Protein kinase G type Iα activity in human ovarian cancer cells significantly contributes to enhanced Src activation and DNA synthesis/cell proliferation

Abstract

Previously, we showed that basal activity of nitric oxide (NO)/cyclic GMP (cGMP)/protein kinase G (PKG) signaling pathway protects against spontaneous apoptosis and confers resistance to cisplatin-induced apoptosis in human ovarian cancer cells. The present study determines whether basal PKG kinase activity regulates Src family kinase (SFK) activity and proliferation in these cells. PKG-Iα was identified as predominant isoform in both OV2008 (cisplatin-sensitive, wild-type p53) and A2780cp (cisplatin-resistant, mutated p53) ovarian cancer cells. In both cell lines, ODQ (inhibitor of endogenous NO-induced cGMP biosynthesis), DT-2 (highly specific inhibitor of PKG-Iα kinase activity), and PKG-Iα knockdown (using small interfering RNA) caused concentration-dependent inhibition of DNA synthesis (assessed by bromodeoxyuridine incorporation), indicating an important role of basal cGMP/PKG-Iα kinase activity in promoting cell proliferation. DNA synthesis in OV2008 cells was dependent on SFK activity, determined using highly selective SFK inhibitor, 4-(4′-phenoxyanilino)-6,7-dimethoxyquinazoline (SKI-1). Studies using DT-2 and PKG-Iα small interfering RNA revealed that SFK activity was dependent on PKG-Iα kinase activity. Furthermore, SFK activity contributed to endogenous tyrosine phosphorylation of PKG-Iα in OV2008 and A2780cp cells. In vitro coincubation of recombinant human c-Src and PKG-Iα resulted in c-Src–mediated tyrosine phosphorylation of PKG-Iα and enhanced c-Src autophosphorylation/activation, suggesting that human c-Src directly tyrosine phosphorylates PKG-Iα and the c-Src/PKG-Iα interaction enhances Src kinase activity. Epidermal growth factor–induced stimulation of SFK activity in OV2008 cells increased PKG-Iα kinase activity (indicated by Ser239 phosphorylation of the PKG substrate vasodilator-stimulated phosphoprotein), which was blocked by both SKI-1 and SU6656. The data suggest an important role of Src/PKG-Iα interaction in promoting DNA synthesis/cell proliferation in human ovarian cancer cells. The NO/cGMP/PKG-Iα signaling pathway may provide a novel therapeutic target for disrupting ovarian cancer cell proliferation. Mol Cancer Res; 8(4); 578–91. ©2010 AACR.