The Integrin-Linked Kinase-PINCH-Parvin Complex Supports Integrin αIIbβ3 Activation

Abstract

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.

Molecular Basis of Kindlin-2 Binding to Integrin-linked Kinase Pseudokinase for Regulating Cell Adhesion

“…Our results demonstrate that ILK had no effect in modulating integrin ␣IIb␤3 activation with or without associating with kindlin-2 and/or talin. We cannot exclude the possibility that ILK may be involved in activating integrins by associating with other binding partners such as PINCH and Parvin as suggested before (35). Nevertheless, our results are consistent with vast majority of studies indicating that ILK is involved in integrin outside-in signaling (for review, see Refs.…”

“…we showed that the interaction is not required for mediating integrin activation/inside-out signaling but is crucial for integrin outside-in signaling involving FA assembly and cytoskeleton reorganization (cell spreading). The former finding is surprising given the previously implicated role of both kindlin-2 (19 -21) and ILK (33)(34)(35) in integrin activation. The kindlin-2-mediated integrin activation is well accepted and confirmed in a large number of studies (for review, see Refs.…”

“…14 -16, 37, and 38), but the role of ILK in mediating integrin activation remains controversial. Although ILK was previously indicated to play a role in integrin ␣IIb␤3 activation (33)(34)(35), a more recent study demonstrated that ILK is not essential for integrin ␣IIb␤3-mediated platelet activation (39). Instead, it may be involved in regulating the rate of platelet activation and spreading (39).…”

“…Given the previously suggested role of ILK in integrin activation (34,35), we also examined whether ILK directly cooperates with talin-H to mediate ␣ IIb ␤ 3 integrin activation. Co-expression of ILK with talin-H did not enhance integrin activation beyond that observed with talin-H alone (Fig.…”

Integrin αIIbβ3 outside-in signaling

“…120 ILK and the IPP complex support a IIb b 3 activation, 134 whereas ILK also supports a IIb b 3 -mediated clot retraction. 22,135,136 Recruitment of the Arp2/3 proteins to the integrin adhesome via interactions with FAK and vinculin could allow local regulation of actin polymerization, which may also be achieved through a/b-PAK-interactive exchange factor regulation of Arp2/3 via Rac/Cdc42.…”