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Protein kinase C-regulated dynamitin-macrophage-enriched myristoylated alanine-rice C kinase substrate interaction is
1Department 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.
Abstract:
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.
Insights
Dynamitin binds to MacMARCKS, a protein kinase C (PKC) substrate, influencing macrophage spreading. This interaction, regulated by PKC and calcium, is crucial for cell adhesion and spreading dynamics.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Macrophage spreading is essential for immune responses and relies on the microtubule cytoskeleton and protein kinase C (PKC).
- The precise roles of microtubules and PKC in macrophage spreading remain unclear.
- Dynamitin, a dynactin subunit, links dynein to membranes, but its function in cell spreading is not well-defined.
Purpose of the Study:
- To investigate the interaction between dynamitin and MacMARCKS (macrophage-enriched myristoylated alanine-rice C kinase substrate).
- To elucidate the role of the dynamitin-MacMARCKS interaction in macrophage cell spreading.
- To explore the regulation of this interaction by PKC and Ca(2+)/calmodulin.
Main Methods:
- Co-purification and co-sedimentation assays to confirm dynamitin-MacMARCKS interaction.
- Identification of interacting domains using protein fragments.
- Immunofluorescence microscopy to assess cellular localization.
- Functional assays using peptides to disrupt the interaction.
Main Results:
- Dynamitin binds directly to MacMARCKS, a PKC substrate involved in cell spreading.
- The effector domain of MacMARCKS interacts with the N-terminal domain of dynamitin.
- Dynamitin and MacMARCKS partially colocalize in macrophages and epithelial cells, with localization affected by phorbol ester treatment.
- Disruption of the dynamitin-MacMARCKS interaction promotes macrophage spreading and flattening.
Conclusions:
- The dynamitin-MacMARCKS interaction is a novel regulator of macrophage cell spreading.
- Regulation of this interaction by PKC and Ca(2+)/calmodulin offers a mechanism for controlling cell adhesion and spreading.
- This finding provides new insights into the molecular mechanisms governing cell motility and integrin activation.