Advertisement

Regulatory Role for Src and Phosphatidylinositol 3-Kinase in Initiation of Fibronectin Matrix Assembly*

  • Iwona Wierzbicka-Patynowski
    Affiliations
    Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544-1014
    Search for articles by this author
  • Jean E. Schwarzbauer
    Correspondence
    To whom correspondence should be addressed. Tel.: 609-258-2893; Fax: 609-258-1035
    Affiliations
    Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544-1014
    Search for articles by this author
  • Author Footnotes
    * This work was supported by National Institutes of Health Training Grants T32 CA09528-16 (to I. W.-P.) and R01 GM 59383 (to J. E. S.). 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 23, 2002DOI:https://doi.org/10.1074/jbc.M200270200
      Fibronectin (FN) matrix assembly is a tightly regulated stepwise process that is initiated by interactions between FN and cell surface integrin receptors. These interactions activate many intracellular signaling pathways that regulate processes such as cell adhesion, migration, and survival. Here we demonstrate that cells lacking Src family kinases showed reduced ability to assemble FN fibrils as detected by immunofluorescence and by analysis of detergent extracts. The amount of FN matrix was further reduced by treatment with the phosphatidylinositol 3 (PI 3-kinase) inhibitor, wortmannin. CHOα5 cells, which are dependent on exogenous FN to initiate fibril formation, also showed significant reductions in matrix when treated with inhibitors of Src and PI 3-kinase. Combination of both inhibitors showed an additive inhibitory effect on assembly, which was concomitant with a loss of focal adhesion kinase phosphorylation. Decreased binding of the 70-kDa amino-terminal FN fragment at matrix assembly sites further supports a role for these kinases early during the process. We propose that these two signaling molecules, which lie downstream of integrins and focal adhesion kinase, are essential for efficient initiation of FN matrix assembly.
      The extracellular matrix (ECM)
      The abbreviations used are: ECM
      extracellular matrix
      FN
      fibronectin
      PI 3-kinase
      phosphatidylinositol 3-kinase
      CHO
      Chinese hamster ovary
      FAK
      focal adhesion kinase
      SYF cells
      cells lacking Src family kinases
      Rh-70 kDa
      rhodamine-labeled 70-kDa fragment
      PBS
      phosphate-buffered saline
      pFN
      plasma FN
      MAP
      mitogen-activated protein
      MEK
      MAP kinase
      ERK
      extracellular signal-regulated kinase
      1The abbreviations used are: ECM
      extracellular matrix
      FN
      fibronectin
      PI 3-kinase
      phosphatidylinositol 3-kinase
      CHO
      Chinese hamster ovary
      FAK
      focal adhesion kinase
      SYF cells
      cells lacking Src family kinases
      Rh-70 kDa
      rhodamine-labeled 70-kDa fragment
      PBS
      phosphate-buffered saline
      pFN
      plasma FN
      MAP
      mitogen-activated protein
      MEK
      MAP kinase
      ERK
      extracellular signal-regulated kinase
      surrounds cells and dynamically regulates cellular functions such as adhesion, migration, growth, and differentiation. Composition of the matrix varies from tissue to tissue; however, FN is a major component of most matrices (

