Akt activation by Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) in ovarian cancer cells

作者：Gocher Angela M; Azabdaftari Gissou; Euscher Lindsey M; Dai Shuhang; Karacosta Loukia G; Franke Thomas F; Edelman Arthur M^*

来源：JOURNAL OF BIOLOGICAL CHEMISTRY, 2017, 292(34): 14188-14204.

DOI：10.1074/jbc.M117.778464

• Akt PKB
• calmodulin (CaM)
• ovarian cancer
• phosphorylation
• protein kinase

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 Ca2+/calmodulin (CaM)-dependent protein kinase kinase 2 () (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(1)/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 Ca2+/CaM-dependent manner. In OVCa cells, p-Akt Thr-308 was significantly inhibited by intracellular Ca-i(2+) chelation or CaM inhibition. Ionomycin-induced Ca2+ 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.

• 出版日期2017-8-25