PhosphoMARCKS drives motility of mouse melanoma cells.
Phosphorylation of myristoylated alanine-rich C-kinase substrate (MARCKS) by protein kinase C alpha (PKC alpha) is known to trigger its release from the plasma membrane/cytoskeleton into the cytoplasm, thereby promoting actin reorganization during migration. This study shows that once released into the cytoplasm, phosphoMARCKS directly promotes motility of melanoma cells. Aggressively motile B16 F10 mouse melanoma cells express high levels of phosphoMARCKS, whereas in weakly motile B16 F1 cells it is undetectable. Following treatment with okadaic acid (OA) (a protein phosphatase inhibitor), F1 cells exhibited a dramatic increase in phosphoMARCKS that was co-incident with a 5-fold increase in motility. Both MARCKS phosphorylation and motility were substantially decreased when prior to OA addition, MARCKS expression was knocked out by a MARCKS-specific shRNA, thereby implicating MARCKS as a major component of the motility pathway. Decreased motility and phosphoMARCKS levels in OA-treated cells were observed with a PKC inhibitor (calphostin C), thus indicating that PKC actively phosphorylates MARCKS in F1 cells but that this reaction is efficiently reversed by protein phosphatases. The mechanistic significance of phosphoMARCKS to motility was further established with a pseudo-phosphorylated mutant of MARCKS-GFP in which Asp residues replaced Ser residues known to be phosphorylated by PKC alpha. This mutant localized to the cytoplasm and engendered three-fold higher motility in F1 cells. Expression of an unmyristoylated, phosphorylation-resistant MARCKS mutant that localized to the cytoplasm, blocked motility by 40-50% of both OA-stimulated F1 cells and intrinsically motile F10 cells. These results demonstrate that phosphoMARCKS contributes to the metastatic potential of melanoma cells, and reveal a previously undocumented signaling role for this cytoplasmic phospho-protein.
Chen
Xiangyu
X
Department of Chemistry and Biochemistry, Queens College, The City University of New York, NY 11367, USA.
Rotenberg
Susan A
SA
D000818:Animals; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D008546:Melanoma, Experimental / Q000378:metabolism* / Q000473:pathology; D008565:Membrane Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D009154:Mutation; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D009362:Neoplasm Metastasis; D019319:Okadaic Acid / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects
D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; C000619711:Marcks protein, mouse; D008565:Membrane Proteins; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D019319:Okadaic Acid