A functional role for mitochondrial protein kinase Cα in Bcl2 phosphorylation and suppression of apoptosis

Phosphorylation of Bc12 at serine 70 may result from activation of a classic protein kinase C (PKC) isoform and is required for functional suppression of apoptosis by Bcl2 in murine growth factor-dependent cell lines (Ito, T., Deng, X, Carr, B,, and May, W. S, (1997) J. Biol, Chem. 272, 11671-11673), Human pre-B REH cells ex press high levels of Bcl2 yet remain sensitive to the chemotherapeutic agents etoposide, cytosine arabinoside, and Adriamycin. In contrast, myeloid leukemia-derived HL60 cells express less than half the level of Bcl-2 but are >10-fold more resistant to apoptosis induced by these drugs, The mechanism responsible for this apparent dichotomy appears to involve a deficiency of mitochondrial PKC alpha since 1) HL60 but not REH cells contain highly phosphorylated Bcl2; 2) PKC alpha is the only classical isoform co-localized with Bcl2 in HL60 but not REH mitochondrial membranes; 3) the natural product and potent PKC activator bryostatin-1 induces mitochondrial localization of PKC alpha in association with Bcl2 phosphorylation and increased REH cell resistance to drug-induced apoptosis; 4) PKC alpha can directly phosphorylate wild-type but not phosphorylation-negative and loss of function S70A Bcl2 in vitro; 5) stable, forced expression of exogenous PKC alpha induces mitochondrial localization of PKC alpha, increased Bcl2 phosphorylation and a >10-fold increase in resistance to drug-induced cell death; and (6) PKC alpha-transduced cells remain highly sensitive to staurosporine, a potent PKC inhibitor. Furthermore, treatment of the PKC alpha transformants with bryostatin-1 leads to even higher levels of mitochondrial PKC alpha, Bcl2 phosphorylation, and REH cell survival following chemotherapy, While these findings strongly support a role for PKC alpha as a functional Bcl2 kinase that can enhance cell resistance to antileukemic chemotherapy, they do not exclude the possibility that another Bcl2 kinase(s) may also exist. Collectively, these findings identify a functional role for PKC alpha in Bcl2 phosphorylation and in resistance to chemotherapy and suggest a novel target for antileukemic strategies.