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ATP allosterically stabilizes integrin-linked kinase for efficient force generation.

ABSTRACT: SignificanceThe pseudokinase integrin-linked kinase (ILK) is a central component of focal adhesions, cytoplasmic multiprotein complexes that integrate and transduce biochemical and mechanical signals from the extracellular environment into the cell and vice versa. However, the precise molecular functions, particularly the mechanosensory properties of ILK and the significance of retained adenosine triphosphate (ATP) binding, are still unclear. Combining molecular-dynamics simulations with cell biology, we establish a role for ATP binding to pseudokinases. We find that ATP promotes the structural stability of ILK, allosterically influences the interaction between ILK and its binding partner parvin at adhesions, and enhances the mechanoresistance of this complex. On the cellular level, ATP binding facilitates efficient traction force buildup, focal adhesion stabilization, and efficient cell migration.

SUBMITTER: Martin IM

PROVIDER: S-EPMC8933812 | biostudies-literature | 2022 Mar

REPOSITORIES: biostudies-literature

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ATP allosterically stabilizes integrin-linked kinase for efficient force generation.

Martin Isabel M IM Nava Michele M MM Wickström Sara A SA Gräter Frauke F

Proceedings of the National Academy of Sciences of the United States of America 20220308 11

SignificanceThe pseudokinase integrin-linked kinase (ILK) is a central component of focal adhesions, cytoplasmic multiprotein complexes that integrate and transduce biochemical and mechanical signals from the extracellular environment into the cell and vice versa. However, the precise molecular functions, particularly the mechanosensory properties of ILK and the significance of retained adenosine triphosphate (ATP) binding, are still unclear. Combining molecular-dynamics simulations with cell bi ...[more]

PMID: 35259013

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Project description:Background and purposeRecombinant osteopontin (rOPN) has been reported to be neuroprotective in stroke animal models. The purpose of this study is to investigate a potential role and mechanism of nasal administration of rOPN on preserving the vascular smooth muscle phenotype in early brain injury after subarachnoid hemorrhage (SAH).MethodsOne hundred and ninety-two male adult Sprague-Dawley rats were used. The SAH model was induced by endovascular perforation. Integrin-linked kinase small interfering RNA was intracerebroventricularly injected 48 hours before SAH. The integrin receptor antagonist fibronectin-derived peptide Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP), focal adhesion kinase inhibitor Fib-14, and Rac-1 inhibitor NSC23766 were administered 1 hour before SAH induction. rOPN was administered via the intracerebroventricular and nasal route after SAH. SAH grade, neurological scores, brain water content, brain swelling, hematoxylin and eosin staining, India ink angiography, Western blots, and immunofluorescence were used to study the mechanisms of rOPN on the vascular smooth muscle phenotypic transformation.ResultsThe marker proteins of vascular smooth muscle phenotypic transformation α-smooth muscle actin decreased and embryonic smooth muscle myosin heavy chain (SMemb) increased significantly at 24 and 72 hours in the cerebral arteries after SAH. rOPN prevented the changes of α-smooth muscle actin and SMemb and significantly alleviated neurobehavioral dysfunction, increased the cross-sectional area and the lumen diameter of the cerebral arteries, reduced the brain water content and brain swelling, and improved the wall thickness of cerebral arteries. These effects of rOPN were abolished by GRGDSP, integrin-linked kinase small interfering RNA, and NSC23766. Intranasal application of rOPN at 3 hours after SAH also reduced neurological deficits.ConclusionsrOPN prevented the vascular smooth muscle phenotypic transformation and improved the neurological outcome, which was possibly mediated by the integrin receptor/integrin-linked kinase/Rac-1 pathway.

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 (αIIbα6Bβ3) and mutant CHO cells expressing inactive αIIbα6Bβ3 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 α-parvin, and transfection of ILK cDNA into the mutant cells increased not only ILK but also PINCH and α-parvin, resulting in the restoration of αIIbα6Bβ3 activation. In the parental cells expressing active αIIbα6Bβ3, ILK, PINCH, and α-parvin were co-immunoprecipitated, indicating the formation of the IPP complex. Moreover, short interfering RNA (siRNA) experiments targeting PINCH-1 or both α- and β-parvin mRNA in the parent cells impaired the αIIbα6Bβ3 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 αIIbα6Bβ3 activation in the mutant cells. Kindlin-2 siRNA in the parental cells impaired αIIbα6Bβ3 activation without disturbing the expression of ILK. For CHO cells stably expressing wild-type αIIbβ3 that is an inactive form, overexpression of a talin head domain (THD) induced αIIbβ3 activation and the THD-induced αIIbβ3 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 αIIbβ3-expressing CHO cells further augmented THD-induced αIIbβ3 activation, whereas they did not induce αIIbβ3 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.

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ATP allosterically stabilizes integrin-linked kinase for efficient force generation.

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ATP allosterically stabilizes integrin-linked kinase for efficient force generation.

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