Filippo Giancotti: Complexity and Specificity of Integrin Signaling

The integrins bind to distinct, although partially overlapping, subsets of extracellular matrix proteins and transmit both mechanical and chemical signals. In addition to imparting polarity to the cell and organizing and remodeling its cytoskeleton during adhesion and migration, these signals exert a stringent control on cell survival and cell proliferation.

The Src Family of tyrosine Kinases (SFKs) are dedicated to integrin signaling. These enzymes localize to focal adhesions, which are sites of integrin-mediated cell attachment to the matrix, and phosphorylate several of their target-effectors at these sites. The Src Family of tyrosine Kinases (SFKs) are dedicated to integrin signaling. These enzymes localize to focal adhesions, which are sites of integrin-mediated cell attachment to the matrix, and phosphorylate several of their target-effectors at these sites.

Most integrins activate Focal Adhesion Kinase (FAK) and thereby Src Family Kinases (SFKs), causing phosphorylation of, and hence signaling from, p130-CAS and paxillin. A subset of integrins — α1β1, 5β1, and αvβ3 — also activate the adaptor protein Shc. Further complexity arises from the existence of integrin-specific mechanisms of signaling, such as those exemplified by α6β4. Despite this complexity and specificity, the essence of integrin signaling — what integrins do — is simple: they promote cell survival and impart positional control to the action of receptor tyrosine kinases (RTKs), determining whether cells proliferate and migrate in response to soluble growth factors and cytokines.

We have elucidated several integrin signaling pathways. Maja Oktay provided evidence that FAK/SFK signaling induces activation of JNK and cell cycle progression through phosphorylation of p130-CAS and recruitment of Crk. Laura Barberis showed that CAS/Crk signaling can lead to activation of ERK if a cell expresses B-Raf.

shared integrin signaling machinery The shared integrin signaling machinery (adapted from Giancotti and Tarone. 2003. Annu. Rev. Cell Dev. Biol. 19:173-206)

Kishore Wary and Fabrizio Mainiero demonstrated for the first time signaling differences between integrins. They showed that a subset of integrins — including α1β1, α5β1, αvβ3, and α6β4 — activate SFKs, and thereby ERK, independently of FAK. Kishore Wary demonstrated that α1β1, α5β1, and αvβ3 activate ERK through the SFK/Shc pathway. In this pathway, caveolin — or another lipid microdomain organizer — couples the transmembrane segment of the integrin β subunit to a palmitoylated SFK. Upon integrin-mediated activation, the palmitoylated SFK undergoes a conformational change and its newly exposed SH-3 domain recruits Shc. Shc is then phosphorylated on tyrosine and combines with the Grb2/SOS complex, causing activation of ERK and thereby AP-1-dependent transcription. In primary fibroblasts and endothelial cells, the integrins that activate this pathway are able to cooperate with RTKs to promote cell survival and cell proliferation, whereas other integrins are not able to do so.

Amel Mettouchi provided evidence that the Shc-linked integrins promote cell cycle progression by inducing — through Rac — translation of the mRNA encoding Cyclin D1.

Integrin-specific signaling Integrin-specific signaling (adapted from Giancotti and Tarone. 2003. Annu. Rev. Cell Dev. Biol. 19:173-206).

Miguel Lopez-Lago examined the underlying mechanism and found that Rac promotes PAK phosphorylation and thereby inactivation of Merlin. Loss of Merlin in turn activates mTORC1 and translation of cyclinD and other mRNAs involved in cell cycle progression and cell survival.

We are currently using affinity purification in conjunction with mass spectrometry to identify membrane-proximal components involved in integrin signaling. After biochemical validation, all novel integrin interactors are subjected to functional analysis. The results of these experiments suggest that the integrin signaling system is characterized by an unanticipated degree of complexity and specificity.

Mainiero F, Pepe A, Wary KK, Spinardi L, Mohammadi M, Schlessinger J, Giancotti FG. Signal transduction by the α6β4 integrin: distinct β4 subunit sites mediate recruitment of Shc/Grb2 and association with the cytoskeleton of hemidesmosomes. EMBO J. 1995, 14(18):4470-81.

