Dynamitin controls Beta 2 integrin avidity by modulating cytoskeletal constraint on integrin molecules.
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.
Jin
Tianquan
T
Department of Oral Biology, College of Dentistry, University of Illinois at Chicago, Chicago, Illinois 60612, USA.
Li
Jianxun
J
D000818:Animals; D018821:CD18 Antigens / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins; D002460:Cell Line; D003598:Cytoskeletal Proteins / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism*; D000072159:Dynactin Complex; D004791:Enzyme Inhibitors / Q000494:pharmacology; D049452:Green Fluorescent Proteins; D006801:Humans; D016023:Integrins / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins; D008024:Ligands; D008164:Luminescent Proteins / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D008840:Microfilament Proteins; D008856:Microscopy, Fluorescence; D018715:Microscopy, Video; D008869:Microtubule-Associated Proteins / Q000502:physiology*; D008870:Microtubules / Q000378:metabolism; D051419:Paxillin; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D019311:Staurosporine / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
D018821:CD18 Antigens; D002148:Calmodulin-Binding Proteins; D003598:Cytoskeletal Proteins; C000606361:DCTN2 protein, human; C000606365:Dctn2 protein, mouse; D000072159:Dynactin Complex; D004791:Enzyme Inhibitors; D016023:Integrins; D047908:Intracellular Signaling Peptides and Proteins; D008024:Ligands; D008164:Luminescent Proteins; C070177:MARCKSL1 protein, human; C512945:Marcksl1 protein, mouse; D008565:Membrane Proteins; D008840:Microfilament Proteins; D008869:Microtubule-Associated Proteins; C496061:PXN protein, human; D051419:Paxillin; D010750:Phosphoproteins; C496062:Pxn protein, mouse; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D014443:Tyrosine; D011493:Protein Kinase C; D020742:rhoA GTP-Binding Protein; D019311:Staurosporine