Fibroblast Migration Is Regulated by Myristoylated Alanine-Rich C-Kinase Substrate (MARCKS) Protein.

Abstract

Myristoylated alanine-rich C-kinase substrate (MARCKS) is a ubiquitously expressed substrate of protein kinase C (PKC) that is involved in reorganization of the actin cytoskeleton. We hypothesized that MARCKS is involved in regulation of fibroblast migration and addressed this hypothesis by utilizing a unique reagent developed in this laboratory, the MANS peptide. The MANS peptide is a myristoylated cell permeable peptide corresponding to the first 24-amino acids of MARCKS that inhibits MARCKS function. Treatment of NIH-3T3 fibroblasts with the MANS peptide attenuated cell migration in scratch wounding assays, while a myristoylated, missense control peptide (RNS) had no effect. Neither MANS nor RNS peptide treatment altered NIH-3T3 cell proliferation within the parameters of the scratch assay. MANS peptide treatment also resulted in inhibited NIH-3T3 chemotaxis towards the chemoattractant platelet-derived growth factor-BB (PDGF-BB), with no effect observed with RNS treatment. Live cell imaging of PDGF-BB induced chemotaxis demonstrated that MANS peptide treatment resulted in weak chemotactic fidelity compared to RNS treated cells. MANS and RNS peptides did not affect PDGF-BB induced phosphorylation of MARCKS or phosphoinositide 3-kinase (PI3K) signaling, as measured by Akt phosphorylation. Further, no difference in cell migration was observed in NIH-3T3 fibroblasts that were transfected with MARCKS siRNAs with or without MANS peptide treatment. Genetic structure-function analysis revealed that MANS peptide-mediated attenuation of NIH-3T3 cell migration does not require the presence of the myristic acid moiety on the amino-terminus. Expression of either MANS or unmyristoylated MANS (UMANS) C-terminal EGFP fusion proteins resulted in similar levels of attenuated cell migration as observed with MANS peptide treatment. These data demonstrate that MARCKS regulates cell migration and suggests that MARCKS-mediated regulation of fibroblast migration involves the MARCKS amino-terminus. Further, this data demonstrates that MANS peptide treatment inhibits MARCKS function during fibroblast migration and that MANS mediated inhibition occurs independent of myristoylation.