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.

Using differential display PCR, we have identified a gene [NOEY2, ARHI (designation by the Human Gene Nomenclature Committee)] with high homology to ras and rap that is expressed consistently in normal ovarian and breast epithelial cells but not in ovarian and breast cancers. Reexpression of NOEY2 through transfection suppresses clonogenic growth of breast and ovarian cancer cells. Growth suppression was associated with down-regulation of the cyclin D1 promoter activity and induction of p21 WAF1͞CIP1 . In an effort to identify mechanisms leading to NOEY2 silencing in cancer, we found that the gene is expressed monoallelically and is imprinted maternally. Loss of heterozygosity of the gene was detected in 41% of ovarian and breast cancers. In most of cancer samples with loss of heterozygosity, the nonimprinted functional allele was deleted. Thus, NOEY2 appears to be a putative imprinted tumor suppressor gene whose function is abrogated in ovarian and breast cancers.

Under resting conditions, the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3K) serves to both stabilize and inactivate the p110 catalytic subunit. The inhibitory activity of p85 is relieved by occupancy of the NH 2 -terminal SH2 domain of p85 by phosphorylated tyrosine. Src family kinases phosphorylate tyrosine 688 in p85, a process that we have shown to be reversed by the activity of the p85-associated SH2 domain-containing phosphatase SHP1. We demonstrate that phosphorylation of the downstream PI3K target Akt is increased in cells lacking SHP1, implicating phosphorylation of p85 in the regulation of PI3K activity. Furthermore, the in vitro specific activity of PI3K associated with tyrosinephosphorylated p85 is higher than that associated with nonphosphorylated p85. Expression of wild-type p85 inhibits PI3K enzyme activity as indicated by PI3K-dependent Akt phosphorylation. The inhibitory activity of p85 is accentuated by mutation of tyrosine 688 to alanine and reversed by mutation of tyrosine 688 to aspartic acid, changes that block and mimic tyrosine phosphorylation, respectively Strikingly, mutation of tyrosine 688 to aspartic acid completely reverses the inhibitory activity of p85 on cell viability and activation of the downstream targets Akt and NFB, indicative of the physiological relevance of p85 phosphorylation. Tyrosine phosphorylation of Tyr 688 or mutation of tyrosine 688 to aspartic acid is sufficient to allow binding to the NH 2 -terminal SH2 domain of p85. Thus an intramolecular interaction between phosphorylated Tyr 688 and the NH 2 -terminal SH2 domain of p85 can relieve the inhibitory activity of p85 on p110. Taken together, the data indicate that phosphorylation of Tyr 688 in p85 leads to a novel mechanism of PI3K regulation. The PI3K1 signaling cascade has been linked to proliferation, cell survival, differentiation, apoptosis, cytoskeletal rearrangement, and vacuolar trafficking. Growth factor-responsive Class IA PI3Ks consist of heterodimers of a 110-kDa catalytic subunit associated with an 85-kDa noncatalytic regulatory subunit designated p85. The p85 adapter subunits are encoded by at least three different genes with splice variation generating multiple proteins potentially serving many different functions (1). Of the known p85 adapter subunits and splice variants, nearly all contain two Src-homology 2 (SH2) domains, which enable p85 to bind phosphotyrosine in an appropriate amino acid context. The p85 SH2 domains most frequently, but not exclusively, recognize phosphotyrosine embedded in a YXXM motif (2). Most p85 gene products also include a Src homology 3 (SH3) domain, as well as other domains involved in protein-protein interactions (3). All p85 family members contain a p110-binding motif located between the two SH2 domains. The diversity of protein interaction domains found among p85 family members likely contributes to the ability of multiple signaling proteins and pathways to activate PI3K. Under resting conditions, p85 serves to both stabilize p110 protein and inhibi...