Akt activation by Ca 2+ /calmodulin-dependent protein kinase kinase 2 (CaMKK2) in ovarian cancer cells

• Published in: The Journal of biological chemistry Vol. 292; no. 34; p. 14188
• Main Authors: Gocher, Angela M, Azabdaftari, Gissou, Euscher, Lindsey M, Dai, Shuhang, Karacosta, Loukia G, Franke, Thomas F, Edelman, Arthur M
• Format: Journal Article
• Language: English
• Published: United States 25.08.2017
• Subjects: Antineoplastic Agents - pharmacology; Apoptosis - drug effects; Calcium Signaling - drug effects; Calcium-Calmodulin-Dependent Protein Kinase Kinase - antagonists & inhibitors; Calcium-Calmodulin-Dependent Protein Kinase Kinase - genetics; Calcium-Calmodulin-Dependent Protein Kinase Kinase - metabolism; Cell Line, Tumor; Cell Proliferation - drug effects; Cell Survival - drug effects; Enzyme Activation - drug effects; Female; G1 Phase - drug effects; Gene Expression Regulation, Neoplastic - drug effects; Humans; Neoplasm Grading; Neoplasm Proteins - agonists; Neoplasm Proteins - antagonists & inhibitors; Neoplasm Proteins - genetics; Neoplasm Proteins - metabolism; Ovarian Neoplasms - drug therapy; Ovarian Neoplasms - metabolism; Ovarian Neoplasms - pathology; Ovary - drug effects; Ovary - metabolism; Ovary - pathology; Phosphorylation - drug effects; Protein Kinase Inhibitors - pharmacology; Protein Processing, Post-Translational - drug effects; Proto-Oncogene Proteins c-akt - agonists; Proto-Oncogene Proteins c-akt - antagonists & inhibitors; Proto-Oncogene Proteins c-akt - metabolism; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; RNA Interference; ovarian cancer; calmodulin (CaM); Akt PKB; phosphorylation; protein kinase
• ISSN: 1083-351X

Hyperactivation of Akt is associated with oncogenic changes in the growth, survival, and chemoresistance of cancer cells. The PI3K/phosphoinositide-dependent kinase (PDK) 1 pathway represents the canonical mechanism for phosphorylation of Akt at its primary activation site, Thr-308. We observed that Ca /calmodulin (CaM)-dependent protein kinase kinase 2 (β) (CaMKK2) is highly expressed in high-grade serous ovarian cancer, and we investigated its role in Akt activation in ovarian cancer (OVCa) cell lines (OVCAR-3, SKOV-3, and Caov-3). Knockdown or pharmacological inhibition of CaMKK2 produced phenotypes expected of Akt inhibition, including reductions in cell growth and cell viability and in the regulation of Akt downstream targets involved in G /S transition and apoptosis. CaMKK2 knockdown or inhibition decreased Akt phosphorylation at Thr-308 and Ser-473 to extents similar to those of PDK1 knockdown or PI3K inhibition. Combined CaMKK2 and PDK1 knockdown or CaMKK and PI3K inhibition, respectively, produced additive effects on p-Akt and cell growth, consistent with direct Akt phosphorylation by CaMKK2. This conclusion was supported by the absence of effects of CaMKK2 knockdown/inhibition on alternative means of activating Akt via p-Akt Thr-450, p-PDK1 Ser-241, or p-IRS1 Ser-636/639. Recombinant CaMKK2 directly activated recombinant Akt by phosphorylation at Thr-308 in a Ca /CaM-dependent manner. In OVCa cells, p-Akt Thr-308 was significantly inhibited by intracellular Ca chelation or CaM inhibition. Ionomycin-induced Ca influx promoted p-Akt, an effect blocked by PDK1, and/or CaMKK2, siRNAs, and by PI3K and/or CaMKK inhibitors. CaMKK2 knockdown potentiated the effects of the chemotherapeutic drugs carboplatin and PX-866 to reduce proliferation and survival of OVCa cells.