Gβγ subunits inhibit Epac-induced melanoma cell migration

Background: Recently we reported that activation of Epac1, an exchange protein activated by cAMP, increases melanoma cell migration via Ca (2+) release from the endoplasmic reticulum (ER). G-protein beta gamma subunits (G beta gamma) are known to act as an independent signaling molecule upon activation of G-protein coupled receptor. However, the role of G beta gamma in cell migration and Ca (2+) signaling in melanoma has not been well studied. Here we report that there is crosstalk of Ca (2+) signaling between G beta gamma and Epac in melanoma, which plays a role in regulation of cell migration. Methods: SK-Mel-2 cells, a human metastatic melanoma cell line, were mainly used in this study. Intracellular Ca (2+) was measured with Fluo-4AM fluorescent dyes. Cell migration was examined using the Boyden chambers. Results: The effect of G beta gamma on Epac-induced cell migration was first examined. Epac-induced cell migration was inhibited by mSIRK, a G beta gamma -activating peptide, but not its inactive analog, L9A, in SK-Mel-2 cells. Guanosine 5', alpha-beta-methylene triphosphate (Gp(CH2)pp), a constitutively active GTP analogue that activates G beta gamma, also inhibited Epac-induced cell migration. In addition, co-overexpression of beta 1 and gamma 2, which is the major combination of G beta gamma, inhibited Epac1-induced cell migration. By contrast, when the C-terminus of beta adrenergic receptor kinase (beta ARK-CT), an endogenous inhibitor for G beta gamma, was overexpressed, mSIRK's inhibitory effect on Epac-induced cell migration was negated, suggesting the specificity of mSIRK for G beta gamma. We next examined the effect of mSIRK on Epac-induced Ca (2+) response. When cells were pretreated with mSIRK, but not with L9A, 8-(4-Methoxyphenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (8-pMeOPT), an Epac-specific agonist, failed to increase Ca (2+) signal. Co-overexpression of beta 1 and gamma 2 subunits inhibited 8-pMeOPT-induced Ca (2+) elevation. Inhibition of G beta gamma with beta ARK-CT or guanosine 5'-O-(2-thiodiphosphate) (GDP beta S), a GDP analogue that inactivates G beta gamma, restored 8-pMeOPT-induced Ca (2+) elevation even in the presence of mSIRK. These data suggested that G beta gamma inhibits Epac-induced Ca (2+) elevation. Subsequently, the mechanism by which G beta gamma inhibits Epac-induced Ca (2+) elevation was explored. mSIRK activates Ca (2+) influx from the extracellular space. In addition, W-5, an inhibitor of calmodulin, abolished mSIRK's inhibitory effects on Epac-induced Ca (2+) elevation, and cell migration. These data suggest that, the mSIRK-induced Ca (2+) from the extracellular space inhibits the Epac-induced Ca (2+) release from the ER, resulting suppression of cell migration. Conclusion: We found the cross talk of Ca (2+) signaling between G beta gamma and Epac, which plays a major role in melanoma cell migration.