A FUNCTIONAL-ROLE FOR MITOCHONDRIAL PROTEIN-KINASE-C-ALPHA IN BCL2 PHOSPHORYLATION AND SUPPRESSION OF APOPTOSIS

Phosphorylation of Bc12 at serine 70 may result from activation of a c lassic protein kinase C (PKC) isoform and is required for functional s uppression of apoptosis by Bcl2 in murine growth factor-dependent cell lines (Ito, T., Deng, X, Carr, B,, and May, W. S, (1997) J. Biol, Che m. 272, 11671-11673), Human pre-B REH cells ex press high levels of Bc l2 yet remain sensitive to the chemotherapeutic agents etoposide, cyto sine arabinoside, and Adriamycin. In contrast, myeloid leukemia-derive d HL60 cells express less than half the level of Bcl-2 but are >10-fol d more resistant to apoptosis induced by these drugs, The mechanism re sponsible for this apparent dichotomy appears to involve a deficiency of mitochondrial PKC alpha since 1) HL60 but not REH cells contain hig hly phosphorylated Bcl2; 2) PKC alpha is the only classical isoform co -localized with Bcl2 in HL60 but not REH mitochondrial membranes; 3) t he natural product and potent PKC activator bryostatin-1 induces mitoc hondrial localization of PKC alpha in association with Bcl2 phosphoryl ation 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 e xpression 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 ce lls 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 phospho rylation, and REH cell survival following chemotherapy, While these fi ndings strongly support a role for PKC alpha as a functional Bcl2 kina se that can enhance cell resistance to antileukemic chemotherapy, they do not exclude the possibility that another Bcl2 kinase(s) may also e xist. Collectively, these findings identify a functional role for PKC alpha in Bcl2 phosphorylation and in resistance to chemotherapy and su ggest a novel target for antileukemic strategies.