Akt Activation by Ca2+/calmodulin-dependent protein Kinase Kinase 2 (CaMKK2) in Ovarian Cancer Cells

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

doi:10.1074/jbc.M117.778464

Akt Activation by Ca2+/calmodulin-dependent protein Kinase Kinase 2 (CaMKK2) in Ovarian Cancer Cells

¹ State University of New York at Buffalo, United States;
² Roswell Park Cancer Institute, United States;
³ New York University School of Medicine, United States

↵* Corresponding author; email: aedelman{at}buffalo.edu

Author contributions: AMG designed the study, performed all of its experiments, analyzed the data and wrote the manuscript. GA evaluated the immunohistochemistry of ovarian cancer specimens. LME contributed to Figure S1, 3 and provided additional technical assistance. SD contributed to Figure S5B, C and provided additional contributions to the design of the study. LGK contributed to the design of the study. TFF provided purified, wild type and mutant, recombinant Akt enzymes. AME designed the study, wrote the manuscript and coordinated the study. All authors approved the final version of the manuscript.

Abstract

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, Thr308. We observed that Ca2+/calmodulin (CaM)-dependent protein kinase kinase 2 (beta) (CaMKK2) is highly expressed in high-grade serous ovarian cancer and investigated its role in Akt activation in ovarian cancer (OVCa) cell lines (OVCAR-3, SKOV-3, 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 the G1/S transition and apoptosis. CaMKK2 knockdown or inhibition, decreased Akt phosphorylation at Thr308 and Ser473 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 Thr450, p-PDK1 Ser241 or p-IRS1 Ser636/639. Recombinant CaMKK2 directly activated recombinant Akt by phosphorylation at Thr308 in a Ca2+/CaM-dependent manner. In OVCa cells, p-Akt Thr308 was significantly inhibited by intracellular Ca2+i -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.