Integrin signaling

seminars in

CANCER BIOLOGY, Vol 7, 1996: pp 111–118

Integrin signaling Susan E. LaFlamme and Kelly L. Auer

Integrins regulate the formation of signaling complexes

Integrins are a family of adhesion receptors used by cells to interact with their extracellular matrix. Integrins also function as signaling receptors, integrating information from the extracellular matrix and other environmental cues including growth factors and hormones. Signals triggered by integrins direct cell adhesion and regulate other aspects of cell behavior including cell proliferation and differentiation, and determine cell survival. The biochemical pathways initiated by integrin engagement are now being identified.

In many cells in culture, integrin-mediated adhesion often results in the formation of specialized adhesion sites, called focal adhesions.2 Both structural and signaling proteins, such as integrins, cytoskeletal proteins, and kinases have been shown to be concentrated at these sites, including the cytoskeletal proteins α-actinin, vinculin, talin, tensin and paxillin, as well as the protein kinases, the focal adhesion kinase (FAK), cSrc, and protein kinase C (PKC). Because of their composition, focal adhesions are believed to function as adhesion-dependent ‘signal transduction organelles’.2 An early event during integrin signaling is the tyrosine phosphorylation of the non-receptor tyrosine kinase, FAK in response either to integrin-mediated cell adhesion or to integrin clustering with antiintegrin antibodies.3,4 The phosphorylation of FAK is believed to initiate a cascade of phosphorylation events and new protein interactions required for adhesion and formation of adhesion-dependent signaling complexes. A major site of FAK phosphorylation is its autophosphorylation site, tyrosine 397. Mutation of this tyrosine to phenylalanine, however, has only a modest effect on FAK’s catalytic activity, suggesting that additional mechanisms may exist to regulate FAK.3,4 Recent evidence suggests that one of these mechanisms may involve Src.5 The autophosphorylation of FAK generates a binding site for the Src homology -2(SH2) domain of Src and Src-family kinases.3,4 Adhesion-dependent complexes containing FAK and Src have been identified.6 Since Src’s SH2 domain is involved in regulating its own activity, the association of FAK and Src may result in Src activation.7 It has recently been shown that, in addition to its autophosphorylation site, FAK also becomes phosphorylated at four additional tyrosine residues. Tyrosines 576 and 577 are contained within FAK’s catalytic domain, and similar to the activation of other tyrosine kinases the phosphorylation of these two residues increases FAK’s catalytic activity.5 Tyrosine 407 and an unidentified tyrosine residue in the C-terminal

Key words: apoptosis / gene expression / integrins / proliferation / signal transduction ©1996 Academic Press Ltd

THE INTERACTION OF CELLS with the extracellular matrix (ECM) affects many aspects of cellular behavior. Integrins are the major class of receptors used by cells to bind to the ECM.1 Integrins are heterodimeric transmembrane receptors that form structural links between the ECM and the cytoskeleton, thereby mediating cell attachment, spreading, migration and fibronectin matrix assembly. It is now known that integrins can also interact with the cellular signaltransduction apparatus. Engagement of integrins during cell adhesion specifically results in release of lipid second messengers, activation of protein kinases, and changes in intracellular pH and Ca2 + , and this has provided a mechanistic link between ECM and the regulation of cell behavior. Integrin signaling regulates cell proliferation, differentiation and survival, as well as cell adhesion. This review will focus on recent advances in the understanding of how ECMtriggered integrin signaling events regulate cell behavior.

From the Department of Physiology and Cell Biology, Albany Medical College, 47 New Scotland Avenue, Albany, NY 12208, USA ©1996 Academic Press Ltd 1044-579X/96/030111 + 08$18.00/0

111

S. E. LaFlamme and K. L. Auer binding sites for SH2-containing proteins. Both Csk and the adaptor protein p47gag-crk can bind to phosphorylated paxillin via their SH2 domains.11,18 The interaction of Crk’s SH3 domain with the guanine nucleotide exchange protein, C3G, provides a pathway by which integrin-triggered signaling may lead to the activation of Ras,19 which is known to be important in regulation growth and differentiation.20 Although many protein–protein interactions involving focal adhesion proteins have been identified (Figure 1), important questions remain. The initial cytoplasmic protein interactions involving integrin receptors themselves need to be identified. The focal adhesion proteins talin, α-actinin,21 paxillin, and FAK22 have all been demonstrated to bind to peptides modeled on the integrin-β1 intracellular domain; however, none of these proteins have been shown to coprecipitate with integrins, making it difficult to determine whether these or unidentified proteins interaction with integrins to initiate signaling. Recently, clustering unoccupied β1 integrins on cells with anti-integrin antibodies attached to polystyrene beads has been shown to co-cluster FAK and tensin, suggesting that they participate in the initial protein interactions involving integrins.23 Since peptides modeled on β intracellular domains can bind to

