Dynamitin controls β2 integrin avidity by modulating cytoskeletal constraint on integrin molecules

Abstract

Dynamitin, a subunit of the microtubule-dependent motor complex, was implicated in cell adhesion by binding to MacMARCKS (Macrophage-enriched myristoylated alanine-rice C kinase substrate). However, how dynamitin is involved in cell adhesion is unclear despite the fact that both MacMARCKS and microtubules regulate β2 integrin activation. We report that dynamitin regulates β2 integrin avidity toward iC3b by modulating the lateral mobility of β2 integrin molecules. Using the single particle tracking method, we found that integrin molecular mobility in cells expressing the fusion protein CFP (cyan fluorescent protein)-dynamitin or CFP-MB (the MacMARCKS binding domain peptide of dynamitin) increased 6-fold over the control cells, suggesting that disturbing dynamitin function dramatically altered the cytoskeletal constraint on β2 integrin molecules. Further mechanistic studies revealed that overexpression of dynamitin stimulated the phosphorylation of endogenous MacMARCKS protein, which lead to the enhanced tyrosine phosphorylation of paxillin. This effect of dynamitin correlates with the observation that higher concentration of PKC inhibitor is required to block β2 integrin mobility in dynamitin-expressing cells. Although dynamitin acts at the point of MacMARCKS phosphorylation, it is upstream of RhoA, because its effect was blocked by RhoA inhibitor. Thus, we conclude that dynamitin is a part of the cytoskeletal constraint that locks β2 integrin in the inactive form.