Abstract 4553: Gβγ subunits inhibit Epac-induced melanoma cell migration

Abstract

Abstract 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 subunits (Gβγ) are known to act as an independent signaling molecule upon activation of G-protein coupled receptor. However, the role of Gβγ in Ca2+ signaling and migration in melanoma has not been well studied. Here we report that there is crosstalk of Ca2+ signaling between Gβγ and Epac1 in melanoma, which results in inhibition of Epac-induced cell migration. Methods: Human metastatic melanoma cell lines, SK-Mel-2, SK-Mel-24, SK-Mel-187, c8161, WM1552C, and WM1361A, were used in this study. Intracellular Ca2+ level was measured with Fura-2AM and/or Fluo-4AM fluorescent dyes. Cell migration was examined with the Boyden chambers. Gβ1 and Gγ2 subunits were overexpressed with plasmid harboring each subunit. C-terminus of β adrenergic receptor kinase (βARK-CT), which inhibits Gβγ, was overexpressed with adenovirus. Results: A Gβγ -activating peptide, mSIRK, increased cytosolic Ca2+, in all melanoma cell lines. mSIRK induced Ca2+ entry was inhibited by depletion of extracellular Ca2+, suggesting that Gβγ activates Ca2+ influx from extracellular space. When cells were pretreated with mSIRK, but not with mSIRK (L9A), a non-functional mSIRK analogue, 8-(4-Methoxyphenylthio)-2′-O-methyladenosine- 3′,5′-cyclic monophosphate (8-pMeOPT), an Epac-specific agonist, failed to increase cytosolic Ca2+. In addition, co-overexpression of β1 and βγ2, which is a major combination of Gβγ, also inhibited 8-pMeOPT-induced Ca2+ elevation. Inhibition of Gβγ with βARK-CT, an inhibiting-peptide for Gβγ, or guanosine 5′-O-(2-thiodiphosphate) (GDPβS), a GDP analogue that inactivates Gβγ, recovered 8-pMeOPT-induced Ca2+ elevation. These data suggested that Gβγ inhibits Epac-induced Ca2+ release. We then examined the effect of Gβγ on Epac-induced cell migration. Epac-induced cell migration was decreased by mSIRK. Gp(CH)2pp (Guanosine 5′, α-β-methylene triphosphate), a constitutively active GTP analogue, also inhibited Epac-induced cell migration. Further, co-overexpression of Gβ1 and Gγ2 subunits inhibited Epac1-induced cell migration. By contrast, when βARK-CT was overexpressed, mSIRK's inhibitory effect on cell migration was negated. These data suggested that Gβγ has an inhibitory effect on Epac-induced melanoma cell migration. Finally, W-5, an inhibitor for calmodulin, negated mSIRK's inhibitory effects on Epac-induced Ca2+ elevation, and cell migration, suggesting involvement of calmodulin in the crosstalk of Ca2+ signaling between Gβγ and Epac. Conclusion: Gβγ evokes Ca2+ entry from extracellular space, which results in suppression of Epac-induced cytosolic Ca2+ elevation and cell migration in melanoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4553. doi:10.1158/1538-7445.AM2011-4553