domain of FAK also become phosphorylated in response to adhesion and these phosphorylated residues may provide binding sites for additional proteins containing SH2 domains.5 Since the integrin-triggered autophosphorylation of FAK can lead to its association with Src and possibly to Src activation. Src or Src-family kinases may be responsible for the further phosphorylation and activation of FAK. Consistent with this possibility, Src has been found to tyrosine phosphorylate FAK in vitro.5 These results suggest that FAK and Src may become activated by the adhesion-dependent formation of a ‘bipartite kinase complex’,4 which complicates the determination of the relative contributions of FAK and Src to adhesiondependent phosphorylation in vivo. In addition to FAK phosphorylation, integrinmediated adhesion also results in an increase in tyrosine phosphorylation of the cytoskeletal proteins, paxillin, tensin and p130.8-10 These adhesion-induced phosphorylations are believed to be important for integrin signaling, since the formation of focal adhesions is inhibited by the addition of pharmacological inhibitors of tyrosine phosphorylation.8 A number of new protein–protein interactions are induced by these tyrosine phosphorylation events. In addition to Src, phosphorylated FAK also binds to the SH2 domain of the carboxyl-terminal Src kinase (Csk), which negatively regulates Src activity,11 as well as to the SH2 domain of phosphatidylinositol 3-kinase (PI-3 kinase), which is activated during growth regulation.12 The interaction between FAK and PI-3 kinase probably occurs away from FAK’s autophosphorylation site.12 In addition to these signaling proteins, FAK has also been found to bind to the cytoskeletal proteins, paxillin and talin.13,14 The functional significance of these interactions has yet to be determined. Paxillin also appears to play a central role in the formation of adhesion-triggered signaling complexes. Paxilin was initially identified as a focal adhesion protein that could bind to vinculin.15 Recently, it has been shown to associate with signaling proteins. In addition to FAK, paxillin can also bind to Src, and this interaction is mediated by Src’s SH3 domain.16 The association of paxillin and Src may be responsible for the localization of Src to focal adhesions, since this depends upon its SH3 domain.17 The interaction of Src’s SH3 domain with paxillin may also be involved in the regulation of Src’s catalytic activity.7 As discussed previously, paxillin becomes phosphorylated in response to integrin-mediated adhesion.8 The tyrosine phosphorylation of paxillin creates

Figure 1. Protein–protein interactions involving focal adhesion proteins.

112

Integrin signaling FAK,22 FAK may be bound to integrins prior to adhesion. Alternatively, FAK–integrin association may be triggered by integrin clustering during the adhesion process. The mechanism by which tensin coclusters with FAK and β1 integrins has not yet been identified; however, tensin may connect the integrinFAK complex to the actin cytoskeleton.2 In contrast to FAK and tensin, the co-clustering of additional focal adhesion proteins, including α-actinin, talin and paxillin, requires the clustering of ligand-occupied β1 integrins, indicating that additional signaling events are needed for their recruitment and suggesting that these events are downstream of integrin-FAK interactions.23

by adhesion is the generation of arachidonic acid. Cell adhesion over integrins signals the phospholipase A2-dependent release of arachidonic acid.31-33 This release is required for cell spreading and it has been suggested that here arachidonic acid is needed as part of a feedback amplification signal.31

Roles of integrin cytoplasmic domains in integrin signaling An important goal in understanding the mechanisms by which integrins function in signal transduction is to identify the protein–integrin interactions that mediate these signals. One obvious place to begin is to analyse the function of integrin intracellular domains in signaling specific events, and then to identify those cytoplasmic proteins that interact with specific integrin intracellular domains to generate these signals. Integrin β-cytoplasmic domains have been demonstrated to be both required and sufficient for signaling the increase in tyrosine phosphorylation of FAK.34-36 Analysis of the ability of recombinant α5β1 fibronectin receptors lacking either an α- or β-cytoplasmic domain to trigger FAK phosphorylation demonstrated that the β1 intracellular domain is required for this signaling event,34 whereas the α5 intracellular domain is not.37 Additionally, experiments using single-subunit chimeric integrins, containing either the interleukin-2 receptor or CD4 receptor as an extracellular reporter domain connected to various integrin cytoplasmic domains, have shown that the β1 intracellular domain is also sufficient for signaling FAK phosphorylation.35,36 Furthermore, the β1, β3 and β5 intracellular domains all contain sufficient information in their primary sequence to signal FAK phosphorylation; however, an isoform of the β3 intracellular domain, β3B, generated by alternative splicing does not.34 A similar splice variant, β1B in the β1 intracellular domain also fails to induce FAK phosphorylation.38 As previously mentioned, FAK binds in-vitro to peptides modeled on β intracellular domains. The amino acid sequence identified to bind FAK22 is present in both the β1B and β3B intracellular domains, suggesting either that the amino acids inserted by the alternative splice interfere with the ability of FAK to bind to the integrin in vivo, or replace an additional ‘clustering motif’ also required for FAK phosphorylation. Since sequences in this C-terminal region of the β intracellular domain are required for

