Protein kinase C-regulated dynamitin-macrophage-enriched myristoylated alanine-rice C kinase substrate interaction is involved in macrophage cell spreading.
Macrophage spreading requires the microtubule cytoskeleton and protein kinase C (PKC). The mechanism of involvement of the microtubules and PKC in this event is not fully understood. Dynamitin is a subunit of dynactin, which is important for linking the microtubule-dependent motor protein dynein to vesicle membranes. We report that dynamitin is a Ca(2+)/calmodulin-binding protein and that dynamitin binds directly to macrophage-enriched myristoylated alanine-rice C kinase substrate (MacMARCKS), a membrane-associated PKC substrate involved in macrophage spreading and integrin activation. Dynamitin was found to copurify with MacMARCKS both during MacMARCKS purification with conventional chromatography and during the immunoabsorption of MacMARCKS using anti-MacMARCKS antibody. Vice versa, MacMARCKS was also found to cosediment with the 20 S dynactin complex. We determined that the effector domain of MacMARCKS is required to interact with the N-terminal domain of dynamitin. MacMARCKS and dynamitin also partially colocalized at peripheral regions of macrophages and in the cell-cell border of 293 epithelial cells. Treatment with phorbol esters abolished this colocalization. Disrupting the interaction with a short peptide derived from the MacMARCKS-binding domain of dynamitin caused macrophages to spread and flatten. These data suggest that the dynamitin-MacMARCKS interaction is involved in cell spreading. Furthermore, the regulation of this interaction by PKC and Ca(2+)/calmodulin provides a possible regulatory mechanism for cell adhesion and spreading.
Yue
L
L
Department of Oral Biology, College of Dentistry, the Cancer Center and Department of Microbiology & Immunology, College of Medicine, University of Illinois at Chicago, Chicago, Illinois 60612, USA.
Lu
S
S
Garces
J
J
Jin
T
T
Li
J
J
D000251:Adenosine Triphosphatases; D000595:Amino Acid Sequence; D001483:Base Sequence; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D002448:Cell Adhesion / Q000502:physiology; D048429:Cell Size; D000072159:Dynactin Complex; D006801:Humans; D008264:Macrophages / Q000166:cytology / Q000502:physiology*; D008565:Membrane Proteins / Q000378:metabolism*; D008840:Microfilament Proteins; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D008870:Microtubules / Q000502:physiology; D008969:Molecular Sequence Data; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D020539:Sequence Analysis, Protein; D015398:Signal Transduction; D020798:Two-Hybrid System Techniques
D002135:Calcium-Binding Proteins; D002148:Calmodulin-Binding Proteins; C000606361:DCTN2 protein, human; D000072159:Dynactin Complex; C070177:MARCKSL1 protein, human; D008565:Membrane Proteins; D008840:Microfilament Proteins; D008869:Microtubule-Associated Proteins; D011493:Protein Kinase C; D000251:Adenosine Triphosphatases