A central multifunctional role of integrin-linked kinase at muscle attachment sites

Abstract

SummaryIntegrin-linked kinase (ILK) is an essential component of a multiprotein complex that links actin to the plasma membrane. Here, we have used a genetic approach to examine the molecular interactions that are essential for the assembly of this ILK-containing complex at Drosophila muscle attachment sites (MASs). We show that, downstream of integrins, talin plays a decisive role in the recruitment of three proteins: ILK, PINCH and paxillin. The accumulation of ILK at MASs appears to follow an amplification mechanism, suggesting that numerous binding sites are generated by minimal levels of the upstream integrin and talin effectors. This property suggests that ILK functions as an essential hub in the assembly of its partner proteins at sites of integrin adhesion. We found that PINCH stability, and its subcellular localization at MASs, depends upon ILK function, but that ILK stability and localization is not dependent upon PINCH. An in vivo structure-function analysis of ILK demonstrated that each ILK domain has sufficient information for its independent recruitment at embryonic MASs, whereas at later developmental stages only the kinase domain was effectively recruited. Our data strengthen the view that the ILK complex is assembled sequentially at sites of integrin adhesion by employing multiple molecular interactions, which collectively stabilize the integrin-actin link.

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

“…This indicates that the PINCH C-terminal tail also participates in localization, even in the context of normal binding to ILK (Xu et al, 2005). A recent study, that is complementary to this report, has probed how various ILK deletion mutants affect protein assembly at muscle attachment sites (Zervas et al, 2011). This work demonstrates that expression and proper localization of the ANKR domain of ILK in an ilk null mutant is insufficient to localize PINCH despite the capacity of PINCH to bind this portion of ILK (Zervas et al, 2011).…”

“…In Drosophila, ilk null mutational analyses have shown that ILK is required for the appropriate targeting of PINCH to integrin-rich adhesion sites (Zervas et al, 2011). In contrast, we have found that PINCH Q38A -Flag, which lacks the ability to bind ILK, assembles properly at muscle attachment sites, supporting the retention of cellular and muscle architecture, as well as viability of the organism.…”

“…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.…”

“…For example, ILK accumulation at muscle attachment sites is compromised in wech mutants whereas PINCH localization is reported to be undisturbed, raising the possibility that PINCH is not completely dependent on ILK for its appropriate subcellular localization (Löer et al, 2008). Consistent with this view, a PINCH variant that lacks LIM1 and cannot bind ILK (or perform any other putative LIM1-dependent functions), retains some capacity to localize to muscle attachment sites (Zervas 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). Parvin, which binds both to ILK and to Actin, is often included in this functional complex.…”