Integrins trigger lipid second messengers Another early event triggered by integrins is the activation of lipid second-messenger pathways. Integrin ligation has been demonstrated to directly regulate the phosphatidylinositol pathway by activating the tyrosine kinase-dependent phospholipase C-γ (PLC-γ).24,25 PLC hydrolyses phosphatidylinositol biphosphate (PIP2) to inositol triphosphate and diacylglycerol which are second messengers involved in the regulation of intracellular Ca2 + and the activation of PKC. Although integrin-triggered cell adhesion has been shown to activate PKC,26 the mechanism of PKC activation and the specific isoform(s) involved have not been determined. Production of PIP2, the substrate for PLC, is also regulated by adhesion.27 This occurs by the activation of PIP 5-kinase via a pathway involving the small GTP binding protein, rho.28 The mechanism by which integrins activate rho is not known. However, the adhesion-dependent production of PIP2 may explain the need for adhesion as a co-signal in pathways requiring activation of PKC via this inositol hydrolysis pathway. For example, in fibroblasts, adhesion is required as a co-signal for the growth factor-triggered PKC-dependent changes in intracellular [Ca2 + ] and pH.29 Levels of PIP2 also regulate other cellular processes: it modulates the function of several actin binding proteins,30 suggesting a mechanism by which integrins may affect actin polymerization and assembly during cell adhesion. PIP2 is also the substrate for PI-3 kinase and is required in the activation of phospholipase D,30 suggesting that integrin signaling may also affect pathways involving these enzymes. Another lipid second-messenger pathway activated 113

S. E. LaFlamme and K. L. Auer Recombinant α2β1 receptors containing either the α2 or α5 intracellular domain mediated gel contraction, whereas recombinant receptors containing the α4 intracellular domain did not.42 However, receptors containing the α4 intracellular domain showed enhanced cell migration.42 Whether these functional differences are due to differences in integrin–cytoskeletal interactions or in integrin–signaling interactions is not yet known. In addition, it is not clear whether α-intracellular domains interact with cytoplasmic proteins directly, or whether they regulate the interactions mediated by β-intracellular domains. Some evidence suggests that α intracellular domains may in fact regulate the intracellular interactions of β-subunits. For example, β-intracellular domains expressed in the absence of their corresponding α-intracellular domain, either in the context of single-subunit chimeric receptors or in the context of a recombinant heterodimer integrin, target their extracellular domains to focal adhesions by a ligand-independent mechanism, and this ability is inhibited by the presence of an α-intracellular domain.21

binding to talin11,21 and signaling FAK phosphorylation,34,35,38 the interaction between talin and integrins may be required for FAK phosphorylation.11 However, integrins clustered with antibodies to β1 integrins that do not co-cluster talin signal the phosphorylation of a protein that may be FAK.23 As previously discussed, specific integrin-triggered signals are required for cell adhesion and cell spreading.26,33 It is possible that integrin intracellular domains may be required to generate these signals, since requirements for β-intracellular domains have been demonstrated in both cell adhesion and cell spreading.21 It is not known whether β-intracellular domains are required in these processes solely for their role in cytoskeletal linkage, or whether they have additional roles in signaling events regulating these processes. Interestingly, expression of single-subunit chimeras containing β-intracellular domains can function as dominant negative mutants and inhibit endogenous function in cell adhesion, cell spreading, cell migration and fibronectin matrix assembly.36,39,40 Some integrin-mediated processes were found to be more easily inhibited than others. For example, higher levels of expression of the single-subunit chimeric integrins were required to inhibit cell attachment compared to those required to inhibit cell spreading, and even lower levels inhibited matrix assembly, suggesting that different protein interactions may be involved in these processes.39 Consistent with this possibility, several cytoplasmic proteins have been found to interact with β-intracellular domains, α-actinin, talin,21 paxillin and FAK22 bind to β intracellular domains in vitro. Again, whether these dominant negative effects are due to the inhibition of specific integrin–cytoskeletal interactions, specific integrin-signaling interactions, or inappropriate integrin signaling is not yet known. In one case, cytoskeletal rearrangement triggered by lysophosphatic acid was required for the dominant negative effect.40 In another case, a single-subunit chimera containing a β3 intracellular domain inhibited α5β1-mediated phagocytosis, in a manner similar to ligation of an endogenous αvβ3. This effect was inhibited by pharmacological agents that block PKC activity, suggesting that β-intracellular domains may signal through PKC.41 Distinct roles for α-intracellular domains have been found in several integrin-mediated processes. For example, integrin α2β1 heterodimers in which the α2 intracellular domain was replaced with the α4 or α5 intracellular domains differ in their abilities to mediate cell migration and collagen gel contraction.42

