Bcl-2 Undergoes Phosphorylation by c-Jun N-terminal Kinase/Stress-activated Protein Kinases in the Presence of the Constitutively Active GTP-binding Protein Rac1

Abstract

We have studied the phosphorylation of the Bcl-2 family of proteins by different mitogen-activated protein (MAP) kinases. Purified Bcl-2 was found to be phosphorylated by the c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) p54-SAPK␤, and this is specific insofar as the extracellular signal-regulated kinase 1 (ERK1) and p38/RK/CSBP (p38) catalyzed only weak modification. Bcl-2 undergoes similar phosphorylation in COS-7 when coexpressed together with p54-SAPK␤ and the constitutive Rac1 mutant G12V. This is seen by both 32 PO 4 labeling and the appearance of five discrete Bcl-2 bands with reduced gel mobility. As anticipated, both intracellular p54-SAPK␤ activation and Bcl-2 phosphorylation are blocked by co-transfection with the MAP kinase specific phosphatase MKP3/PYST1. MAP kinase specificity is also seen in COS-7 cells as Bcl-2 undergoes only weak phosphorylation when co-expressed with enzymatically activated ERK1 or p38. Four critical residues undergoing phosphorylation in COS-7 cells were identified by expression of the quadruple Bcl-2 point mutant T56A,S70A,T74A,S87A. Sequencing phosphopeptides derived from tryptic digests of Bcl-2 indicates that purified GST-p54-SAPK␤ phosphorylates identical sites in vitro. This is the first report of Bcl-2 phosphorylation by the JNK/SAPK class of MAP kinases and could indicate a key modification allowing control of Bcl-2 function by cell surface receptors, Rho family GTPases, and/or cellular stresses.

Stress signals for apoptosis: ceramide and c-Jun kinase

“…One report suggests that JNK can directly inactivate Bcl-2 by phosphorylation of one or all of four serine and threonine residues within the¯exible loop that resides N-terminal to the BH3 domain (Maundrell et al, 1997). Consistent with this hypothesis in WEHI-231 B cells, full-length Bcl-2 is hyperphosphorylated and ine ective in preventing anti-IgM-induced death while a loop-deletion mutant blocked apoptosis.…”

Serine protease inhibitor TPCK prevents Taxol-induced cell death and blocks c-Raf-1 and Bcl-2 phosphorylation in human breast carcinoma cells

“…JNK has been implicated in the implementation of programmed cell death in response to a variety of stressful stimuli, but the downstream e ectors involved in mediating its apoptotic e ects have not been identi®ed. A recent report demonstrated that JNK was capable of phosphorylating Bcl-2 (Maundrell et al, 1997), suggesting the possibility that JNK could play a role in Taxol-induced Bcl-2 phosphorylation. However, our ®nding that TPCK did not a ect the level of JNK activity seen in response to Taxol treatment although it completely prevented Bcl-2 phosphorylation, argue against this possibility.…”

ZBP-89-induced apoptosis is p53-independent and requires JNK

“…Bcl-2 survival proteins are known to be inhibited by JNK. [58][59][60][61] In the case of Bcl-2 and Mcl-1, JNK inactivates these survival factors by direct phosphorylation. 24,62 However, ZBP-89 expression had no effect on Bcl-2.…”