LAUSR

Background-The canonical PI3K/Akt pathway is hyperactive in a variety of cancers including ovarian cancer (OVCa). Using baculovirus-expressed, FPLC-purified enzymes, we previously demonstrated that Calcium/Calmodulin-dependent CaM Kinase Kinase 2 (CaMKK2) activates the oncogene Akt by direct phosphorylation of its primary activation site T308. In OVCAR-3 and other OVCa cells, RNAi-mediated knockdown of CaMKK2 decreased basal Akt phosphorylation at T308 in a Phosphoinositide-Dependent Kinase-1 (PDK1)-independent manner. CaMKK2 knockdown also decreased Akt phosphorylation at its secondary activation site, S473 by an apparently indirect mechanism. Results- In this study we show that CaMKK2 mediates downstream effects of epidermal growth factor (EGF) on the Akt cascade in OVCa cells. Treatment of OVCAR3 cells with EGF produced a prominent enhancement of p-Akt T308 which was reduced ~2-fold, by either CaMKK2 or PDK1 knockdowns. Knockdown of CaMKK2 but not PDK1 reduced p-Akt S473 after EGF treatment. Similar results were obtained with Insulin-like growth factor 1 (IGF1). To discern how CaMKK2 is engaged by EGF action we examined whether, in vitro, CaMKK2 phosphorylation of WT Akt or a synthetic peptide encompassing T308 is stimulated by Calcium/CaM and/or Phosphatidylinositol (3,4,5)-trisphosphate (PIP3). CaMKK2 activity was enhanced by Calcium/CaM but not by soluble PIP3. CaMKK2 did not bind to a dot blot of various phosphoinositides, although strong binding to PIP3 was observed with PDK1. Alternatively, Akt regulation by CaMKK2 may occur via a Calcium/CaM-dependent pathway. Increased p-Akt T308 and S473 upon treatment of cells with the calcium-ionophore, ionomycin, was significantly reduced by CaMKK2 knockdown. EGF stimulation of Akt phosphorylation at both T308 and S473 was significantly reduced by the intracellular calcium chelator, BAPTA-AM and by the CaM inhibitor, W7. We also probed the mechanism by which optimal cellular Akt phosphorylation at S473 requires CaMKK2. mTOR complex 2 (mTORC2) and DNA-dependent protein kinase (DNA-PKcs) are considered candidate kinases for p-Akt S473. CaMKK2 knockdown regulated p-Akt S473 in M059J cells, a DNA-PKcs null cell line similarly to that observed with OVCAR3 cells, suggesting that mTORC2 may be regulated by CaMKK2. Using an MS/MS Protein-protein interaction (PPI) screen we observed CaMMK2 interaction with multiple ribosomal proteins an observation consistent with suggested mTORC2 ribosomal regulation of Akt S473 phosphorylation. Conclusions-This study documents Akt activating phosphorylation by CaMKK2 in response to EGF in a Calcium/CaM-dependent, PI3K/PIP3 independent, manner. Besides directly phosphorylating Akt at T308, CaMKK2 regulates p-Akt S473 potentially through the regulation of mTORC2 activity. Our results highlight the importance of CaMKK2 activated by growth factor signals as a parallel system to the oncogenic PI3K/PDK1 pathway. Citation Format: Shuhang Dai, Angela M. Gocher, Arthur M. Edelman. CaMKK2 regulates EGF-dependent activation of oncogenic Akt in ovarian cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1430.