Integrins regulate cell proliferation In addition to growth factors and nutrients, many normal cells require anchorage to their underlying ECM to proliferate.43 Integrin signaling has been shown to regulate cell-cycle progression. In fibroblasts, the requirement for adhesion maps to the G0/G1 transition and to mid-to-late G1 before the initiation of DNA synthesis.44,45 The signal at mid-tolate G1 requires post-integrin binding events including changes in cell shape, such as cell spreading.44,46 Since the mitogen-activated protein (MAP) kinases are known to regulate cell proliferation,47 and integrin-mediated adhesion and changes in cell shape are required for their activation,6,46,48,49 MAP kinases have been suggested to regulate this point in the cell cycle.46 Cyclin A expression, which is required for entry into S phase, is regulated by adhesion50 and by c-Myc,51 a transcription factor known to be regulated by MAP kinases.47 Consistent with their importance in pathways regulating adhesion-dependent cell growth, ectopic expression of either cyclin A or c-myc by transfection leads to anchorage-independent cell growth.50,51 The adhesion-dependent activation of MAP kinases appears to be important in the pathway by which integrins regulate cell proliferation. Adhesion may regulate the formation of signaling complexes that tie 114

Integrin signaling intracellular domain,21 suggesting these integrins may affect proliferation by a distinct pathway. Recently, the expression of an alternatively spliced isoform of the β1 subunit, β1C, was shown to inhibit proliferation.55 Expression of β1C inhibited both cyclin A expression and DNA synthesis.55 The β1C isoform contains a 48-amino acid insertion in place of the normal 21 C-terminal amino acids of the β1 intracellular domain. The ability of the β1C subunit to inhibit DNA synthesis required the C-terminal 13 amino acids of its intracellular domain.55 Therefore, β-intracellular domains can have both stimulatory and inhibitory effects on proliferation.

Figure 2. Integrin engagement to the MAP kinase pathway.

Integrins regulate apoptosis Programmed cell death or apoptosis is a normal part of development, homeostasis, and the pathogenesis of some diseases.56 Adhesion to the extracellular matrix is required for growth and survival of many types of cells. Experimental evidence has now accumulated indicating that integrin engagement triggers signals that inhibit apoptosis.57-62 What these signals are is as yet not known: however, it appears that ligand occupancy of integrins is not sufficient to generate them; changes in cell shape, as occur during cell spreading, may also be required.58 In some instances, the integrin-triggered signal may involve the tyrosine phosphorylation of particular proteins, since treatment of cells with vanadate, phosphotyrosine phosphatase inhibitor, can overcome the requirement for adhesion.57 Prevention of apoptosis by the ECM is dependent upon the expression and function of particular integrin heterodimers and these requirements appear to be cell-type specific. Inhibition of αvβ3-function during angiogenesis with anti-αvβ3 antibodies both inhibited proliferation and induced apoptosis, whereas treatment with antibodies that block β1 integrin function did not.59 (see Marshall and Hart this issue). Similarly, prevention of interactions of mammary epithelium with the extracellular matrix by anti-β1 integrin antibodies both inhibited proliferation and induced apoptosis.61,62 The pro-proliferation and survival signal in this case may also require specific integrin heterodimers since it arises from specific ECM components: adhesion of mammary epithelial cells to Engelbreth-Holm-Swarm (EHS) matrix prevented apoptosis, whereas their adhesion to fibronectin or type I collagen did not.61 In mammary epithelial cells, engagement of specific β1 integrins

integrin engagement to the MAP kinase pathway via phosphorylation of FAK (Figure 2).6 As discussed previously, integrin-mediated adhesion signals the autophosphorylation of FAK on Tyr 397, generating a binding site on FAK for SH2 domains of Src and related kinases.3,4 The further phosphorylation of FAK at Tyr 925, presumably by Src, generates a binding site for the SH2 domain of the adaptor protein, GRB2.6 The involvement of GRB2 provides a pathway linking adhesion to the MAP-kinase pathway via Ras.20 GRB2 usually signals through the rasGDP/ GTP exchange protein SOS, which has also been found in the adhesion-dependent signaling complexes containing FAK, GRB2 and cSrc.6 MAP kinase in turn activates a number of transcription factors, including cMyc, that are involved in regulating growth and differentiation.47 In some cases, however, the requirement for FAK phosphorylation may be bypassed or potentiated with alternative integrintriggered pathways. For example, adhesion of insulinstimulated cells to vitronectin results in the association of αvβ3 with insulin receptor substrate-1 (IRS1) and increased proliferation.52 IRS1 also binds to GRB2 and can thereby connect to the MAP kinase pathway.52 Since integrin β-intracellular domains signal FAK phosphorylation, it is likely that they play a role in regulating cell proliferation. The β1-cytoplasmic domain has been demonstrated to stimulate cell proliferation.53 The β6 cytoplasmic domain has also been shown to have a growth stimulatory effect, which requires the 11-amino acid sequence at its carboxyl terminus.54 This sequence is not present in the β1 115

