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The structural basis of integrin-linked kinase-PINCH interactions.

ABSTRACT: The heterotrimeric complex between integrin-linked kinase (ILK), PINCH, and parvin is an essential signaling platform, serving as a convergence point for integrin and growth-factor signaling and regulating cell adhesion, spreading, and migration. We report a 1.6-A crystal structure of the ILK ankyrin repeat domain bound to the PINCH1 LIM1 domain, revealing the molecular basis of ILK-PINCH interactions and providing a structural description of this region of ILK. This structure identifies 5 ankyrin repeats in ILK, explains previous deletion mutagenesis data, permits identification of ILK and PINCH1 point mutations that disrupt the interaction, shows how zincs are coordinated by PINCH1 LIM1, and suggests that conformational flexibility and twisting between the 2 zinc fingers within the LIM1 domain may be important for ILK binding. These data provide an atomic-resolution description of a key interaction in the ILK-PINCH-parvin scaffolding complex.

SUBMITTER: Chiswell BP

PROVIDER: S-EPMC2634877 | biostudies-literature | 2008 Dec

REPOSITORIES: biostudies-literature

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The structural basis of integrin-linked kinase-PINCH interactions.

Chiswell Brian P BP Zhang Rong R Murphy James W JW Boggon Titus J TJ Calderwood David A DA

Proceedings of the National Academy of Sciences of the United States of America 20081212 52

The heterotrimeric complex between integrin-linked kinase (ILK), PINCH, and parvin is an essential signaling platform, serving as a convergence point for integrin and growth-factor signaling and regulating cell adhesion, spreading, and migration. We report a 1.6-A crystal structure of the ILK ankyrin repeat domain bound to the PINCH1 LIM1 domain, revealing the molecular basis of ILK-PINCH interactions and providing a structural description of this region of ILK. This structure identifies 5 ankyr ...[more]

PMID: 19074270

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Project description:Integrin-linked kinase (ILK) is an important signaling regulator that assembles into the heteroternary complex with adaptor proteins PINCH and parvin (termed the IPP complex). We recently reported that ILK is important for integrin activation in a Chinese hamster ovary (CHO) cell system. We previously established parental CHO cells expressing a constitutively active chimeric integrin (?IIb?6B?3) and mutant CHO cells expressing inactive ?IIb?6B?3 due to ILK deficiency. In this study, we further investigated the underlying mechanisms for ILK-dependent integrin activation. ILK-deficient mutant cells had trace levels of PINCH and ?-parvin, and transfection of ILK cDNA into the mutant cells increased not only ILK but also PINCH and ?-parvin, resulting in the restoration of ?IIb?6B?3 activation. In the parental cells expressing active ?IIb?6B?3, ILK, PINCH, and ?-parvin were co-immunoprecipitated, indicating the formation of the IPP complex. Moreover, short interfering RNA (siRNA) experiments targeting PINCH-1 or both ?- and ?-parvin mRNA in the parent cells impaired the ?IIb?6B?3 activation as well as the expression of the other components of the IPP complex. In addition, ILK mutants possessing defects in either PINCH or parvin binding failed to restore ?IIb?6B?3 activation in the mutant cells. Kindlin-2 siRNA in the parental cells impaired ?IIb?6B?3 activation without disturbing the expression of ILK. For CHO cells stably expressing wild-type ?IIb?3 that is an inactive form, overexpression of a talin head domain (THD) induced ?IIb?3 activation and the THD-induced ?IIb?3 activation was impaired by ILK siRNA through a significant reduction in the expression of the IPP complex. In contrast, overexpression of all IPP components in the ?IIb?3-expressing CHO cells further augmented THD-induced ?IIb?3 activation, whereas they did not induce ?IIb?3 activation without THD. These data suggest that the IPP complex rather than ILK plays an important role and supports integrin activation probably through stabilization of the active conformation.

Project description:UNLABELLED:Infectious spleen and kidney necrosis virus (ISKNV) is the type species of the Megalocytivirus genus, Iridoviridae family, causing a severe systemic disease with high mortality in mandarin fish (Siniperca chuatsi) in China and Southeast Asia. At present, the pathogenesis of ISKNV infection is still not fully understood. Based on a genome-wide bioinformatics analysis of ISKNV-encoded proteins, we found that ISKNV open reading frame 119L (ORF119L) is predicted to encode a three-ankyrin-repeat (3ANK)-domain-containing protein, which shows high similarity to the dominant negative form of integrin-linked kinase (ILK); i.e., viral ORF119L lacks the ILK kinase domain. Thus, we speculated that viral ORF119L might affect the host ILK complex. Here, we demonstrated that viral ORF119L directly interacts with particularly interesting Cys-His-rich protein (PINCH) and affects the host ILK-PINCH interaction in vitro in fathead minnow (FHM) cells. In vivo ORF119L overexpression in zebrafish (Danio rerio) embryos resulted in myocardial dysfunctions with disintegration of the sarcomeric Z disk. Importantly, ORF119L overexpression in zebrafish highly resembles the phenotype of endogenous ILK inhibition, either by overexpressing a dominant negative form of ILK or by injecting an ILK antisense morpholino oligonucleotide. Intriguingly, ISKNV-infected mandarin fish develop disorganized sarcomeric Z disks in cardiomyocytes. Furthermore, phosphorylation of AKT, a downstream effector of ILK, was remarkably decreased in ORF119L-overexpressing zebrafish embryos. With these results, we show that ISKNV ORF119L acts as a domain-negative inhibitor of the host ILK, providing a novel mechanism for the megalocytivirus pathogenesis. IMPORTANCE:Our work is the first to show the role of a dominant negative inhibitor of the host ILK from ISKNV (an iridovirus). Mechanistically, the viral ORF119L directly binds to the host PINCH, attenuates the host PINCH-ILK interaction, and thus impairs ILK signaling. Intriguingly, ORF119L-overexpressing zebrafish embryos and ISKNV-infected mandarin fish develop similar disordered sarcomeric Z disks in cardiomyocytes. These findings provide a novel mechanism for megalocytivirus pathogenesis.

Dataset Information

The structural basis of integrin-linked kinase-PINCH interactions.

Publications

The structural basis of integrin-linked kinase-PINCH interactions.

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