Expression of the protein kinase C substrate pleckstrin in macrophages: association with phagosomal membranes.
Despite evidence suggesting that protein kinase C (PKC) isoforms are important in phagocytosis by Fcgamma receptors, the mechanisms by which the substrates of these kinases act are largely unknown. We have investigated the role of one PKC substrate, pleckstrin, in cells of the monocyte/macrophage lineage. Pleckstrin expression in mouse macrophages was induced severalfold in response to bacterial LPS and IFN-gamma. In unstimulated cells, the protein was largely confined to the cytosol. Upon ingestion of IgG-opsonized zymosan particles (OPZ), however, pleckstrin accumulated on the phagosomal membrane. This association was transient, being maximal after 15 min and declining thereafter. Similar kinetics of association was also seen for both filamentous actin and the delta isoform of PKC. Ingestion of OPZ was found to induce phosphorylation of pleckstrin. To examine whether phosphorylation was required for phagosomal association, pleckstrin was expressed in CHO-IIA cells that stably express the FcgammaRIIA receptor and are competent for phagocytosis of OPZ. In these cells, both wild-type pleckstrin and mutants in which the phosphoacceptor sites had been mutated to either alanine (nonphosphorylatable) or glutamine (pseudophosphorylated) were found to accumulate on OPZ phagosomes. Thus, association of pleckstrin with phagosomes is independent of its phosphorylation. Our findings suggest that pleckstrin may serve as an intracellular adaptor/targeting protein in response to particulate stimuli. By targeting interacting ligands to the phagosomal compartment, pleckstrin may serve to regulate phagocytosis and/or early steps during maturation of the phagosome.
Brumell
J H
JH
Programme in Molecular Biology and Cancer, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada.
Howard
J C
JC
Craig
K
K
Grinstein
S
S
Schreiber
A D
AD
Tyers
M
M
D000818:Animals; D001792:Blood Platelets / Q000378:metabolism; D001798:Blood Proteins / Q000096:biosynthesis* / Q000378:metabolism; D016466:CHO Cells; D002460:Cell Line; D019070:Cell Lineage; D006224:Cricetinae; D007074:Immunoglobulin G / Q000378:metabolism; D007371:Interferon-gamma / Q000494:pharmacology; D007425:Intracellular Membranes / Q000201:enzymology / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000494:pharmacology; D008262:Macrophage Activation / Q000276:immunology; D008264:Macrophages / Q000187:drug effects / Q000201:enzymology / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D009000:Monocytes / Q000378:metabolism; D009895:Opsonin Proteins / Q000378:metabolism; D010587:Phagocytosis; D010588:Phagosomes / Q000201:enzymology / Q000378:metabolism*; D010750:Phosphoproteins; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D013379:Substrate Specificity; D015054:Zymosan / Q000378:metabolism
D001798:Blood Proteins; D007074:Immunoglobulin G; D008070:Lipopolysaccharides; D009895:Opsonin Proteins; D010750:Phosphoproteins; C039077:platelet protein P47; D007371:Interferon-gamma; D015054:Zymosan; D011493:Protein Kinase C