biol. 1994; 5: 413-421
        • Schlaepfer D.D.
        • Hanks S.K.
        • Hunter T.
        • van der Geer P.
        Nature. 1994; 372: 786-791
        • Schwartz M.A.
        • Schaller M.D.
        • Ginsberg M.H.
        Annu. Rev. Cell Biol. 1995; 11: 549-600
        • Chen H.-C.
        • Guan J.-L.
        Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10148-10152
        • Guinebault C.
        • Payrastre B.
        • Racaud-Sultan C.
        • Mazarguil H.
        • Breton M.
        • Mauco G.
        • Plantavid M.
        • Chap H.
        J. Cell Biol. 1995; 129: 831-842
        • Hruska K.A.
        • Rolnick F.
        • Huskey M.
        • Alvarez U.
        • Cheresh D.
        Endocrinology. 1995; 136: 2984-2992
        • Miyauchi A.
        • Alvarez J.
        • Greenfield R.M.
        • Teti A.
        • Grano M.
        • Colucci S.
        • Zambonin-Zallone A.
        • Ross F.P.
        • Teitelbaum S.L.
        • Cheresh D.
        • Hruska K.A.
        J. Biol. Chem. 1991; 266: 20369-20374
        • Chellaiah M.
        • Hruska K.A.
        Mol. Biol. Cell. 1996; 7: 743-753
        • Janmey P.A.
        • Stossel T.P.
        Nature. 1987; 325: 362-364
        • Lassing I.
        • Lindberg U.
        Nature. 1985; 314: 472-474
        • Fukami K.
        • Furuhashi K.
        • Inagaki M.
        • Endo T.
        • Hatano S.
        • Takenawa T.
        Nature. 1992; 359: 150-152
        • Fukami K.
        • Endo T.
        • Imamura M.
        • Takenawa T.
        J. Biol. Chem. 1994; 269: 1518-1522
        • Johnson R.P.
        • Craig S.W.
        Biochem. Biophys. Res. Commun. 1995; 210: 159-164
        • Pleiman C.M.
        • Hertz W.M.
        • Cambier J.C.
        Science. 1994; 263: 1609-1612
        • Escobedo J.A.
        • Navankassatusas K.
        • Kavanaugh W.M.
        • Milfay D.
        • Fried V.
        • Williams L.T.
        Cell. 1991; 65: 75-82
        • Otsu M.
        • Hiles I.
        • Gout I.
        • Fry M.J.
        • Ruizlarrea F.
        • Panayotou G.
        • Thompson A.
        • Dhand R.
        • Hsuan J.
        • Totty N.
        • Smith A.D.
        • Morgan S.J.
        • Ciurtneidge S.A.
        • Parker P.J.
        • Waterfield M.A.
        Cell. 1991; 65: 91-104
        • Liu X.
        • Marengere L.E.M.
        • Koch C.A.
        • Pawson T.
        Mol. Cell. Biol. 1993; 13: 5226-5232
        • Gutkind J.S.
        • Lacal P.M.
        • Robbins K.C.
        Mol. Cell. Biol. 1990; 10: 3806-3809
        • Fukui Y.
        • Kornbluth S.
        • Jong S.M.
        • Wang L.H.
        • Hanafusa H.
        Oncogene Res. 1989; 4: 283-292
        • Fukui Y.
        • Hanafusa H.
        Mol. Cell. Biol. 1991; 11: 1972-1979
        • Horne W.C.
        • Neff L.
        • Chatterjee D.
        • Lomri A.
        • Levy J.B.
        • Baron R.
        J. Cell Biol. 1992; 119: 1003-1013
        • Lowe C.
        • Yoneda T.
        • Boyce B.F.
        • Chen H.
        • Mundy G.R.
        • Soriano P.
        Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4485-4489
        • Boyce B.F.
        • Yoneda T.
        • Lowe C.
        • Soriano P.
        • Mundy G.R.
        J. Clin. Invest. 1992; 90: 1622-1627
        • Nakamura I.
        • Takahashi N.
        • Sasaki T.
        • Tanaka S.
        • Udagawa N.
        • Murakami H.
        • Kimura K.
        • Kubuyama Y.
