Interaction of Hic-5, A Senescence-related Protein, with Focal Adhesion Kinase

Abstract

Integrin-mediated cell adhesion induces various biological events such as cell proliferation, survival, migration, cytokine production, and cytoskeleton reorganization (1-7). Despite the significance of these events, signal transduction pathways of integrins have not been fully understood. One of the approaches for integrin-mediated signal transduction is to study the proteins that are localized to focal adhesions.Focal adhesions are the sites where cells attach to substrata via integrin-extracellular matrix binding (7,8). Various cytoskeletal proteins and signaling molecules such as focal adhesion kinase (FAK), 1 paxillin, vinculin, talin, p130 Cas , and tensin, are accumulated in the cytoplasmic part of focal adhesions (7-10). More interestingly, focal adhesions are strongly stained by immunohistochemical analysis with anti-phosphotyrosine mAb (7, 10). Several proteins localized to focal adhesions such as FAK, paxillin, and p130Cas , are tyrosine phosphorylated following integrin stimulation (7, 9, 10). When tyrosine phosphorylated, these proteins bind to signaling molecules such as Crk, Nck, GRB2, Src, which contain Src homology (SH) 2 domain (7, 10). Paxillin, a 68-kDa cytoskeletal protein, is one of these focal adhesion proteins (11-13) and has two functions in integrin-mediated signal transduction. First, paxillin appears to be the major entity that recruits two binding proteins, FAK,, to focal adhesions (16,17). Second, paxillin is tyrosine phosphorylated following integrin stimulation in a FAK-dependent manner, and then binds to the Crk SH2 domain (15,18). Thus, paxillin is involved in recruitment of FAK to focal adhesions, tyrosine phosphorylated by FAK and/or a kinase(s) that is associated with FAK, and then works as a docking protein in integrin-mediated signal transduction. Hydrogen peroxide-inducible clone (hic)-5 was initially identified as a gene which was induced by transforming growth factor ␤1 or by H 2 O 2 (19). The expression of hic-5 mRNA was augmented in the in vitro senescent process of human diploid fibroblasts (19). The forced overexpression of Hic-5 induced growth retardation, senescence-like morphology such as the enlarged and flattened morphology, and the increased expression of p21/WAF1/Cip1/sdi1 and extracellular matrix-related proteins such as fibronectin (FN), collagen, and collagenase (20). These findings suggest that Hic-5 is involved in very interesting function in the senescent process and in transforming growth factor ␤1-mediated signal transduction. However, the nature of Hic-5 function has not been clarified yet. Structurally, Hic-5 contains four LIM domains in the C-terminal half and LD domains in the N-terminal half (19,21). These domains are conserved in paxillin, and the four LIM domains of Hic-5 and paxillin share 62% homology (17, 19 -21). These structural homology with paxillin suggested that Hic-5 is localized to focal adhesions and involved in integrin-mediated signal transduction. Recently, Matsuya et al. (21) reported that Hic-5 bound to Cak ␤, a FAKrelated tyro...

Hic-5 mediates endothelial sprout initiation by regulating a key surface metalloproteinase

“…We found that loss of Hic-5 decreased the formation of the complex between MT1-MMP and FAK, supporting that Hic-5 bridges MT1-MMP and FAK. Hic-5 is well-suited for this role as an adaptor protein given that the N-terminal LD domain of Hic-5 binds FAK (Fujita et al, 1998), and the C-terminal LIM2 and LIM3 domains are required for interaction with MT1-MMP (shown here). Hic-5 is known to localize to integrin-mediated sites of adhesion, where it coordinates cell migration in 2D (Brown and Turner, 2004;Tumbarello and Turner, 2007;Deakin and Turner, 2008).…”

“…Because Hic-5 is a known focal adhesion scaffold protein that forms a complex with FAK (Fujita et al, 1998), and the fine punctate staining pattern within 3D sections (Fig. 4A) appears to be similar to those reported for other focal adhesion proteins in 3D (Cukierman et al, 2001;Petroll and Ma, 2003), we further investigated whether Hic-5 colocalized with activated FAK.…”

“…Hic-5 is highly similar to paxillin and contains four N-terminal Leu-and Asp-rich (LD) domains and four C-terminal LIM domains. The N-terminal LD domains of Hic-5 interact with focal adhesion kinase (FAK, also known as PTK2) (Fujita et al, 1998) vinculin (Deakin et al, 2012), paxillin (Deakin et al, 2012), c-Src kinase (Csk) (Thomas et al, 1999), protein tyrosine kinase 2 β (PYK2, also known as PTK2B) (Matsuya et al, 1998) and Arf GAP1 (GIT1) (Nishiya et al, 2002). The LIM domains at the C-terminus of Hic-5 mediate binding to PTP-PEST (also known as PTPN12) (Nishiya et al, 1999) and Hsp27 (also known as HSPB1) (Jia et al, 2001).…”

Regulation of contractility by Hsp27 and Hic-5 in rat mesenteric small arteries

“…During the current study, we identified in small arteries a high expression of Hic-5, a homologue of paxillin (51). Hic-5, in addition to having a similar domain structure as paxillin, also localizes to the same sites and shares binding partners with paxillin (16,51). However, they must not entirely duplicate each others functions since paxillin knockout is embryonic lethal (18), and unlike paxillin, Hic-5 is not activated by integrin engagement or phosphorylation by p125FAK (16,51).…”

“…However, it is phosphorylated by PYK2, a calcium-dependent homologue of p125FAK (16,51), suggesting different cellular functions for the two proteins. Hic-5 was shown to be a substrate for PYK2 (23), and in COS cells and rat fibroblasts, it was shown to interact with PYK2 (31), whereas in rat kidney epithelial cells (24), Hic-5 was shown to associate with Hsp27.…”