Molecular dissection of the ILK-PINCH-parvin triad reveals a fundamental role for the ILK kinase domain in the late stages of focal-adhesion maturation

Abstract

Cell adhesion receptors of the integrin family assume fundamental roles throughout development and in adult organisms by mediating interactions between cells and the extracellular matrix that control migration, survival, proliferation, differentiation and matrix assembly (Hynes, 2002;van der Flier and Sonnenberg, 2001). To accomplish these diverse functions, integrins assemble a host of ancillary proteins into specialized adhesive structures with distinct morphologies, subcellular localization and signaling potential . Focal complexes (FXs), focal adhesions (FAs) and fibrillar adhesions (FBs) are among the most widely studied, with FXs being the smallest adhesions that emerge from highly dynamic nascent adhesions (Choi et al., 2008) at the edge of extending lamellipodia and guide cell spreading. Shortly after their formation, FXs can either dissolve or connect to actin stress fibers and enlarge to form FAs that provide firm anchorage to the substrate. FBs, which are sites of fibronectin (FN) fibrillogenesis characterized by their elongated shape and more central location, are formed by translocation of α5β1 integrin dimers out of FAs along nascent FN fibers on the cell surface (Pankov et al., 2000;Zamir et al., 2000).Several reports have described the diversity of these adhesive structures in terms of their composition, life span, integrin density and turnover, and dependence on acto-myosin-generated tension (Ballestrem et al., 2001;Zaidel-Bar et al., 2003;Zamir et al., 1999). With regard to their molecular content, the formation of FXs, FAs and FBs is accompanied by the hierarchical recruitment of different integrins and integrin-associated proteins (Zaidel-Bar et al., 2003). Typically, paxillin is recruited early into FXs, followed by vinculin, whereas zyxin and tensin are recruited after the complete maturation of FAs. The actin-binding protein tensin is particularly abundant in FBs, whereas levels of phosphotyrosine (PY) on resident proteins are significantly lower in these matrix-forming adhesions (Zamir et al., 1999).Unraveling the signals that regulate the dynamic conversion of FXs to FAs and FBs is of primary importance for understanding integrin function. Previous studies from our laboratory have identified integrin-linked kinase (ILK) as a key regulator of the stability and/or turnover of FAs and FBs in fibroblasts and endothelial cells (Boulter et al., 2006;Vouret-Craviari et al., 2004). ILK is a modular scaffolding protein comprising an N-terminal array of ankyrin repeats, a short pleckstrin-homology (PH) domain and a C-terminal Ser/Thr kinaselike domain (Delcommenne et al., 1998). By means of its N-terminal ankyrin repeat, ILK binds to the first of five LIM domains in the adaptor protein PINCH1 (Chiswell et al., 2008;Li et al., 1999). In addition to integrin β subunits, the kinase domain interacts directly with other components of integrin-based adhesion plaques, including paxillin and the most C-terminal of the two calponin-homology (CH) domains of the adaptor protein α-parvin (also known as actopa...

A critical role for Ras Suppressor-1 (RSU-1) revealed when PINCH-Integrin-linked Kinase (ILK) binding is disrupted

“…In agreement with data from mammalian cell culture and in zebrafish, we show that loss of PINCH affects ILK protein levels and vice versa, indicating that this is an evolutionarily conserved characteristic of proteins in this adhesion complex (Fukuda et al, 2003;Stanchi et al, 2009;Meder et al, 2011). Contrary to these data, a recent publication has claimed that ILK stabilizes PINCH, but that PINCH does not stabilize ILK, basing this conclusion on qualitative immunofluorescence images, where consistent with other published data ILK localization is preserved in a stck null mutant (Clark et al, 2003;Zervas et al, 2011).…”

“…Stabilization within this complex is modulated not only by proteins with direct or primary interactions (PINCH-RSU-1 or PINCH-ILK), but by secondary protein interactions as well (ILK-RSU-1). This secondary regulation is also observed in other studies where loss of PINCH reduces Parvin and Tensin protein levels, which are both direct partners of ILK (Fukuda et al, 2003;Stanchi et al, 2009). Further work will be required to reveal the extent of this stabilization phenomenon within adhesion complexes in Drosophila.…”

“…In cell culture, reductions in both ILK and PINCH protein levels are observed when either ILK or PINCH levels are depleted either by RNA interference or targeted gene disruption (Fukuda et al, 2003;Xu et al, 2005;Stanchi et al, 2009). A recent publication has also demonstrated mutual stabilization of PINCH and ILK in vivo using antisense morpholinos in zebrafish (Meder et al, 2011).…”

“…ILK and PINCH have both been implicated in survival signaling via AKT, and it will be important to determine the contributions of RSU-1 (Fukuda et al, 2003;Xu et al, 2005;Meder et al, 2011). Many defects observed from disruption of integrin adhesion complexes are attributed to improper ILK-PINCH-Parvin complex formation, function, and downstream signaling (Zhang et al, 2002;Legate et al, 2006;Stanchi et al, 2009;Wickström et al, 2010;Zervas et al, 2011). Here, we propose that the components of this integrin effector complex be expanded to include RSU-1.…”

“…First, the N-terminal LIM domain of PINCH interacts directly with the Nterminal ankyrin repeat domain (ANKR) of ILK Tu et al, 1999). Second, depletion of either ILK or PINCH results in reduction in the levels of the other, indicating a mutual stabilization of these two proteins (Fukuda et al, 2003;Stanchi et al, 2009;Meder et al, 2011). Third, targeted disruption of the interaction between PINCH and ILK in mammalian cell culture experiments by a point mutation in LIM1 of PINCH results in disturbances in cell spreading and survival, as well as reduced stability of both PINCH and ILK (Velyvis et al, 2001;Zhang et al, 2002;Xu et al, 2005).…”