S. E. LaFlamme and K. L. Auer prevented apoptosis by inhibiting the expression of interleukin-1β converting enzyme (ICE), a known inducer of apoptosis in mammalian cells.61

lated transcription factors that interact at this site have not yet been identified.

Integrins regulate gene expression

Summary

There are many examples where ECM has been shown to affect gene expression.1,63 Here we describe three pathways in which a direct role for integrins has been established. Signaling in monocytes via α4β1 is known to regulate the production of inflammatory mediators such as IL-1 β.64 The signaling pathway from integrins to the induction of IL-1 β mRNA requires tyrosine phosphorylation.65 The major phosphorylated protein that is observed after integrin engagement in monocytes is a 76 kDa protein;65 however, it has not been established whether it is involved in the integrin-triggered production of IL1β. Integrin signaling has also been shown to regulate metalloproteinase expression. Production of metalloproteinases in synovial fibroblasts is regulated by opposing signals from integrins α5β1 and α4β1.66 These integrins bind to distinct domains on fibronectin; α5β1 to the RGDS-cell-binding domain and α4β1 to the CS1 region.63 Engagement of α5β1 activates transcription of the metalloproteinases; however, this signal is suppressed if α4β1 is also engaged.66 It is possible that fragments of fibronectin lacking CS1 are generated by proteolysis at sites of inflammation or during wound repair; such fragments would activate transcription of metalloproteinase genes. In contrast, intact fibronectin would suppress their expression. The complete cascade of signaling events by which integrins regulate metalloproteinase expression is not yet fully understood. However, it is known that the regulation of collagenase expression by integrins requires the induction of c-fos expression and the presence of AP1 and PEA3 response elements in the promoter of the collagenase gene.67 In some cases, coordinated signals from the ECM and other environmental cues are required for the tissue-specific regulation of gene expression. In mammary epithelial cells, signals from both β1 integrins and specific hormones are required to regulate β casein gene expression.68 Recently, it has been demonstrated that the interaction of β1-integrins with the E3 region of laminin is important in this regulation, suggesting a role for integrin α3β1.69 In addition, an ECM response element has been identified in the β-casein promoter.70 However, the integrin β1-regu-

Integrin-mediated adhesion triggers a variety of signaling pathways that regulate cell behavior. It is becoming increasingly clear that the signaling pathways triggered by integrins are intertwined with those triggered by growth factors and hormones in a complex network. Furthermore, signals generated by different integrin heterodimers on the same cell must also be integrated in order for the cell to respond appropriately to its extracellular environment. Although much has been learned recently about integrin signaling, important questions remain concerning the circuitry responsible for the crosstalk among signals triggered by different integrins, as well as the integration of signals from integrins and other environmental stimuli.

Acknowledgements We thank Drs Sottile, Vincent, Longo and Morse for their helpful comments during the preparation of this manuscript and Timothy Driscoll and Byron Olsen for their useful editorial suggestions. This work was supported in part by NIH grant GM51540 and AHA-NYS Affiliate grant #940-002 to SEL. KLA is supported by NIH grant T32-HL-07529.

References 1. Hynes RO (1992) Integrins: versatility, modulation, and signaling in cell adhesion. Cell 69:11-25 2. Lo SH, Chen LB (1994) Focal adhesion as a signal transduction organelle. Cancer Metastas Rev 13:9-24 3. Schaller MD, Parsons JT (1994) Focal adhesion kinase and associated proteins. Curr Opin Cell Biol 6:705-710 4. Richardson A, Parsons JT (1995) Signal transduction through integrins: a central role for focal adhesion kinase? BioEssays 17:229-236 5. Calalb MB, Polte TR, Hanks SK (1995) Tyrosine phosphorylation of focal adhesion kinase at sites in the catalytic domain regulates kinase activity: a role for Src family kinases. Mol Cell Biol 15:954-963 6. Schlaepfer DD, Hanks SK, Hunter T, van der Geer P (1994) Integrin-mediated signal transduction linked to ras pathway by GRB2 binding to the focal adhesion kinase. Nature 372:786-791 7. Thorsten E, Courtneidge SA (1995) Src family protein tyrosine kinases and cellular signal transduction pathways. Curr Opin Cell Biol 7:176-182