      Mosher, D. F. (ed) (1989) Fibronectin, Academic Press, New York

      ,
      • Hynes R.O.
      ). FN influences diverse processes including inflammation, wound repair, malignant metastasis, microorganism attachment, and thrombosis. It does this not as a soluble protein but as a major component of a fibrillar network. FN is assembled into fibrils through a regulated stepwise process (
      • Schwarzbauer J.E.
      • Sechler J.L.
      ). The initiation of matrix assembly depends on interaction of FN with its cell surface receptor α5β1 integrin. FN binding induces integrin interactions with the actin cytoskeleton inside the cell, whereas addition of FN dimers to growing multimers results in elongation of fibrils outside. Fibrils are then gradually converted into detergent-insoluble stable matrix.
      Integrin-mediated interactions with FN-coated substrates induce reorganization of the actin cytoskeleton and associated proteins and lead to the formation of focal adhesions (
      • Geiger B.
      • Bershadsky A.
      • Pankov R.
      • Yamada K.M.
      ). These sites contain a multitude of cytoskeletal and adapter proteins such as vinculin, paxillin, and talin as well as signal transduction molecules such as focal adhesion kinase (FAK), Src family kinases, and protein kinase C. Many of these focal adhesion components are multidomain molecules that can interact with several distinct partners. Focal adhesions have been shown to serve both structural and functional roles as the sites where activation of intracellular pathways takes place. Some of these pathways have been implicated in FN matrix assembly. For example, treatment of fibroblasts with phorbol esters or other protein kinase C activators resulted in increased FN binding to the cell surface (
      • Somers C.E.
      • Mosher D.F.
      ). An FN fragment containing the first type III repeat, which can modify FN matrix assembly (
      • Hocking D.C.
      • Smith R.K.
      • McKeown-Longo P.J.
      ,
      • Mercurius K.O.
      • Morla A.O.
      ), activates ERK/MAP kinase signaling (
      • Mercurius K.O.
      • Morla A.O.
      ) and affects vascular smooth muscle cell proliferation (
      • Mercurius K.O.
      • Morla A.O.
      ,
      • Bourdoulous S.
      • Orend G.
      • MacKenna D.A.
      • Pasqualini R.
      • Ruoslahti E.
      ).
      Interaction of FN with integrins also results in activation of FAK, which then binds to the signaling proteins Src (
      • Schaller M.D.
      • Parsons J.T.
      ,
      • Xing Z.
      • Chen H.C.
      • Nowlen J.K.
      • Taylor S.J.
      • Shalloway D.
      • Guan J.L.
      ), PI 3-kinase (
      • Chen H.C.
      • Appeddu P.A.
      • Isoda H.
      • Guan J.L.
      ), and the Grb7 adapter protein (
      • Han D.C.
      • Guan J.L.
      ). Src associates with FAK through direct interaction of its Src homology (SH)2 domain with the major autophosphorylation site Tyr-397 on FAK. Association of Src with FAK is believed to be important for reciprocal activation of those two kinases (
      • Guan J.L.
      ). Furthermore, Src binding to FAK leads to phosphorylation of additional tyrosine residues on FAK that not only create new binding sites but also increase FAK catalytic activity (
      • Calalb M.B.
      • Polte T.R.
      • Hanks S.K.
      ). Src/FAK complex formation results in phosphorylation of other proteins such as paxillin, tensin, and p130cas. The Tyr-397 site on FAK also serves as a binding site for PI 3-kinase. Integrin-induced PI 3-kinase association with FAK has been demonstrated in platelets (
      • Guinebault C.
      • Payrastre B.
      • Racaud-Sultan C.
      • Mazarguil H.
      • Breton M.
      • Mauco G.
      • Plantavid M.
      • Chap H.
      ) and fibroblasts (
      • Chen H.C.
      • Guan J.L.
      ).
      We have demonstrated previously that a mutant FN, which forms a structurally altered FN matrix, inhibited phosphorylation of FAK and blocked cell cycle progression (
      • Sechler J.L.
      • Schwarzbauer J.E.
      ). The role of signaling effectors downstream of FAK in the assembly of FN into fibrils has not been extensively studied. Here we investigated the requirement for active Src and PI 3-kinase in FN matrix assembly. We demonstrate that both Src and PI 3-kinase regulate early stages of matrix assembly, suggesting that FAK acts through multiple pathways to regulate matrix formation.

