The protooncogene Bcl-2 functions as a suppressor of apoptosis in growth factor-dependent cells, but a postreceptor signaling mechanism is not known. We recently reported that interleukin 3 (IL-3) and erythropoietin, or the protein kinase C activator bryostatin-1 (Bryo), not only suppresses apoptosis but also stimulates the phosphorylation of Bcl-2 (May, W. S., Tyler, P. G., Ito, T., Armstrong, D. K., Qatsha, K. A., and Davidson, N. E. (1994) J. Biol. Chem. 269, 26865-26870). To test whether phosphorylation is required for Bcl-2 function, conservative serine 3 alanine mutations were produced at the seven putative protein kinase C phosphorylation sites in Bcl-2. Results indicate that the S70A Bcl-2 mutant fails to be phosphorylated after IL-3 or Bryo stimulation and is unable to support prolonged cell survival either upon IL-3 deprivation or etoposide treatment when compared with wild-type Bcl-2. In contrast, a Ser 3 Glu mutant, S70E, which may mimic a potential phosphate charge, more potently suppressed the etoposide-induced apoptosis than wild type in the absence of IL-3. Since the loss of function S70A mutant can heterodimerize with its partner protein and death effector Bax, these findings demonstrate that Bcl-2:Bax heterodimerization is not sufficient and Bcl-2 phosphorylation is required for full Bcl-2 death suppressor signaling activity.
The double-stranded (ds) RNA-dependent protein kinase (PKR) regulates protein synthesis by phosphorylating the ␣ subunit of eukaryotic initiation factor-2. PKR is activated by viral induced dsRNA and thought to be involved in the host antiviral defense mechanism. PKR is also activated by various nonviral stresses such as growth factor deprivation, although the mechanism is unknown. By screening a mouse cDNA expression library, we have identified an ubiquitously expressed PKR-associated protein, RAX. RAX has a high sequence homology to human PACT, which activates PKR in the absence of dsRNA. Although RAX also can directly activate PKR in vitro, overexpression of RAX does not induce PKR activation or inhibit growth of interleukin-3 (IL-3)-dependent cells in the presence of IL-3. However, IL-3 deprivation as well as diverse cell stress treatments including arsenite, thapsigargin, and H 2 O 2 , which are known to inhibit protein synthesis, induce the rapid phosphorylation of RAX followed by RAX-PKR association and activation of PKR. Therefore, cellular RAX may be a stress-activated, physiologic activator of PKR that couples transmembrane stress signals and protein synthesis.
). The potent apoptotic agent ceramide can activate a PP2A, suggesting that one potential component of the ceramide-induced death signal may involve the inactivation of Bcl2. Results indicate that C2-ceramide but not inactive C2-dihydroceramide, was found to specifically activate a mitochondrial PP2A, which rapidly and completely induced Bcl2 dephosphorylation and correlated closely with ceramide-induced cell death. Using a genetic approach, the gain-of-function S70E Bcl2 mutation, which mimics phosphorylation, fails to undergo apoptosis even with the addition of high doses of ceramide (IC 50 > 50 M). In contrast, cells overexpressing exogenous wild-type Bcl2 were sensitive to ceramide at dosages where PP2A is fully active and Bcl2 would be expected to be dephosphorylated (IC 50 ؍ 14 M). These findings indicate that in cells expressing functional Bcl2, the mechanism of death action for ceramide may involve, at least in part, a mitochondrial PP2A that dephosphorylates and inactivates Bcl2.
We have cloned and characterized a novel zinc finger protein, termed JAZ. JAZ contains four C 2 H 2 -type zinc finger motifs that are connected by long (28 -38) amino acid linker sequences. JAZ is expressed in all tissues tested and localizes in the nucleus, primarily the nucleolus. JAZ preferentially binds to double-stranded (ds) RNA or RNA/DNA hybrids rather than DNA. Mutation of individual zinc finger motifs reveals that the zinc finger domains are not only essential for dsRNA binding but are also required for its nucleolar localization, which demonstrates a complex trafficking mechanism dependent on the nucleic acid-binding capability of the protein. Furthermore, forced expression of JAZ potently induces apoptosis in murine fibroblast cells. Thus, JAZ may belong to a class of zinc finger proteins that features dsRNA binding and may regulate cell growth via the unique dsRNA binding properties.
Interleukin 3 (IL-3) stimulates the net growth of murine factor-dependent NSF/N1.H7 and FDC-P1/ER myeloid cells by stimulating proliferation and suppressing apoptosis. Recently, we discovered that Bcl2 is phosphorylated at an evolutionarily conserved serine residue (Ser 70 ) after treatment with the survival agonists IL-3 or bryostatin 1, a potent activator of protein kinase (Ito, T., Deng, X., Carr, B., and May, W. S. (1997) J. Biol. Chem. 272, 11671-11673). In addition, an intact Ser 70 was found to be required for Bcl2's ability to suppress apoptosis after IL-3 withdrawal or toxic chemotherapy. We now show that phosphorylation of Bcl2 occurs rapidly after the addition of agonist to IL-3-deprived cells and can be reversed by the action of an okadaic acid (OA)-sensitive phosphatase. A role for protein phosphatase (PP) 2A as the Bcl2 regulatory phosphatase is supported by several observations: 1) dephosphorylation of Bcl2 is blocked by OA, a potent PP1 and PP2A inhibitor; 2) intracellular PP2A, but not PP1, co-localizes with Bcl2; 3) the purified PP2Ac catalytic subunit directly dephosphorylates Bcl2 in vitro in an OA-sensitive manner; 4) the purified PP2Ac catalytic subunit preferentially dephosphorylates Bcl2 in vitro compared with PP1 and PP2B; 5) reciprocal immunoprecipitation studies indicate a direct interaction between PP2A and hemagglutinin (HA)-Bcl2; and 6) treatment of factor-deprived cells with bryostatin 1 dramatically increases the association between PP2A and Bcl2. Increased association between Bcl2 and PP2A occurs 15 min after agonist stimulation when Bcl2 phosphorylation has peaked and immediately before dephosphorylation. An agonist-induced increased association of PP2A and Bcl2 fails to occur in cells expressing the inactive, phosphorylation-negative S70A Bcl2 mutant, which indicates that an intact Ser 70 site is necessary and sufficient for the interaction to occur. Functional phosphorylation of Bcl2 at Ser 70 is proposed to be a dynamic process regulated by the sequential action of an agonist-activated Bcl2 kinase and PP2A.
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