116

Integrin signaling 26. Vuori K, Ruoslahti E (1993) Activation of protein kinase C precedes α5β1 integrin-mediated cell spreading on fibronectin. J Biol Chem 268:21459-21462 27. McNamee HP, Ingber DE, Schwartz MA (1993) Adhesion of fibronectin stimulates inositol lipid synthesis and enhances PDGF-induced inositol lipid breakdown. J Cell Biol 121:673-678 28. Chong LD, Traynor-Kaplan A, Bokoch GM, Schwartz MA (1994) The small GTP-binding protein rho regulates a phosphatidylinositol 4-phosphate 5-kinase in mammalian cells. Cell 79:507-513 29. Schwartz MA (1994) Integrins as signal transducing receptors, in Integrins: Molecular and Biological Responses to the Extracellular Matrix (Cheresh DA, Mecham RP, eds) pp 33-48. Academic Press, New York 30. Lee SB, Rhee SG (1995) Significance of PIP2 hydrolysis and regulation of phospholipase C isozymes. Curr Opin Cell Biol 7:183-189 31. Chun J-S, Jacobson BS (1993) Requirement of diacylglycerol and protein kinase C in Hela cell-substratum adhesion and their feedback amplification of arachidonic acid production for optimum cell spreading. Mol Biol Cell 4:271-281 32. Cybulsky AV, Carbonetto S, Cyr MD, Mctavish AJ, Huang Q (1993) Extracellular matrix-stimulated phospholipase activation is mediated by β1-integrin. Am J Physiol 264:C323-332 33. Auer KL, Jacobson BS (1995) β1 integrins signal lipid second messengers during cell adhesions. Mol Biol Cell 6:1305-1313 34. Guan J-L, Trevithick JE, Hynes RO (1991) Fibronectin/integrin interaction induces tyrosine phosphorylation of a 120-kDa protein. Cell Regul 2:951-964 35. Akiyama SK, Yamada SS, Yamada KM, LaFlamme SE (1994) Transmembrane signal transduction by integrin cytoplasmic domains expressed in single-subunit chimeras. J Biol Chem 269:15961-15964 36. Lukashev ME, Sheppard D, Pytela R (1994) Disruption of integrin function and induction of tyrosine phosphorylation by the autonomously expressed β1 integrin cytoplasmic domain. J Biol Chem 269:18311-18314 37. Bauer JS, Varner J, Schreiner C, Kornberg L, Nickolas R, Juliano RL (1993) Functional role of the cytoplasmic domain of the integrin α5 subunit. J Cell Biol 122:209-221 38. Balzac F, Retta SF, Albini A, Melchiorri A, Koteliansky VE, Geuna M, Silengo L, Tarone G (1994) Expression of β1B integrin isoform in CHO cells results in a dominant negative effect on cell adhesion and motility. J Cell Biol 127:557-565 39. LaFlamme SE, Thomas LA, Yamada SS, Yamada KM (1994) Single-subunit chimeric integrins as mimics and inhibitors of endogenous integrin functions in receptor localization, cell spreading, and migration, and matrix assembly. J Cell Biol 126:1287-1298 40. Smilenov L, Briesewitz R, Marcantonio EE (1994) Integrin β1 cytoplasmic domain dominant negative effects revealed by lysophosphatidic acid treatment. Mol Biol Cell 5:1215-1223 41. Blystone SD, Lindberg FP, LaFlamme SE, Brown EJ (1995) Integrin β3 cytoplasmic tail is necessary and sufficient for regulation of α5β1 phagocytosis by αvβ3 and integrin-associated protein. J Cell Biol 130:745-754 42. Chan BMC, Kassner PD, Schiro JA, Byers HR, Kupper TS, Hemler ME (1992) Distinct cellular functions mediated by different VLA integrin α subunit cytoplasmic domains. Cell 68:1051-1060 43. Stoker M, O’Neill C, Berryman S, Waxman V (1968) Anchorage and growth regulation in normal and virus transformed cells. Int J Cancer 3:683-693 44. Hansen LK, Mooney DJ, Vacanti JP, Ingber DE (1994) Integrin binding and cell spreading on extracellular matrix act at different points in the cell cycle to promote hepatocyte growth. Mol Biol Cell 5:967-975