Wary KK, Mainiero F, Isakoff S, Marcantonio EE, Giancotti FG. The adaptor protein Shc couples a class of integrins to the control of cell cycle progression. Cell 1996, 87:733-743.

Mainiero F, Pepe A, Yeon M, Ren Y, Giancotti FG. The intracellular functions of α6β4 integrin are regulated by EGF. J Cell Biol. 1996, 134(1):241-53.

Mainiero F, Murgia C, Wary KK, Curatola AM, Pepe A, Blumemberg M, Westwick JK, Der CJ, Giancotti FG. The coupling of α6β4 integrin to Ras-MAP kinase pathways mediated by Shc controls keratinocyte proliferation. EMBO J. 1997, 16(9):2365-75.

Murgia C, Blaikie P, Kim N, Dans M, Petrie HT, Giancotti FG. Cell cycle and adhesion defects in mice carrying a targeted deletion of the integrin β4 cytoplasmic domain. EMBO J. 1998, 17(14):3940-51.

Pozzi A, Wary KK, Giancotti FG, Gardner HA. Integrin α1β1 mediates a unique collagen dependent proliferation pathway in vivo. J. Cell Biol. 1998, 142:587-594.

Wary KK, Mariotti A, Zurzolo C, Giancotti FG. A requirement for caveolin-1 and associated kinase Fyn in integrin signaling and anchorage-dependent cell growth. Cell 1998, 94:625-634.

Oktay M, Wary KK, Dans M, Birge RB, Giancotti FG. Integrin-mediated activation of FAK is required for signaling to JNK and progression through the G1 phase of the cell cycle. J. Cell Biol. 1999, 145:1461-1469.

Barberis L, Wary, KK, Liu, F, Hirsch, E, Brancaccio, M, Altruda, F, Tarone, G, Giancotti, FG. Distinct roles of the adaptor protein Shc and Focal Adhesion Kinase in integrin signaling to ERK. J. Biol. Chem. 2000, 275:36532-36540.

Mettouchi A, Klein S, Guo W, Lopez-Lago M, Lemichez E, Westwick JK, Giancotti FG. Integrin-specific activation of Rac controls progression through the G(1) phase of the cell cycle. Mol. Cell 2001, 8:115-127 [Previews by Ridley, A.J. in Dev. Cell 1(2):160-161, 2001; Research Roundup by Powledge, T.M. in J. Cell Biol. 154:677, 2001; Must Read F1000].

Hirsch E, Barberis L, Brancaccio M, Azzolino O, Xu D, Kyriakis JM, Silengo L, Giancotti FG, Tarone G, Fässler R, Altruda F. Defective Rac-mediated proliferation and survival after targeted mutation of the β1 integrin cytodomain. J Cell Biol. 2002, 157(3):481-92.

Klein S, de Fougerolles AR, Blaikie P, Khan L, Pepe A, Green CD, Koteliansky V, Giancotti FG. α5β1 integrin activates an NF-kappa B-dependent program of gene expression important for angiogenesis and inflammation. Mol Cell Biol. 2002, 22(16):5912-22.

Gagnoux-Palacios L, Dans M, van't Hof W, Mariotti A, Pepe A, Meneguzzi G, Resh MD, Giancotti FG. Compartmentalization of integrin α6β4 signaling in lipid rafts. J Cell Biol. 2003, 29;162(7):1189-96.

Nikolopoulos SN, Blaikie P, Yoshioka T, Guo W, Puri C, Tacchetti C, Giancotti FG. Targeted deletion of the integrin β4 signaling domain suppresses laminin-5-dependent nuclear entry of mitogen-activated protein kinases and NF-kappaB, causing defects in epidermal growth and migration. Mol Cell Biol. 2005, 25(14):6090-102.

López-Lago MA, Okada T, Murillo MM, Socci N, Giancotti FG. Loss of the tumor suppressor gene NF2, encoding merlin, constitutively activates integrin-dependent mTORC1 signaling. Mol Cell Biol. 2009, 29(15):4235-49.