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Lecture 7

Lecture 7 FAK and actin dynamics Jan 31,2011.doc

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Cell and Systems Biology

1 Lecture 7 Focal adhesion kinase and regulation of actin dynamics by Rac1 January 31,2011 Sensing the topography of the microenvironment: Focal adhesion kinase a regulator of cell adhesion, migration and anchorage dependent growth Cell-matrix adhesion complexes The role of protein-tyrosine kinases (PTKs) regulating focal adhesion One type of signaling event stimulated by integrins is tyrosine phosphorylation of cytoskeletal as well as signaling proteins. However, since integrins do not possess catalytic activity, the signals initiated by ECM-integrin interactions must be transduced into cells through the activation of integrin- associated proteins. A number of different intracellular protein-tyrosine kinases (PTKs) such as focal adhesion kinase (FAK) and Src-family PTKs are activated by integrin clustering or cell binding to ECM proteins. FAK is a multifunction protein that affects a variety of cellular processes; the focus was on how FAK is linked to intracellular signaling pathways controlling cell motility. Structural organization of FAK FAK FAK is often referred to in the literature as pp125 , reflecting that FAK is a phosphoprotein with a molecular weight of 125 kDa. FAK is a cytoplasmic PTK that localizes to focal contacts and adhesion as the name implies. FAK is localized to sites of integrin clustering at focal adhesions through indirect protein interactions of the C-terminal focal adhesion targeting (FAT) regions – a region containing binding sites for integrin-associated proteins such as paxillin and talin. This domain therefore plays a critical role in critical role in the localization of FAK to focal adhesions sites. The proline-rich domains (SH3 docking sites) of the FAT domain recruit several proteins, including p130cas, GRAF, and ASAP1. The FAK N-terminal region harbors a FERM (band 4.1, erzin, radixin and meosin) homology motifs followed by a SH3 binding site for the Src-family of PTPs and a highly conserved central kinase domain. Hence, think of FAK as a scaffold as well as a kinase. Below is modified text taken from a review article that summarizes our understanding FAK activation and its contributions to GEF/GAP-mediated RhoGTPase regulation of focal adhesion and turnover. Tomar and Schlaepfer (2009) Current Opinion Cell Biology 21:676-683 FAK activation Integrin clustering results in FAK activation at nascent FA sites. However, a recent study demonstrated that FAK can also activate integrins, resulting in increased integrin-ECM interaction and adhesion strengthening. Therefore, during the initial steps of cell spreading, there is a cycle where integrin activation (outside-in signaling) causes FAK activation and FAK can further enhance the pool of activated integrins (inside-out signaling. The interaction of α5β1 integrins to fibronectin (FN), an important ECM component occurs in two stages. First, under low cell contractility (less tension) α5β1 binds to the RGD (arginine glycine aspartate) motif of FN and second, under high cell contractility (high tension), α5β1 integrins interact with a synergy site on FN, causing increased adhesion strengthening which was found to be important for full FAK activation. Thus, there is a complex interplay and potentially sequential series of events initiated by integrins leading to FAK activation and resulting in v leading edge cytoskeletal organization. These findings are summarized in a simplistic model whereby FAK facilitates the formation of a stable leading cell edge. The cytoskeletal protein talin binds to FAK and integrin cytoplasmic tails both initiating and 2 also enhancing integrin activation. This integrin-talin complex promotes localized increase in cell tension resulting in unfolding of talin rod domain, binding of vinculin and actin filaments to talin, and promoting the assembly of nascent FAs. Paxillin is also an important cytoskeletal and scaffolding protein recruited early to FAs. FAK is recruited to nascent FAs by its FAT (focal adhesion targeting) domain that binds to both talin and paxillin. Structural and mutagenesis studies have shown that FAK exists in an auto-inhibited conformation, where the N-terminal FERM domain of FAK interacts with the FAK kinase domain. In this auto-inhibited conformation, it is possible that the FAK FERM domain may also bind to the actin nucleating protein Arp3 and promote the recruitment of the Arp2/3 complex to nascent adhesions in a kinase-independent manner. Thus, specific FAK FAT and FAK FERM-mediated protein interactions can link integrins with the actin polymerizing cell machinery; thereby facilitating leading edge protrusion. Alternative mechanisms regulating FAK activation have also been elucidated recently. In addition to C-terminal domain mediated clustering of FAK within integrins, the FAK FERM Auto-inhibited conformation can be disrupted by FERM domain binding to phospholipids such as PIP2 (phosphoinositol 4,5 bisphosphate) through a basic amino acid enriched patch within the FERM. FAK is maintained in an inactive state by the binding of the FERM domain to its kinase domain –an autoinhibited state. Hence, activation of FAK requires the separation of the FERM domain from the kinase. Engagement of integrins with ECM components begins the activation cascade. FAK is anchored indirectly to the cytoplasmic tail of the β subunit of integrins via adapter molecules such as paxillin and talin. Upon binding to these adapters, there is some evidence that FAK can autophosphorylate 397 itsel397t tyrosine (mainly by trans phosophorylation of adjacent FAK). Phosphorylation of Tyr- does not fully activate the kinase activity of FAK due to conformational constraints. As matrix-mediated integrin clustering continues, FAK undergoes a conformational change that exposes Tyr -PO re4idue, which serves as an SH2 docking sites for members of the Src family of tyrosine 397 kinases (Src-PTKs). Interestingly, phosphorylation at Tyr destabilizes the FAK-Arp2/3 complex, resulting in the release of Arp2/3 from sites of FAs, and allowing for Arp2/3 recruitment to the extended lamellipodia. Tyr 397FAK phosphorylation promotes FAK-Src complex formation, resulting in complete FAK activation. Once the FAK-Src complex is formed, FAK phosphorylates Src, facilitating the activation of Src. Activated Src in turn further phosphorylates FAK at tyrosine residues 576 and 577 (located within the kinase domain), fully activating the catalytic activity of FAK. In essence, a bipartite kinase complex has been created. Src phosphorylates additional sites within the FAK C-terminal domain, creating additional SH2 docking sites for regulatory factors such as the guanine nucleotide exchange factor (GEF) and GTPase-activating proteins (GAPs). The striking complexity of the signaling events initiated by the FAK-Src complex has lead researcher to refer to FAK as “an activatable” scaffold. FAK: a key regulator of localized GAP and GEF a
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