8. Burridge K, Turner CE, Romer LH (1992) Tyrosine phosphorylation of paxillin and pp125FAK accompanies cell adhesion to extracellular matrix: a role in cytoskeletal assembly. J Cell Biol 119:893-903 9. Bockholt SM, Burridge K (1993) Cell spreading on extracellular matrix proteins induces tyrosine phosphorylation of tensin. J Biol Chem 268:14565-14567 10. Petch LA, Bockholt SM, Bouton A, Parsons JT, Burridge K (1995) Adhesion-induced tyrosine phosphorylation of the p130 SRC substrate. J Cell Sci 108:1371-1379 11. Sabe H, Hata A, Okada M, Nakagawa H, Hanafusa H (1994) Analysis of binding of the src homology 2 domain of Csk to tyrosine-phosphorylated proteins in the suppression and mitotic activation of c-src. Proc Natl Acad Sci USA 91:3984-3988 12. Chen H-C, Guan J-L (1994) Association of focal adhesion kinase with its potential substrate phosphatidylinositol 3-kinase. Proc Natl Acad Sci USA 91:10148-10152 13. Hildebrand JD, Schaller MD, Parsons JT (1995) Paxillin, a tyrosine phosphorylated focal adhesion-associated protein binds to the carboxyl terminal domain of the focal adhesion kinase. Mol Biol Cell 6:637-647 14. Chen H-C, Appeddu PA, Parsons JT, Hildebrand JD, Schaller MD, Guan J-L (1995) Interaction of focal adhesion kinase with cytoskeletal protein talin. J Biol Chem 270:16995-17016 15. Turner CE, Glenney JT, Burridge K (1990) Paxillin: a new vinculin-binding protein present in focal adhesions. J Cell Biol 111:1059-1068 16. Weng Z, Taylor JA, Turner CE, Brugge JS, Seidel-Dugan C (1993) Detection of Src homology 3-binding proteins, including paxillin, in normal and v-Src-transformed Balb/c 3T3 cells. J Biol Chem 268:14956-14963 17. Kaplan KB, Bibbins KB, Swedlow JR, Arnaud M, Morgan DO, Varmus HE (1994) Association of the amino-terminal half of c-Src with focal adhesions alters their properties and is regulated by phosphorylation of tyrosine 527. EMBO J 13:4745-4556 18. Birge RB, Fajardo JE, Reichman C, Shoelson SE, Songyang Z, Cantley LC, Hanafusa H (1993) Identification and characterization of a high-affinity interaction between v-crk and tyrosinephosphorylated paxillin in CT10-transformed fibroblasts. Mol Cell Biol 13:4648-4656 19. Tanaka S, Morishita T, Hashimoto Y, Hattori S, Nakamura S, Shibuya M, Matuoka K, Takenawa T, Kurata T, Nagashima K, Matsuda M (1994) C3G, a guanine nucleotide-releasing protein expressed ubiquitously, binds to the Src homology 3 domains of CRK and GRB2/ASH proteins. Proc Natl Acad Sci USA 91:3443-3447 20. Burgering BMT, Bos JL (1995) Regulation of Ras-mediated signalling: more than one way to skin a cat. Trends Biochem Sci 20:18-22 21. Sastry SK, Horwitz AF (1993) Integrin cytoplasmic domains: mediators of cytoskeletal linkages and extra- and intracellular initiated transmembrane signaling. Curr Opin Cell Biol 5:819-831 22. Schaller MD, Otey CA, Hildebrand JD, Parsons JT (1995) Focal adhesion kinase and paxillin bind to peptides mimicking the β integrin cytoplasmic domains. J Cell Biol 130:1181-1187 23. Miyamoto S, Akiyama SK, Yamada KM (1995) Synergistic roles for receptor occupancy and aggregation in integrin transmembrane function. Science 267:883-885 24. Kanner SB, Grosmaire LS, Ledbetter JA, Damle NK (1993) β2-integrin LFA-1 signaling through phospholipase C-γ1 activation. Proc Natl Acad Sci USA 90:7099-7103 25. Daniel JL, Dangelmaier C, Smith JB (1992) Evidence for a role for tyrosine phosphorylation of phospholipase Cγ2 in collageninduced platelet cytosolic mobilization. Biochem J 302:617-622

