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Protein-protein interactions and/or signaling activities at focal adhesions, where integrin-mediated adhesion to extracellular matrix occurs, are critical for the regulation of adhesion-dependent cellular functions. Although the phosphorylation and activities of focal adhesion molecules have been intensively studied, the effects of the O-GlcNAc modification of their Ser/Thr residues on cellular functions have been largely unexplored. We investigated the effects of O-GlcNAc modification on actin reorganization and morphology of rat insulinoma INS-1 cells after glucosamine (GlcN) treatment. We found that paxillin, a key adaptor molecule in focal adhesions, could be modified by O-GlcNAc in INS-1 cells treated with GlcN and in pancreatic islets from mice treated with streptozotocin. Ser-84/85 in human paxillin appeared to be modified by O-GlcNAc, which was inversely correlated to Ser-85 phosphorylation (Ser-83 in rat paxillin). Integrin-mediated adhesion signaling inhibited the GlcN treatment-enhanced O-GlcNAc modification of paxillin. Adherent INS-1 cells treated with GlcN showed restricted protrusions, whereas untreated cells showed active protrusions for multiple-elongated morphologies. Upon GlcN treatment, expression of a triple mutation (S83A/S84A/S85A) resulted in no further restriction of protrusions. Together these observations suggest that murine pancreatic β cells may have restricted actin organization upon GlcN treatment by virtue of the O-GlcNAc modification of paxillin, which can be antagonized by a persistent cell adhesion process.

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.