        • Kurokawa T.
        • Suda T.
        • Fukui Y.
        FEBS Lett. 1995; 361: 79-84
        • Alvarez J.I.
        • Teitelbaum S.L.
        • Blair H.C.
        • Greenfield E.M.
        • Athanasou N.A.
        • Ross F.P.
        Endocrinology. 1991; 128: 2324-2335
        • Chellaiah M.
        • Fitzgerald C.
        • Filardo E.J.
        • Cheresh D.A.
        • Hruska K.A.
        Endocrinology. 1996; 137: 2432-2440
        • Takeya T.
        • Hanafusa H.
        Cell. 1983; 32: 881-890
        • Duncan R.L.
        • Kizer N.
        • Barry E.L.R.
        • Friedman P.A.
        • Hruska K.A.
        Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1864-1869
        • Barry E.
        • Gesek F.
        • Friedman P.
        Biofeedback. 1993; 15: 1019-1020
        • Graves D.D.
        • Lucas S.C.
        • Alexander D.R.
        • Cantrell D.A.
        Biochem. J. 1990; 190: 407-413
        • Jackson T.R.
        • Stephens L.R.
        • Hawkins P.T.
        J. Biol. Chem. 1992; 267: 16627-16636
        • Whitman M.
        • Kaplan D.R.
        • Schaffhausen B.
        • Cantley L.
        • Roberts T.
        Nature. 1985; 315: 239-242
        • Cooper J.A.
        Carraway K.L. Carraway C.A.C. The Cytoskeleton: A Practical Approach. Oxford University Press, New York1992: 47-71
        • Brown P.D.
        • Benya P.D.
        J. Cell Biol. 1988; 106: 171-179
        • Auger K.R.
        • Serunian L.A.
        • Soltoff S.P.
        • Libby P.
        • Cantley L.C.
        Cell. 1989; 57: 167-175
        • Boyce B.F.
        • Chen H.
        • Soriano P.
        • Mundy G.R.
        Bone. 1993; 14: 335-340
        • Burridge K.
        • Fath K.
        • Kelly T.
        • Nuckolls G.
        • Turner C.
        Annu. Rev. Cell Biol. 1988; 4: 487-525
        • Schaller M.
        • Parsons J.T.
        Curr. Opin. Cell. Biol. 1994; 6: 705-710
        • Richardson A.
        • Parsons T.
        Nature. 1996; 380: 538-540
        • Schwartz M.A.
        • Schaller M.D.
        • Ginsberg M.H.
        Annu. Rev. Cell Biol. 1995; 11: 549-600
        • Guan J.L.
        • Trevithick J.
        • Hynes R.O.
        Cell Regul. 1991; 2: 951-964
        • Bockholt S.M.
        • Burridge K.
        J. Biol. Chem. 1993; 268: 14565-14567
        • Schlaepfer D.D.
        • Hunter T.
        Mol. Cell. Biol. 1996; 16: 5623-5633
        • Chong L.D.
        • Traynor-Kaplan A.
        • Bokoch G.M.
        • Schwartz M.A.
        Cell. 1994; 79: 507-513
        • Chang J.H.
        • Gill S.
        • Settleman J.
        • Parsons S.J.
        J. Cell Biol. 1995; 130: 355-368
        • Fincham V.J.
        • Unlu M.
        • Brunton V.G.
        • Pitts J.D.
        • Wyke J.A.
        • Frame M.C.
        J. Cell. Biol. 1996; 135: 1551-1564
        • Suda T.
        • Takahashi N.
        • Martin T.J.
        Endocr. Rev. 1992; 13: 66-80
        • Hall T.J.
        • Schaeublin M.
        • Missbach M.
        Biochem. Biophys. Res. Commun. 1994; 199: 1237-1244
        • Hawkins P.T.
        • Jackson T.R.
        • Stephens L.R.
        Nature. 1992; 358: 157-159
        • DeCorte V.
        • Gettemans J.
        • Vandekerckhove J.
        FEBS Lett. 1997; 401: 191-196
        • Rameh L.E.
        • Chen C.S.
        • Cantley L.C.
        Cell. 1995; 83: 821-830