117

S. E. LaFlamme and K. L. Auer 60. Frisch SM, Francis H (1994) Disruption of epithelial cell-matrix interactions induces apoptosis. J Cell Biol 124:619-626 61. Boudreau N, Sympson CJ, Werb Z, Bissell MJ (1995) Suppression of ICE and apoptosis in mammary epithelial cells by extracellular matrix. Science 267:891-893 62. Howlett AR, Bailey N, Damsky C, Petersen OW, Bissell MJ (1995) Cellular growth and survival are mediated by β1 integrins in normal human breast epithelium but not in breast carcinoma. J Cell Sci 108:1945-1957 63. Adams JC, Watt FM (1993) Regulation and development and differentiation by extracellular matrix. Development 117:1183-1198 64. Yurochko AD, Liu DY, Eierman D, Haskill S (1992) Integrins as a primary signal transduction molecule regulating monocyte immediate-early gene induction. Proc Natl Acad Sci USA 89:9034-9038 65. Lin TH, Yurochko A, Kornberg L, Morris J, Walker JJ, Haskill S, Juliano RL (1994) The role of protein tyrosine phosphorylation in integrin-mediated gene induction in monocytes. J Cell Biol 126:1585-1593 66. Huhtala P, Humphries MJ, McCarthy JB, Tremble PM, Werb Z, Damsky CH (1995) Cooperative signaling by α5β1 and α4β1 integrins regulates metalloproteinase gene expression in fibroblasts adhering to fibronectin. J Cell Biol 129:867-879 67. Tremble P, Damsky CH, Werb Z (1995) Components of the nuclear signaling cascade that regulate collagenase gene expression in response to integrin-derived signals. J Cell Biol 129:1707-1720 68. Streuli CH, Bailey N, Bissell MJ (1991) Control of mammary epithelial differentiation: basement membrane induces tissuespecific gene expression in the absence of cell-cell interaction and morphological polarity. J Cell Biol 115:1383-1395 69. Streuli CH, Schmidhauser C, Bailey N, Yurchenco P, Skubitz APN, Roskelley C, Bissell MJ (1995) Laminin mediates tissuespecific gene expression in mammary epithelia. J Cell Biol 129:591-603 70. Schmidhauser C, Casperson GF, Myers CA, Sanzo KT, Bolten S, Bissell MJ (1992) A novel transcriptional enhancer is involved in the prolactin- and extracellular matrix-dependent regulation of beta-casein gene expression. Mol Biol Cell 3:699-709

45. Guadagno TM, Assoian RK (1991) G1/S control of anchorageindependent growth in the fibroblast cell cycle. J Cell Biol 115:1419-1425 46. Zhu X, Assoian RK (1995) Integrin-dependent activation of MAP kinase: a link to shape-dependent cell proliferation. Mol Biol Cell 6:273-282 47. Davis RJ (1993) The mitogen-activated protein kinase signal transduction pathway. J Biol Chem 268:14553-14556 48. Chen Q, Kinch MS, Lin TH, Burridge K, Juliano RL (1994) Integrin-mediated cell adhesion activates mitogen-activated protein kinases. J Biol Chem 269:26602-26605 49. Morino N, Mimura T, Hamasaki K, Tobe K, Ueki K, Kikuchi K, Takehara K, Kadowaki T, Yazaki Y, Nojima Y (1995) Matrix/ integrin interaction activates the mitogen-activated protein kinase, p44erk-1 and p42erk-2. J Biol Chem 270:269-273 50. Guadagno TM, Ohtsubo M, Roberts JM, Assoian RK (1993) A link between cyclin A expression and adhesion-dependent cell cycle progression. Science 262:1572-1575 51. Barrett JF, Lewis BC, Hoang AT, Alvarez RJ, Dang CV (1995) Cyclin A links c-Myc to adhesion-independent cell proliferation. J Biol Chem 270:15923-15925 52. Vuori K, Ruoslahti E (1994) Association of insulin receptor substrate-1 with integrins. Science 266:1576-1578 53. Pasqualini R, Hemler ME (1994) Contrasting roles for integrins β1 and β5 cytoplasmic domains in subcellular localization, cell proliferation, and cell migration. J Cell Biol 125:447-460 54. Agrez M, Chen A, Cone RI, Pytela R, Sheppard D (1994) The αvβ6 integrin promotes proliferation of colon carcinoma cells through a unique region of the β6 cytoplasmic domain. J Cell Biol 127:547-556 55. Meredith J, Takada Y, Fornaro M, Languino LR, Schwartz MA (1995) Inhibition of cell cycle progression by the alternatively spliced integrin β1C. Science 269:1570-1572 56. Steller H (1995) Mechanisms and genes of cellular suicide. Science 267:1445-1449 57. Meredith JE, Fazeli B, Schwartz MA (1993) The extracellular matrix as a cell survival factor. Mol Biol Cell 4:953-961 58. Re F, Zanetti A, Sironi M, Palentarutti N, Lanfrancone L, Dejana E, Colotta F (1994) Inhibition of anchorage-dependent cell spreading triggers apoptosis in cultured human endothelial cells. J Cell Biol 127:537-546 59. Brooks PC, Montgomery AMP, Rosenfeld M, Reisfeld RA, Hu T, Klier G, Cheresh DA (1994) Integrin αvβ3 antagonists promote tumor regression by inducing apoptosis of angiogenic blood vessels. Cell 79:1157-1164

118