Sensing the osmolarity of the environment is a critical response for all organisms. Whereas bacteria will migrate away from high osmotic conditions, most eukaryotic cells are not motile and use adaptive metabolic responses for survival. The p38 MAPK pathway is a crucial mediator of survival during cellular stress. We have discovered a novel scaffold protein that binds to actin, the GTPase Rac, and the upstream kinases MEKK3 and MKK3 in the p38 MAPK phospho-relay module. RNA interference (RNAi) demonstrates that MEKK3 and the scaffold protein are required for p38 activation in response to sorbitol-induced hyperosmolarity. FRET identifies a cytoplasmic complex of the MEKK3 scaffold protein that is recruited to dynamic actin structures in response to sorbitol treatment. Through its ability to bind actin, relocalize to Rac-containing membrane ruffles and its obligate requirement for p38 activation in response to sorbitol, we have termed this protein osmosensing scaffold for MEKK3 (OSM). The Rac-OSM-MEKK3-MKK3 complex is the mammalian counterpart of the CDC42-STE50-STE11-Pbs2 complex in Saccharomyces cerevisiae that is required for the regulation of p38 activity.

Bax induces mitochondrial-dependent cell death signals in mammalian cells. However, the mechanism of how Bax is kept inactive has remained unclear. Yeast-based functional screening of Bax inhibitors from mammalian cDNA libraries identified Ku70 as a new Bax suppressor. Bax-mediated apoptosis was suppressed by overexpression of Ku70 in mammalian cells, but enhanced by downregulation of Ku70. We found that Ku70 interacts with Bax, and that the carboxyl terminus of Ku70 and the amino terminus of Bax are required for this interaction. Bax is known to translocate from the cytosol to mitochondria when cells receive apoptotic stimuli. We found that Ku70 blocks the mitochondrial translocation of Bax. These results suggest that in addition to its previously recognized DNA repair activity in the nucleus, Ku70 has a cytoprotective function in the cytosol that controls the localization of Bax.

The mitogen-activated protein kinase (MAPK) 1 pathways transduce various extracellular stimuli into distinct intracellular responses. The core component of such a MAPK module is a set of three sequential kinases that are evolutionarily conserved in eukaryotes from unicellular yeast to plants and animals. In mammalian cells, three distinguishable MAPK modules have been well described; they are the extracellular signalregulated kinases 1 and 2 (ERK1/2), the c-Jun N-terminal kinase (JNK), and the p38 pathways. The MAPK pathways regulate cell growth, differentiation, adaptation to the environment, and apoptosis in response to a great number of stimuli, including cytokines and various stresses (1-3). The MAPKs also control numerous regulatory processes during development and homeostasis (4 -6).Our laboratory previously cloned two MAPK kinase kinases, designated MEKK2 and MEKK3 (7). MEKK2 and MEKK3 are extremely homologous (94% conserved) in their catalytic domains, but their regulatory N-terminal sequences are quite divergent, with only 65% homology, predicting that they perform similar as well as different cellular tasks. In this regard, Schaefer et al. (8) showed that T cell MEKK2 but not MEKK3 is activated and translocates to the plasma membrane at the contact with antigen-loaded presenting cells. Thus, although MEKK2 and MEKK3 are both mediators of signal transduction to the MAPK pathways, they are subject to stimulus-and cell type-specific regulation. A differential involvement of MEKK2 and MEKK3 in cellular signaling has also been demonstrated by our recent finding that MEKK2 but not MEKK3 regulates the activity of the protein kinase C-related kinase PRK2 (9).Big mitogen-activated protein kinase 1 (BMK1)/ERK5 was recently cloned as a novel member of the MAPK family (10, 11). Like ERK1/2, BMK1/ERK5 has a TEY sequence in its dual phosphorylation motif; however, other structural features such as a large regulatory C terminus and a unique loop 12 domain distinguish BMK1/ERK5 from ERK1/2 and other MAP kinases (10). This predicts that the regulation and function of BMK1/ ERK5 are distinct from those of other MAPKs. In this regard, Zhou et al. (11) showed that BMK1/ERK5 interacts specifically with MEK5 but not its closely related MAPK kinases MEK1 and MEK2, suggesting that MEK5/BMK1 represents a separate signaling module. Indeed, MEK5 selectively phosphorylates and activates BMK1/ERK5 (12, 13), and its activity is required for the activation of BMK1/ERK5 induced by extracellular signals, including growth factors, serum, oxidative stress, and hyperosmolarity (13,14). Consistent with the notion that MEK5/BMK1 lies in a signal transduction pathway

IntroductionPlatelet endothelial cell adhesion molecule-1 (PECAM-1; also known as CD31) is a 130-kDa member of the immunoglobulin gene (Ig) superfamily expressed on the surface of circulating platelets and leukocytes and at the intercellular junctions of all continuous endothelium. 1-3 Extracellular Ig homology domain 1 possesses homophilic binding properties 4,5 and functions to mediate leukocyte transendothelial migration 6,7 and angiogenesis, 8 while the cytoplasmic domain harbors a functional 9-11 immunoreceptor tyrosine-based inhibitory motif (ITIM) that, when tyrosine phosphorylated, has been shown to recruit and activate the protein-tyrosine phosphatase, SHP-2, in a number of cellular systems, including human platelets, 12 bovine aortic vascular endothelial cells, 13 and rat basophilic leukemia cells. 14 Owing, in part, to its cytoplasmic ITIM, PECAM-1 has recently been assigned to the Ig-ITIM family of inhibitory receptors. 15 In addition to its role in vascular cell adhesion and signaling, there is growing evidence that PECAM-1 may be able to transduce signals that suppress programmed cell death. The first evidence of a role for PECAM-1 in apoptosis was provided by the studies of Noble et al, 16 who found that monocytes promoted the survival of serum-starved endothelial cells. Interestingly, when the anti-PECAM-1 monoclonal antibody (mAb), PECAM-1.3, was included in the monocyte/endothelial cell coculture, the cytoprotective role of added monocytes was lost. Because PECAM-1.3 inhibits PECAM-1 homophilic interactions, 4,17 the authors speculated that monocyte PECAM-1-endothelial cell PECAM-1 homophilic interactions might contribute to endothelial cell survival. Further evidence that engagement of PECAM-1 can result in the transduction of a survival signal was provided by studies showing that the rate and extent of serum deprivation-induced apoptosis of endothelial cells is lessened if endothelial cells are first attached to immobilized PECAM-1/IgG 18 or treated with an anti-PECAM-1 monoclonal antibody. 19 While the latter investigation, like the study of Noble et al, found a correlation between PECAM-1-induced cell survival and increased transcript levels of the antiapoptotic gene, A1, the molecular mechanisms by which PECAM-1 might exert its cytoprotective effects have not to date been examined.Two major cell death pathways-termed the extrinsic and intrinsic pathways of apoptosis-exist in mammalian cells (reviewed by Hengartner 20 ). The extrinsic pathway is initiated by engagement and aggregation of tumor necrosis factor (TNF) family death receptors (such as The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ''advertisement'' in accordance with 18 U.S.C. section 1734. CD95/Fas) which, through a series of death domain-containing adaptor molecules, recruit and directly activate cytosolic caspase 8, which in turn converts procaspase 3 to caspase 3-the central executioner of the apoptotic process. Th...