Molecular Dissection of PINCH-1 Reveals a Mechanism of Coupling and Uncoupling of Cell Shape Modulation and Survival

Abstract

How cells couple and uncouple regulation of cellular processes such as shape change and survival is an important question in molecular cell biology. PINCH-1, a widely expressed protein consisting of five LIM domains and a C-terminal tail, is an essential focal adhesion protein with multiple functions including regulation of the integrin-linked kinase (ILK) level, cell shape, and survival signaling. We show here that the LIM1-mediated interaction with ILK regulates all these three processes. By contrast, the LIM4-mediated interaction with Nck-2, which regulates cell morphology and migration, is not required for the control of the ILK level and survival. Remarkably, a short 15-residue tail C-terminal to LIM5 is required for both cell shape modulation and survival, albeit it is not required for the control of the ILK level. The C-terminal tail not only regulates PINCH-1 localization to focal adhesions but also functions after it localizes there. These findings suggest that PINCH-1 functions as a molecular platform for coupling and uncoupling diverse cellular processes via overlapping but yet distinct domain interactions. Cell-extracellular matrix (ECM)1 adhesion is a fundamental process that is critically involved in embryonic development and numerous physiological and pathological processes including injury repair, cancer, and organ (e.g. heart and renal) failure. Among key regulators of cell-ECM adhesion and signaling are proteins localized at the membrane-actin cytoskeleton junctions in cell-ECM contacts (e.g. focal adhesions). PINCH-1 is a widely expressed focal adhesion protein consisting of five LIM domains and a short residue C-terminal tail (for review, see Refs. 1-4). Recent biochemical, cell biological, and genetic studies have demonstrated that PINCH-1 is crucial for cell shape modulation and signal transduction (5-13). Furthermore, there is evidence suggesting that PINCH-1 could serve as a useful target for therapeutic intervention of human diseases such as cancer and organ (e.g. heart and renal) failure (2, 4, 14 -19). Elucidating the molecular mechanism whereby PINCH-1 functions, therefore, is important for understanding the general principle that governs the regulation of cell-ECM adhesion and signaling as well as for the development of new therapeutic approaches that control these processes.PINCH-1 interacts with ILK (5, 20, 21), an equally widely expressed focal adhesion component that is essential for integrin-mediated cell-ECM adhesion and signaling (for review, see Refs. 2-4, 22, and 23). The ILK-binding site has been mapped to the second zinc finger of the N-terminal-most LIM1 domain that contains 21,24). In addition, PINCH-1 interacts with Nck-2 (25, 26), an SH2/SH3-containing adaptor protein that is involved in both cell adhesion-and growth factormediated signaling and actin cytoskeleton remodeling (27-29). The Nck-2-binding site is located in the first zinc finger of the PINCH-1 C-terminal LIM4 domain (25,26). Recently, we have suppressed the expression of PINCH-1 and ILK, respectively, in ...

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

“…1B). In mammalian cells, a variant of PINCH in which a universally conserved amino acid (glutamine 40) is mutated to alanine (Q40A) does not bind to ILK, fails to localize at focal adhesions, and causes defects in cell spreading and adhesion (Zhang et al, 2002;Xu et al, 2005). We engineered and expressed Drosophila PINCH wt -His or PINCH Q38A -His (the fly mutation corresponding to PINCH Q40A ) in Drosophila S2 cells, captured His-tagged proteins with their partners under native conditions using Ni-NTA agarose, and performed western blots for known binding partners.…”

“…It has been demonstrated that the physical interaction of PINCH with ILK is critical for their mutual stability in cell culture (Xu et al, 2005). To test directly whether the ability of PINCH to bind ILK is required for their stability in vivo, we performed western blots of PINCH wt -Flag and PINCH Q38A -Flag rescued embryos and observed that levels of PINCH and ILK are comparable in multiple transgenic lines (Fig.…”

“…We have shown that disrupting the PINCH-ILK interaction does not compromise viability, wing adhesion, or muscle structure or function. This was surprising given the prevailing idea that PINCH-ILK complexes form a functional unit, and from data in cell culture demonstrating loss of adhesion upon disruption of the PINCH-ILK interaction (Xu et al, 2005). Despite their capacity for physical association, tight co-localization at integrin-rich sites, and strikingly similar null phenotypes, the direct interaction between PINCH and ILK is not required 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).…”

“…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). Fourth, ILK is required for localizing PINCH at integrin-rich sites (Zervas et al, 2011).…”