      DISCUSSION

      In this report, we show using two different cell systems that inhibition of Src by mutation of Src family kinase genes in SYF cells or with Src-specific inhibitors in CHOα5 cells results in significant reduction of FN matrix assembly. A further reduction in matrix was seen with concomitant inhibition of PI 3-kinase activity. The major effects of blocking kinase action occurred early during fibril formation and could be demonstrated by detergent insolubility as well as by monitoring development of matrix assembly sites with 70-kDa binding. Both Src and PI 3-kinase lie immediately downstream of FAK. Inhibition of MAP kinase signaling, a third pathway downstream of FAK, had no effect on initiation of fibril formation. Therefore, we propose that a subset of signaling pathways activated by FAK are essential for proper initiation of FN matrix assembly.
      Src family kinases govern many cellular functions such as growth factor-induced proliferation and gene expression, ECM-promoted adhesion, spreading, migration, and protection from apoptosis (
      • Brown M.T.
      • Cooper J.A.
      ,
      • Lowell C.A.
      • Soriano P.
      ,
      • Parsons J.T.
      • Parsons S.J.
      ,
      • Thomas S.M.
      • Brugge J.S.
      ). Mutation of three ubiquitous members (Src, Yes, and Fyn) leads to severe developmental defects and lethality by mid-gestation. Interestingly, the SYF triple mutant embryonic defects resemble those of FN-null embryos, suggesting overlapping functions during development (
      • Klinghoffer R.A.
      • Sachsenmaier C.
      • Cooper J.A.
      • Soriano P.
      ,
      • George E.L.
      • Georges-Labouesse E.N.
      • Patel-King R.S.
      • Rayburn H.
      • Hynes R.O.
      ). SYF cells are able to adhere to FN substrata but show dramatically reduced FN-dependent tyrosine phosphorylation (
      • Klinghoffer R.A.
      • Sachsenmaier C.
      • Cooper J.A.
      • Soriano P.
      ). Similarly, we found that FAK phosphorylation was ablated in SYF cells during matrix assembly and that this reduction in phosphorylation correlated with decreased fibril formation. In contrast, SYF cells overexpressing c-Src showed FN-induced tyrosine phosphorylation of focal adhesion proteins (including FAK) as well as normal levels of FN fibrils. These findings provide evidence for a link between Src kinase activity and FN matrix assembly.
      PI 3-kinase phosphorylates phosphatidylinositol and together with its lipid derivatives acts as a second messenger in a variety of signaling pathways including cell survival through activation of PKB/Akt (
      • King W.G.
      • Mattaliano M.D.
      • Chan T.O.
      • Tsichlis P.N.
      • Brugge J.S.
      ,
      • Khwaja A.
      • Rodriguez-Viciana P.
      • Wennstrom S.
      • Warne P.H.
      • Downward J.
      ,
      • Frisch S.M.
      • Vuori K.
      • Kelaita D.
      • Sicks S.
      ,
      • Kennedy S.G.
      • Wagner A.J.
      • Conzen S.D.
      • Jordan J.
      • Bellacosa A.
      • Tsichlis P.N.
      • Hay N.
      ) and cell migration (
      • Shaw L.M.
      • Rabinovitz I.
      • Wang H.H.
      • Toker A.
      • Mercurio A.M.
      ,
      • Reiske H.R.
      • Kao S.C.
      • Cary L.A.
      • Guan J.L.
      • Lai J.F.
      • Chen H.C.
      ,
      • Kundra V.
      • Escobedo J.A.
      • Kazlauskas A.
      • Kim H.K.
      • Rhee S.G.
      • Williams L.T.
      • Zetter B.R.
      ,
      • Wennstrom S.
      • Siegbahn A.
      • Yokote K.
      • Arvidsson A.K.
      • Heldin C.H.
      • Mori S.
      • Claesson-Welsh L.
      ). PI 3-kinase binds to the same site on FAK as Src does (
      • Chen H.C.
      • Appeddu P.A.
      • Isoda H.
      • Guan J.L.
      ) and has been shown to associate with FAK upon integrin activation in platelets (
      • Guinebault C.
      • Payrastre B.
      • Racaud-Sultan C.
      • Mazarguil H.
      • Breton M.
      • Mauco G.
      • Plantavid M.
      • Chap H.
      ) and fibroblasts (
      • Chen H.C.
      • Guan J.L.
      ). Our results indicate that PI 3-kinase also regulates FN fibril formation. In combination, inhibition of both Src and PI 3-kinase dramatically reduced FN matrix assembly. At 2 h the effect was twice as potent as with either inhibitor used alone, suggesting that the effects are additive. Interestingly, however, we consistently observed that Src activity was required at the earliest times tested, although PI 3-kinase inhibition had its major impact after 2 h. This suggests that although both kinases participate in this process, their roles are not identical.
