Protein kinase C regulates MARCKS cycling between the plasma membrane and lysosomes in fibroblasts.
MARCKS is a protein kinase C (PKC) substrate that is phosphorylated during neurosecretion, phagocyte activation and growth factor-dependent mitogenesis. MARCKS binds calcium/calmodulin and crosslinks F-actin, and both these activities are regulated by PKC-dependent phosphorylation. We present evidence here that PKC-dependent phosphorylation also regulates the cycling of MARCKS between the plasma membrane and Lamp-1-positive lysosomes. Immuno-fluorescence and immunoelectron microscopy, and subcellular fractionation, demonstrated that MARCKS was predominantly associated with the plasma membrane of resting fibroblasts. Activation of PKC resulted in MARCKS phosphorylation and its displacement from the plasma membrane to Lamp-1-positive lysosomes. MARCKS phosphorylation is required for its translocation to lysosomes since mutating either the serine residues phosphorylated by PKC (phos-) or the PKC inhibitor staurosporine, prevented MARCKS phosphorylation, its release from the plasma membrane, and its subsequent association with lysosomes. In the presence of lysosomotropic agents or nocodazole, MARCKS accumulated on lysosomes and returned to the plasma membrane upon drug removal, further suggesting that the protein cycles between the plasma membrane and lysosomes. In contrast to wild-type MARCKS, the phos- mutant did not accumulate on lysosomes in cells treated with NH4Cl, suggesting that basal phosphorylation of MARCKS promotes its constitutive cycling between these two compartments.
Allen
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Laboratory of Signal Transduction, Rockefeller University, New York, NY 10021, USA.
Aderem
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D000470:Alkaloids / Q000494:pharmacology; D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D015703:Antigens, CD; D001692:Biological Transport / Q000187:drug effects; D002462:Cell Membrane / Q000378:metabolism* / Q000648:ultrastructure; D004789:Enzyme Activation; D005347:Fibroblasts / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins; D051907:Lysosomal Membrane Proteins; D008247:Lysosomes / Q000378:metabolism* / Q000648:ultrastructure; D008562:Membrane Glycoproteins / Q000032:analysis; D008565:Membrane Proteins; D051379:Mice; D008813:Mice, Inbred ICR; D008870:Microtubules / Q000187:drug effects; D009154:Mutation / Q000502:physiology; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D015739:Nocodazole / Q000494:pharmacology; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D019311:Staurosporine
D000470:Alkaloids; D015703:Antigens, CD; D047908:Intracellular Signaling Peptides and Proteins; D051907:Lysosomal Membrane Proteins; C000619711:Marcks protein, mouse; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D011506:Proteins; D000643:Ammonium Chloride; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D011493:Protein Kinase C; D019311:Staurosporine; D015739:Nocodazole