Dynamitin Controls β2 Integrin Avidity by Modulating Cytoskeletal Constraint on Integrin Molecules

Tianquan Jin,Jianxun Li

doi:10.1074/jbc.m201525200

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 beta(2) integrin activation. We report that dynamitin regulates beta(2) integrin avidity toward iC3b by modulating the lateral mobility of beta(2) 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 beta(2) 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 beta(2) 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 beta(2) integrin in the inactive form.

Highlights

In resting leukocytes, ␤2 integrin molecules are immobilized on the cell membrane in a diffused distribution, because they are constrained by cytoskeletal complex [13,14,15]
We found that the increased avidity is likely the result of the disruption of cytoskeletal constraint on integrin molecules and subsequent enhancement of integrin molecular mobility, which is linked to protein kinase C (PKC) and Rho GTPase in the signal transduction pathway leading to integrin activation
Overexpression of Dynamitin Modulates Protein Phosphorylation in the Integrin Activation Pathways—To investigate the mechanism of how dynamitin is involved in regulating the cytoskeletal constraint on integrin molecules, we examined the effect of dynamitin expression on protein phosphorylation related to integrin activation

Summary

Introduction

In resting leukocytes, ␤2 integrin molecules are immobilized on the cell membrane in a diffused distribution, because they are constrained by cytoskeletal complex [13,14,15]. How dynamitin is involved in cell adhesion is unclear despite the fact that both MacMARCKS and microtubules regulate ␤2 integrin activation. Small GTP-binding proteins are clearly involved in the microtubule-dependent integrin activation and cell spreading (34 –37).

Results

Conclusion