      A major mechanism of activation of Src and PI 3-kinase is through binding to phosphorylated FAK in response to integrin ligation by FN (
      • Schwartz M.A.
      ,
      • Zhao J.H.
      • Guan J.L.
      ,
      • Shen T.L.
      • Guan J.L.
      ). FAK itself has been implicated in regulatory responses to FN matrix in that alterations in FN matrix structure modulate the level of FAK tyrosine phosphorylation (
      • Sechler J.L.
      • Schwarzbauer J.E.
      ). In addition, FAK-null cells show a dramatically reduced ability to assemble FN matrix
      D. Ilic and C. Damsky, manuscript in preparation; I. Wierzbicka-Patynowski, unpublished data.
      although the ability to attach to immobilized FN is only slightly impaired (
      • Ilic D.
      • Furuta Y.
      • Kanazawa S.
      • Takeda N.
      • Sobue K.
      • Nakatsuji N.
      • Nomura S.
      • Fujimoto J.
      • Okada M.
      • Yamamoto T.
      ). One plausible model for regulation of integrin-mediated FN fibril initiation is that active FAK recruits Src and PI 3-kinase, which in turn transduce downstream signals required to initiate and maintain propagation of FN fibrils. Inhibition of any one of these kinases, either through mutation or with specific inhibitors, reduces the extent of fibril formation. There is evidence that association of Src with FAK may further stimulate both kinases (
      • Guan J.L.
      ). Similarly, the inhibition of PI 3-kinase leads to partial inhibition of FAK tyrosine phosphorylation in COS7 cells (
      • King W.G.
      • Mattaliano M.D.
      • Chan T.O.
      • Tsichlis P.N.
      • Brugge J.S.
      ). These observations suggest that there may be a feedback loop between FAK, Src, and PI 3-kinase that results in mutual activation of these signaling components through direct interactions. Both Src and PI 3-kinase bind to phosphorylated Tyr-397 on FAK, presenting an attractive idea that cells require phosphorylation of this tyrosine to regulate matrix assembly.
      The initial interaction of FN dimers with cell surface receptors leads to receptor clustering, which not only promotes FN self-association but also connects FN to the actin cytoskeleton. Both an intact cytoskeleton and the β integrin cytoplasmic domain are required for FN matrix assembly (
      • Solowska J.
      • Guan J.L.
      • Marcantonio E.E.
      • Trevithick J.E.
      • Buck C.A.
      • Hynes R.O.
      ,
      • Wu C.
      • Kevins V.
      • O'Toole T.E.
      • McDonald J.A.
      • Ginsberg M.H.
      ). Choquet et al. (
      • Choquet D.
      • Felsenfeld D.P.
      • Sheetz M.P.
      ) showed that initial connections between FN, α5β1 integrin, and actin filaments are relatively weak in adherent cells but become reinforced in response to applied force. This strengthening mechanism appears to require phosphoproteins, although the specific components have not been reported. It seems likely that reinforcement of cell-FN connections is also required during fibril assembly. FAK, Src, and/or PI 3-kinase may contribute to reinforcement of transmembrane connections because integrins assemble FN fibrils and may thus represent some of the necessary phosphoproteins. We did not observe any major effects of Src and PI 3-kinase inhibitors on actin stress fibers, but it remains possible that these kinases exert effects by strengthening existing links between integrins and actin.
      The deposition of FN into the ECM is a complex, integrin-dependent, and highly regulated process. The ECM has important effects on cell morphology, growth, and gene expression. Defects in matrix organization contribute to disease and developmental defects. Therefore, it is important to understand regulation of FN matrix formation. The data presented here demonstrate that Src and PI 3-kinase, two downstream effectors of FAK, are important in this regulation, particularly during initiation. Assembly is a multistep process, and other signaling molecules have been implicated in both early and late stages, further demonstrating the complexities of regulating FN fibril formation. It remains possible that other signaling molecules downstream of FAK, such as Grb7 (
      • Han D.C.
      • Shen T.L.
      • Guan J.L.
      ) or the γ1 isoform of phospholipase C (
      • Zhang X.
      • Chattopadhyay A.
      • Ji Q.S.
      • Owen J.D.
      • Ruest P.J.
      • Carpenter G.
      • Hanks S.K.
      ), may also participate. Clearly, further investigation is needed to sort out the intracellular components that control each step of FN matrix assembly.

