Solution Structure of the Focal Adhesion Adaptor PINCH LIM1 Domain and Characterization of Its Interaction with the Integrin-linked Kinase Ankyrin Repeat Domain

Abstract

PINCH is a recently identified adaptor protein that comprises an array of five LIM domains. PINCH functions through LIM-mediated protein-protein interacProteins often function through domains or recurring motifs. The LIM domain is a common protein-protein interaction motif that was originally discovered in the products of the lin-11, isl-1, and mec-3 genes and hence given the acronym "LIM" (1, 2). The domain consists of a loosely conserved cysteine-rich consensus sequence (CX 2 CX 16 -23 HX 2 CX 2 CX 2 CX 16 -21 CX 2-3 (H/ D/C)) that encodes two separate zinc fingers (shown in underlined and boldface type, respectively) (3-5). Frequently occurring as an array of one to five copies, the double zinc finger LIM domains have been found in a variety of proteins with diverse functions, either alone or associated with other functional domains (5). Based on sequence similarity, LIM-containing proteins are classified into three groups (5): Group 1 includes the LHX (LIM homeodomain protein), LMO, and LIMK (LIM kinase) subfamilies; Group 2 contains the CRP 1 and CRIP subfamilies; and Group 3 is heterogeneous, and the sequences in this group are very divergent from those in Groups 1 and 2. Although genetic and biochemical studies have shown that LIM domains can interact with diverse target proteins, the molecular basis of how the domains confer specificity and/or coordinate with other functional domains remains elusive. Structural studies geared toward understanding the LIM functions so far have been performed on avian CRPs, related mammalian intestinal protein (CRIP), and a fragment of Lasp-1 protein (6 -12), which belong to Group 2 of the LIM subfamilies with CCHC and CCCC zinc finger types. These studies revealed a conserved fold of LIM domains in which two zinc ions orchestrate the formation of separate zinc fingers that stack together through a shared hydrophobic core. Each finger consists of two orthogonally packed antiparallel ␤-sheets, and the C-terminal CCCC module is terminated by a ␣-helix. The conserved tetrahedral zinc coordination and hydrophobic core appear to determine the overall fold of LIM domains, whereas other variable regions in the domains may confer the specificities for binding diverse target proteins. The exact mode of LIM domain binding to various target proteins remains essentially uncharacterized.PINCH (particularly interesting new Cys-His protein) is a widely expressed adaptor protein comprising five LIM domains that belong to Group 3 of the LIM protein subfamilies (5). Originally identified from screening of a human cDNA library with antibodies recognizing senescent erythrocytes (13), PINCH has become increasingly interesting because of its involvement in mediating integrin signaling (14, 15). Specifically, PINCH interacts with a membrane-proximal integrin-linked kinase (ILK), which colocalizes with ␤ 1 -integrin in the focal adhesion plaques (16,17). PINCH-ILK interaction is essential for the focal adhesion localization of ILK (17) and integrin signaling as evidenced by genetic studies in whi...

Paxillin: Adapting to Change

“…The structures of several LIM domains have been solved and reveal that LIM domains are arranged such that each individual zinc finger is comprised of two antiparallel ␤-sheets that are separated by a tight turn. In addition, the two zinc fingers pack together due to hydrophobic interactions, and each LIM domain ends with a short ␣-helix (48, 203,289). Interestingly, although it is now generally accepted that the primary function for LIM domains is in mediating protein-protein interactions (235), the CRIP and CRP structures are nearly identical to the DNAbinding domains of GATA-1 and the steroid hormone receptor family (203).…”

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

“…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).…”

Integrin-linked kinase activity regulates Rac- and Cdc42-mediated actin cytoskeleton reorganization via α-PIX

“…These proteins include paxillin Turner, 2001, 2002), PINCH Wu, 1999;Tu et al, 1999;Velyvis et al, 2001;Zhang et al, 2002a;Fukuda et al, 2003) and the calponin homology (CH) domain-containing actin binding proteins a-parvin/CH-ILKBP/actopaxin (herein referred to as a-parvin) (Tu et al, 2001) and b-parvin/ affixin (herein referred to as b-parvin) (Yamaji et al, 2001). These protein-protein interactions provide a framework for the formation of ILK signaling complexes that couple integrins and growth factor receptors to the regulation of actin cytoskeletal dynamics (Wu and Dedhar, 2001).…”