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.
Highlights
Myristoylated alanine-rich C-kinase substrate (MARCKS) is a ubiquitously expressed protein kinase C (PKC) substrate that binds both actin and calmodulin (CaM) and regulates actin dynamics
As a positive control for inhibition of migration, fibroblasts were incubated with the phosphoinositide 3-kinase (PI3K) inhibitor wortmannin, which has been shown previously to inhibit NIH3T3 fibroblast migration [28]
These results are the first to demonstrate that the MANS peptide inhibits fibroblast migration and suggest that the amino-terminus of MARCKS may be involved in regulating this process
Summary
Myristoylated alanine-rich C-kinase substrate (MARCKS) is a ubiquitously expressed protein kinase C (PKC) substrate that binds both actin and calmodulin (CaM) and regulates actin dynamics. Phosphorylation of MARCKS by PKC, or CaM binding, results in the release of MARCKS from the plasma membrane into the cytosol in a process called the ‘‘myristoyl-electrostatic switch’’ mechanism [3]. Dephosphorylation or release of CaM results in the ability of MARCKS to return to the plasma membrane This membrane to cytosol shuttling, or bi-lateral translocation of MARCKS, has been associated with the reorganization of the actin cytoskeleton [4,5], with various cellular processes regulated by MARCKS, including: endo- [6], exo- [7], and phagocytosis [8,9], as well as cell migration [10,11]. Expression of a mutated MARCKS in which the myristoylelectrostatic switch mechanism is altered ( inhibiting MARCKS bi-lateral translocation) resulted in abrogated cell adhesion and spreading [12,13]. MARCKS is localized to focal adhesions during a5 integrin myoblast attachment and spreading and silencing of MARCKS resulted in decreased myoblast spreading [21]
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