title: Gβγ subunits inhibit Epac-induced melanoma cell migration.
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 βγ subunits (Gβγ) are known to act as an independent signaling molecule upon activation of G-protein coupled receptor. However, the role of Gβγ 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 Gβγ 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 Gβγ on Epac-induced cell migration was first examined. Epac-induced cell migration was inhibited by mSIRK, a Gβγ -activating peptide, but not its inactive analog, L9A, in SK-Mel-2 cells. Guanosine 5', α-β-methylene triphosphate (Gp(CH2)pp), a constitutively active GTP analogue that activates Gβγ, also inhibited Epac-induced cell migration. In addition, co-overexpression of β1 and γ2, which is the major combination of Gβγ, inhibited Epac1-induced cell migration. By contrast, when the C-terminus of β adrenergic receptor kinase (βARK-CT), an endogenous inhibitor for Gβγ, was overexpressed, mSIRK's inhibitory effect on Epac-induced cell migration was negated, suggesting the specificity of mSIRK for Gβγ. 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 β1 and γ2 subunits inhibited 8-pMeOPT-induced Ca 2+ elevation. Inhibition of Gβγ with βARK-CT or guanosine 5'-O-(2-thiodiphosphate) (GDPβS), a GDP analogue that inactivates Gβγ, restored 8-pMeOPT-induced Ca 2+ elevation even in the presence of mSIRK. These data suggested that Gβγ inhibits Epac-induced Ca 2+ elevation. Subsequently, the mechanism by which Gβγ 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.
  
  
  CONCLUSIONS
  We found the cross talk of Ca 2+ signaling between Gβγ and Epac, which plays a major role in melanoma cell migration.
authors.affiliation: Department of Cell Biology and Molecular Medicine, New Jersey Medical School-University of Medicine and Dentistry of New Jersey, 185 South Orange Avenue, Newark 07103, USA.
authors.forename: Erdene
authors.initials: E
authors.lastname: Baljinnyam
authors.forename: Masanari
authors.initials: M
authors.lastname: Umemura
authors.forename: Mariana S
authors.initials: MS
authors.lastname: De Lorenzo
authors.forename: Lai-Hua
authors.initials: LH
authors.lastname: Xie
authors.forename: Martha
authors.initials: M
authors.lastname: Nowycky
authors.forename: Mizuka
authors.initials: M
authors.lastname: Iwatsubo
authors.forename: Suzie
authors.initials: S
authors.lastname: Chen
authors.forename: James S
authors.initials: JS
authors.lastname: Goydos
authors.forename: Kousaku
authors.initials: K
authors.lastname: Iwatsubo
mesh_terms: D000595:Amino Acid Sequence; D002121:Calcium Channel Blockers / Q000494:pharmacology; D020013:Calcium Signaling / Q000502:physiology*; D002147:Calmodulin / Q000502:physiology; D045744:Cell Line, Tumor / Q000187:drug effects; D002465:Cell Movement / Q000187:drug effects; D044387:GTP-Binding Protein beta Subunits / Q000235:genetics / Q000502:physiology*; D019204:GTP-Binding Proteins / Q000235:genetics / Q000502:physiology*; D020662:Guanine Nucleotide Exchange Factors / Q000037:antagonists & inhibitors* / Q000502:physiology; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D008545:Melanoma / Q000473:pathology* / Q000556:secondary; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors / Q000502:physiology*; D010446:Peptide Fragments / Q000494:pharmacology; D010455:Peptides / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000502:physiology; D011994:Recombinant Proteins / Q000494:pharmacology; D013873:Thionucleotides / Q000494:pharmacology; D051552:beta-Adrenergic Receptor Kinases / Q000037:antagonists & inhibitors
chemical_list: D002121:Calcium Channel Blockers; D002147:Calmodulin; C516790:GNB1 protein, human; C410342:GNG2 protein, human; D044387:GTP-Binding Protein beta Subunits; D020662:Guanine Nucleotide Exchange Factors; D009363:Neoplasm Proteins; D010446:Peptide Fragments; D010455:Peptides; C116122:RAPGEF3 protein, human; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D013873:Thionucleotides; C445259:beta-adrenergic receptor kinase inhibitory peptide; D006153:Guanosine Diphosphate; C025675:guanosine 5'-(alpha,beta-methylene)triphosphate; C023427:guanosine 5'-O-(2-thiodiphosphate); D006160:Guanosine Triphosphate; D051552:beta-Adrenergic Receptor Kinases; D019204:GTP-Binding Proteins