Abstract
Integrin-linked kinase (ILK) is involved in signal transduction by integrin-mediated cell adhesion that leads to dynamic actin reorganization. Actin (de)polymerization is regulated by cofilin, the Ser(3) phosphorylation (pS(3)cofilin) of which inhibits its actin-severing activity. To determine how ILK regulates pS(3)cofilin, we examined the effects of ILK on pS(3)cofilin using normal RIE1 cells. Compared with suspended cells, fibronectin-adherent cells showed enhanced pS(3)cofilin, depending on ILK expression and c-Src activity. The ILK-mediated pS(3)cofilin in RIE1 cells did not involve Rho-associated kinase, LIM kinase, or testicular protein kinases, which are known to be upstream of cofilin. The kinase domain of ILK, including proline-rich regions, appeared to interact physically with the Src homology 3 domain of c-Src. In vitro kinase assay revealed that ILK immunoprecipitates phosphorylated the recombinant glutathione S-transferase-cofilin, which was abolished by c-Src inhibition. Interestingly, epidermal growth factor treatment abolished the ILK effects, indicating that the linkage from ILK to cofilin is biologically responsive to extracellular cues. Altogether, this study provides evidence for a new signaling connection from ILK to cofilin for dynamic actin polymerization during cell adhesion, depending on the activity of ILK-associated c-Src.
Highlights
Integrin-mediated interaction with the extracellular matrix triggers intracellular signal cascades that regulate the activity and localization of numerous signaling molecules that modulate diverse cellular functions including actin reorganization [1, 2]
Integrin-linked kinase (ILK) is a Ser/Thr kinase located at focal adhesions by binding to the 1 integrin cytoplasmic tail, the overexpression of which inhibits intestinal epithelial cell adhesion to integrin substrates [8]
ILK-mediated Cofilin Phosphorylation upon Cell Adhesion— ILK and cofilin are both known to be involved in actin organization, their functional linkage remains largely unknown
Summary
Integrin-mediated interaction with the extracellular matrix triggers intracellular signal cascades that regulate the activity and localization of numerous signaling molecules that modulate diverse cellular functions including actin reorganization [1, 2]. We performed an in vitro pulldown assay using recombinant GST-c-Src proteins and RIE1-␣5 extracts to determine whether ILK directly interacts with c-Src. ILK from only Fn-adherent (but not suspended) cells bound to the WT GST-c-Src, which was abolished by PP2 treatment (Fig. 3B, left panel).
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