A Functional Role for Mitochondrial Protein Kinase Cα in Bcl2 Phosphorylation and Suppression of Apoptosis

Abstract

Phosphorylation of Bcl2 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 express high levels of Bcl2 yet remain sensitive to the chemotherapeutic agents etoposide, cytosine arabinoside, and Adriamycin. In contrast, myeloid leukemiaderived 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␣ since 1) HL60 but not REH cells contain highly phosphorylated Bcl2; 2) PKC␣ 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␣ in association with Bcl2 phosphorylation and increased REH cell resistance to drug-induced apoptosis; 4) PKC␣ can directly phosphorylate wild-type but not phosphorylation-negative and loss of function S70A Bcl2 in vitro; 5) stable, forced expression of exogenous PKC␣ induces mitochondrial localization of PKC␣, increased Bcl2 phosphorylation and a >10-fold increase in resistance to drug-induced cell death; and (6) PKC␣-transduced cells remain highly sensitive to staurosporine, a potent PKC inhibitor. Furthermore, treatment of the PKC␣ transformants with bryostatin-1 leads to even higher levels of mitochondrial PKC␣, Bcl2 phosphorylation, and REH cell survival following chemotherapy. While these findings strongly support a role for PKC␣ 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␣ in Bcl2 phosphorylation and in resistance to chemotherapy and suggest a novel target for antileukemic strategies.

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“…28,34,35,47,49 These results indicate that paclitaxel induces a multisite, hyperphosphorylated form of Bcl2 vs the mono-site phosphorylation that is associated with cell survival (Figure 2). Further, the paclitaxel-induced shifted band Bcl2 is stable while IL-3 or bryoinduced phosphorylation is dynamic.…”

“…26,27 Interestingly, a comparison of the human pre-B acute leukemia cell line REH with the myeloid HL60 cell line, derived from a patient with acute promyelocytic leukemia, reveals that the REH cells are at least 10-fold more sensitive to the clinically useful chemotherapeutic drugs adriamycin, ara-C, and etoposide. 28 The relative chemosensitivity of the REH cells (compared to the HL60 cells) initially appears inconsistent with expectations based on Bcl2 expression since REH cells express over threefold more Bcl2. In addition to such experimental data, recent clinical data also indicate that increased expression of Bcl2 can apparently not suppress drug-induced apoptosis and Bcl2 expression levels also cannot explain the chemoresistance observed in patients with small cell lung carcinoma 29 or breast cancer.…”

“…34,35 Certain protein kinase inhibitors relatively specific for PKC, including staurosporine 37,38 and the methylpiperazine, H7, 38,39 were found to partially inhibit Bcl2 phosphorylation in association with blocking the anti-apoptotic effect of Bcl2. 28,34,35 PKC was found to be a direct Bcl2 kinase when it was shown that a mixture of pan-PKC was able to phosphorylate Bcl2 in vitro exclusively on serine residues, the same site(s) phosphorylated in vivo. 34 Mutating each of the seven common and conserved PKC-consensus serine phosphorylation sites found in murine and human Bcl2 (ie residues 24, 70, 102, 158, 164, 202, and 213), 35,40 revealed that only the mutation at serine 70 (S70A) resulted in a nonphosphorylatable Bcl2.…”

“…49 However, the effect of the taxane, paclitaxel, appears to be opposite to that of IL-3, bryo, ATA, or retinoic acid in that paclitaxelinduced Bcl2 phosphorylation was initially reported to be associated with cell death. [41][42][43][44][45][46][47] The morphological pattern of paclitaxel-induced Bcl2 phosphorylation [41][42][43][44][45][46][47] is dramatically different from that observed following growth factor, 34,35 bryo, 28,34,35 retinoic acid 48 or ATA-induced phosphorylation. 49 Only paclitaxel (or the other antimititoic agents) stimulated formation of a slower migrating, band-shifted form of Bcl2 detected by denaturing gel electrophoresis with a Mr 28 000 daltons, as compared to unmodified Bcl2 at Mr 26 000 daltons ( Figure 2).…”

“…31 For example, in the comparison between REH and HL60 cells, while the expression levels of Bax were essentially equivalent, the ratio of Bcl2 to Bax was significantly higher (Ͼ three-fold) in the more chemosensitive REH cells. 28 Although current clinical data indicate that the Bcl2:Bax ratio may be important for cell survival in patients with AML, 32 there is no clear correlation between chemoresistance and the ratio of Bcl2:Bax expressed in other (eg pediatric ALL) acute leukemias. 33 These observations then suggest that there are also other mechanisms in addition to expression or interaction with other protein partners, such as Bax, that contribute to Bcl2's function and role in chemoresistance.…”