      ACKNOWLEDGEMENTS

      We thank Drs. Hisaaki Kawakatsu and Dusko Ilic from UCSF for helpful discussions and Nedra Guckert for technical assistance.

      REFERENCES

      1. Mosher, D. F. (ed) (1989) Fibronectin, Academic Press, New York

        • Hynes R.O.
        Fibronectins. Springer-Verlag, New York1990
        • Schwarzbauer J.E.
        • Sechler J.L.
        Curr. Opin. Cell Biol. 1999; 11: 622-627
        • Geiger B.
        • Bershadsky A.
        • Pankov R.
        • Yamada K.M.
        Nat. Rev. Mol. Cell. Biol. 2001; 2: 793-805
        • Somers C.E.
        • Mosher D.F.
        J. Biol. Chem. 1993; 268: 22277-22280
        • Hocking D.C.
        • Smith R.K.
        • McKeown-Longo P.J.
        J. Cell Biol. 1996; 133: 431-444
        • Mercurius K.O.
        • Morla A.O.
        Circ. Res. 1998; 82: 548-556
        • Mercurius K.O.
        • Morla A.O.
        BMC Cell Biol. 2001; 2: 18-30
        • Bourdoulous S.
        • Orend G.
        • MacKenna D.A.
        • Pasqualini R.
        • Ruoslahti E.
        J. Cell Biol. 1998; 143: 267-276
        • Schaller M.D.
        • Parsons J.T.
        Curr. Opin. Cell Biol. 1994; 6: 705-710
        • Xing Z.
        • Chen H.C.
        • Nowlen J.K.
        • Taylor S.J.
        • Shalloway D.
        • Guan J.L.
        Mol. Biol. Cell. 1994; 5: 413-421
        • Chen H.C.
        • Appeddu P.A.
        • Isoda H.
        • Guan J.L.
        J. Biol. Chem. 1996; 271: 26329-26334
        • Han D.C.
        • Guan J.L.
        J. Biol. Chem. 1999; 274: 24425-24430
        • Guan J.L.
        Int. J. Biochem. Cell Biol. 1997; 29: 1085-1096
        • Calalb M.B.
        • Polte T.R.
        • Hanks S.K.
        Mol. Cell. Biol. 1995; 15: 954-963
        • Guinebault C.
        • Payrastre B.
        • Racaud-Sultan C.
        • Mazarguil H.
        • Breton M.
        • Mauco G.
        • Plantavid M.
        • Chap H.
        J. Cell Biol. 1995; 129: 831-842
        • Chen H.C.
        • Guan J.L.
        Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10148-10152
        • Sechler J.L.
        • Schwarzbauer J.E.
        J. Biol. Chem. 1998; 273: 25533-25536
        • Sechler J.L.
        • Takada Y.
        • Schwarzbauer J.E.
        J. Cell Biol. 1996; 134: 573-583
        • Engvall E.
        • Ruoslahti E.
        Int. J. Cancer. 1977; 20: 1-5
        • Aguirre K.M.
        • McCormick R.J.
        • Schwarzbauer J.E.
        J. Biol. Chem. 1994; 269: 27863-27868
        • Klinghoffer R.A.
        • Sachsenmaier C.
        • Cooper J.A.
        • Soriano P.
        EMBO J. 1999; 18: 2459-2471
        • King W.G.
        • Mattaliano M.D.
        • Chan T.O.
        • Tsichlis P.N.
        • Brugge J.S.
        Mol. Cell. Biol. 1997; 17: 4406-4418
        • Shaw L.M.
        • Rabinovitz I.
        • Wang H.H.
        • Toker A.
        • Mercurio A.M.
        Cell. 1997; 91: 949-960
        • Reiske H.R.
        • Kao S.C.
        • Cary L.A.
        • Guan J.L.
        • Lai J.F.
        • Chen H.C.
        J. Biol. Chem. 1999; 274: 12361-12366
        • Wymann M.P.
        • Bulgarelli-Leva G.
        • Zvelebil M.J.
        • Pirola L.
        • Vanhaesebroeck B.
        • Waterfield M.D.
        • Panayotou G.
        Mol. Cell. Biol. 1996; 16: 1722-1733
        • Hanke J.H.
        • Gardner J.P.
