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Raf-1 protects cells from apoptosis, independently of its signals to MEK and ERK, by translocating to the mitochondria where it binds Bcl-2 and displaces BAD. However, the answer to the question of how Raf-1 is normally lured to the mitochondria and becomes activated remains elusive. p21-activated protein kinases (Paks) are serine/ threonine protein kinases that phosphorylate Raf-1 at Ser-338 and Ser-339. Here we elucidate the molecular mechanism through which Pak1 signals to BAD through a Raf-1-activated pathway. Upon phosphorylation by Pak1, Raf-1 translocates to mitochondria and phosphorylates BAD at Ser-112. Moreover, the mitochondrial translocation of Raf-1 and the interaction between Raf-1 and Bcl-2 are regulated by Raf-1 phosphorylation at Ser-338/Ser-339. Notably, we show that formation of a Raf-1-Bcl-2 complex coincides with loss of an interaction between Bcl-2 and BAD. These signals are specific for Pak1, because Src-activated Raf-1 only stimulates the MAP kinase cascade. Thus, our data identify the molecular connections of a Pak1-Raf-1-BAD pathway that is involved in cell survival signaling.

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Differentiation of 3T3-L1 preadipocytes into adipocytes is induced by a combination of inducers, including a glucocorticoid, an agent that elevates cellular cAMP, and a ligand of the insulin-like growth factor-1 receptor. Previous studies have implicated protein-tyrosine phosphatase (PTPase) HA2, a homologue of PTPase 1B, in the signaling cascade initiated by the differentiation inducers. Vanadate, a potent PTPase inhibitor, blocks adipocyte differentiation at an early stage in the program, but has no effect on the mitotic clonal expansion required for differentiation. Exposure of preadipocytes to vanadate along with the inducing agents led to the accumulation of pp35, a phosphotyrosyl protein that is a substrate for PTPase HA2. pp35 was purified to homogeneity and shown by amino acid sequence and mass analyses of tryptic peptides to be c-Crk, a known cytoplasmic target of the insulin-like growth factor-1 receptor tyrosine kinase. Transfection of 3T3-L1 preadipocytes with a c-Crk antisense RNA expression vector markedly reduced c-Crk levels and prevented differentiation into adipocytes. Studies with C3G, a protein that binds to the SH3 domain in c-Crk, showed that phosphorylation of c-Crk rendered the SH3 domain inaccessible to C3G. Taken together, these findings indicate that locking c-Crk in the phosphorylated state with vanadate prevents its participation in the signaling system that initiates adipocyte differentiation.Adipocytes serve an important function in the energy economy of higher organisms, providing a large energy reserve that can be mobilized when needed. Thus, when caloric intake exceeds expenditure, metabolite flux is diverted into triglyceride synthesis for storage in adipocytes. Conversely, when caloric expenditure exceeds intake, this triglyceride reserve is mobilized as free fatty acids to provide physiological fuel for use by other tissue/cell types. The need for an energy reserve begins at birth when the newborn must be prepared to survive periods of energy deprivation. Adipocytes, which provide this reserve, develop late in embryonic life, with major expansion of this cell population occurring after birth. The adipose lineage arises from the same multipotent stem cells of mesodermal origin that give rise to the muscle and cartilage lineages.Established preadipocyte cell lines, e.g. the 3T3-L1 preadipocytes, which can be induced to differentiate into adipocytes in cell culture, provide faithful models with which to investigate the adipocyte differentiation program (1-6). When exposed to the appropriate differentiation inducers, including IGF-1 1 (or insulin at a non-physiologically high concentration), dexamethasone (a glucocorticoid), and 1-methyl-3-isobutylxanthine (MIX; a cAMP phosphodiesterase inhibitor that increases intracellular cAMP), 3T3-L1 preadipocytes differentiate into cells that express the adipocyte phenotype (6). Induction of the adipocyte differentiation program involves at least three different signal transduction systems, including those mediated by the glucocorticoid rec...

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