        • Dow R.L.
        • Changelian P.S.
        • Brissette W.H.
        • Weringer E.J.
        • Pollok B.A.
        • Connelly P.A.
        J. Biol. Chem. 1996; 271: 695-701
        • Kwon H.J.
        • Yoshida M.
        • Fukui Y.
        • Horinouchi S.
        • Beppu T.
        Cancer Res. 1992; 52: 6926-6930
        • Vlahos C.J.
        • Matter W.F.
        • Hui K.Y.
        • Brown R.F.
        J. Biol. Chem. 1994; 269: 5241-5248
        • Schwarzbauer J.E.
        J. Cell Biol. 1991; 113: 1463-1473
        • Dzamba B.J.
        • Bultmann H.
        • Akiyama S.K.
        • Peters D.M.
        J. Biol. Chem. 1994; 269: 19646-19652
        • Sottile J.
        • Wiley S.
        J. Biol. Chem. 1994; 269: 17192-17198
        • Brown M.T.
        • Cooper J.A.
        Biochim. Biophys. Acta. 1996; 1287: 121-149
        • Lowell C.A.
        • Soriano P.
        Genes Dev. 1996; 10: 1845-1857
        • Parsons J.T.
        • Parsons S.J.
        Curr. Opin. Cell Biol. 1997; 9: 187-192
        • Thomas S.M.
        • Brugge J.S.
        Annu. Rev. Cell Dev. Biol. 1997; 13: 513-609
        • George E.L.
        • Georges-Labouesse E.N.
        • Patel-King R.S.
        • Rayburn H.
        • Hynes R.O.
        Development. 1993; 119: 1079-1091
        • Khwaja A.
        • Rodriguez-Viciana P.
        • Wennstrom S.
        • Warne P.H.
        • Downward J.
        EMBO J. 1997; 16: 2783-2793
        • Frisch S.M.
        • Vuori K.
        • Kelaita D.
        • Sicks S.
        J. Cell Biol. 1996; 135: 1377-1382
        • Kennedy S.G.
        • Wagner A.J.
        • Conzen S.D.
        • Jordan J.
        • Bellacosa A.
        • Tsichlis P.N.
        • Hay N.
        Genes Dev. 1997; 11: 701-713
        • Kundra V.
        • Escobedo J.A.
        • Kazlauskas A.
        • Kim H.K.
        • Rhee S.G.
        • Williams L.T.
        • Zetter B.R.
        Nature. 1994; 367: 474-476
        • Wennstrom S.
        • Siegbahn A.
        • Yokote K.
        • Arvidsson A.K.
        • Heldin C.H.
        • Mori S.
        • Claesson-Welsh L.
        Oncogene. 1994; 9: 651-660
        • Schwartz M.A.
        Trends Cell Biol. 2001; 11: 466-470
        • Zhao J.H.
        • Guan J.L.
        Prog. Mol. Subcell. Biol. 2000; 25: 37-55
        • Shen T.L.
        • Guan J.L.
        FEBS Lett. 2001; 499: 176-181
        • Ilic D.
        • Furuta Y.
        • Kanazawa S.
        • Takeda N.
        • Sobue K.
        • Nakatsuji N.
        • Nomura S.
        • Fujimoto J.
        • Okada M.
        • Yamamoto T.
        Nature. 1995; 377: 539-544
        • Solowska J.
        • Guan J.L.
        • Marcantonio E.E.
        • Trevithick J.E.
        • Buck C.A.
        • Hynes R.O.
        J. Cell Biol. 1989; 109: 853-861
        • Wu C.
        • Kevins V.
        • O'Toole T.E.
        • McDonald J.A.
        • Ginsberg M.H.
        Cell. 1995; 83: 715-724
        • Choquet D.
        • Felsenfeld D.P.
        • Sheetz M.P.
        Cell. 1997; 88: 39-48
        • Han D.C.
        • Shen T.L.
        • Guan J.L.
        J. Biol. Chem. 2000; 275: 28911-28917
        • Zhang X.
        • Chattopadhyay A.
        • Ji Q.S.
        • Owen J.D.
        • Ruest P.J.
        • Carpenter G.
        • Hanks S.K.
        Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9021-9026