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Oncogenic K-Ras proteins, such as K-Ras(G12D), accumulate in the active, guanosine triphosphate (GTP)-bound conformation and stimulate signaling through effector kinases. The presence of the K-Ras(G12D) oncoprotein at a similar abundance to that of endogenous wild-type K-Ras results in only minimal phosphorylation and activation of the canonical Raf-mitogen-activated or extracellular signal-regulated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) signaling cascades in primary hematopoietic cells, and these pathways remain dependent on growth factors for efficient activation. We showed that phospholipase C-γ (PLC-γ), PI3K, and their generated second messengers link activated cytokine receptors to Ras and ERK signaling in differentiated bone marrow cells and in a cell population enriched for leukemia stem cells. Cells expressing endogenous oncogenic K-Ras(G12D) remained dependent on the second messenger diacylglycerol for the efficient activation of Ras-ERK signaling. These data raise the unexpected possibility of therapeutically targeting proteins that function upstream of oncogenic Ras in cancer.

Deregulation of the phosphoinositide-3-OH kinase (PI(3)K) pathway has been implicated in numerous pathologies including cancer, diabetes, thrombosis, rheumatoid arthritis and asthma. Recently, small-molecule and ATP-competitive PI(3)K inhibitors with a wide range of selectivities have entered clinical development. In order to understand the mechanisms underlying the isoform selectivity of these inhibitors, we developed a new expression strategy that enabled us to determine to our knowledge the first crystal structure of the catalytic subunit of the class IA PI(3)K p110 delta. Structures of this enzyme in complex with a broad panel of isoform- and pan-selective class I PI(3)K inhibitors reveal that selectivity toward p110 delta can be achieved by exploiting its conformational flexibility and the sequence diversity of active site residues that do not contact ATP. We have used these observations to rationalize and synthesize highly selective inhibitors for p110 delta with greatly improved potencies.

Deregulation of the phosphoinositide-3-OH kinase (PI(3)K) pathway has been implicated in numerous pathologies including cancer, diabetes, thrombosis, rheumatoid arthritis and asthma. Recently, small-molecule and ATP-competitive PI(3)K inhibitors with a wide range of selectivities have entered clinical development. In order to understand the mechanisms underlying the isoform selectivity of these inhibitors, we developed a new expression strategy that enabled us to determine to our knowledge the first crystal structure of the catalytic subunit of the class IA PI(3)K p110 delta. Structures of this enzyme in complex with a broad panel of isoform- and pan-selective class I PI(3)K inhibitors reveal that selectivity toward p110 delta can be achieved by exploiting its conformational flexibility and the sequence diversity of active site residues that do not contact ATP. We have used these observations to rationalize and synthesize highly selective inhibitors for p110 delta with greatly improved potencies.

The pathways implicated in the control of epithelial [Na.sup.+] channel (ENaC)-dependent [Na.sup.+] transport in renal collecting duct cells share substantial parallels with those implicated in insulin-regulated glucose metabolism. Notably, both are inhibited by wortmannin and LY294002 and signal through phosphatidylinositol-3-kinase (PI3K)-dependent kinases SGK1 and Akt. The inhibitor pattern is thought to reflect dependence on PI3K activity since wortmannin and LY294002 are both effective inhibitors of this kinase. However, these inhibitors block a variety of kinases from different families and lack specificity within the PI3K family. To begin to dissect more precisely the pathways required for signaling and for control of [Na.sup.+] transport in renal collecting duct cells, we have examined the effect of a set of PI3K inhibitors, which selectively block distinct subsets of PI3K catalytic subunit isoforms. We have found that ENaC-dependent [Na.sup.+] transport was blocked by inhibitors of the p 110-[alpha] isoform of PI3K, but not by inhibitors of p110-[beta], -[gamma] or -[delta] Inhibitors that block [Na.sup.+] current also blocked SGK1 and Akt phosphorylation. In contrast to insulin-stimulated glucose uptake in muscle cells, p110-[beta] inhibition did not enhance sensitivity to p110-[alpha] inhibition. These data support the conclusion that ENaC-dependent [Na.sup.+] current is controlled exclusively by p110-[alpha], the same isoform that is the principal mediator of insulin effects on glucose metabolism, and lacks any dependence on p110-[beta]. These findings further underscore the extent to which [Na.sup.+] and glucose regulation are intertwined and provide additional insight into the interconnections between diabetes and hypertension. phosphoinositide; sgk; phosphatidylinositol-3-phosphokinase; insulin; aldosterone

An approach is described to pharmacologically interrogate the PI3-K family. It is found that p110alpha is the primary insulin-responsive PI3-K in cultured cells, whereas p110beta is dispensable but sets a phenotypic threshold for p110alpha activity.

The pathways implicated in the control of epithelial Na~ channel (ENaC)-dependent Na+ transport in renal collecting duct cells share substantial parallels with those implicated in insulin-regulated glucose metabolism. Notably, both are inhibited by wortmannin and LY294002 and signal through phosphatidylinositol-3-kinase (P13K)-dependent kinases SGK1 and Akt. The inhibitor pattern is thought to reflect dependence on P13K activity since wortmannin and LY294002 are both effective inhibitors of this kinase. However, these inhibitors block a variety of kinases from different families and lack specificity within the P13K family. To begin to dissect more precisely the pathways required for signaling and for control of Na+ transport in renal collecting duct cells, we have examined the effect of a set of P13K inhibitors, which selectively block distinct subsets of P13K catalytic subunit isoforms. We have found that ENaC-dependent Na+ transport was blocked by inhibitors of the p110-α isoform of P13K, but not by inhibitors of p110-β,-γ, or -δ. Inhibitors that block Na+ current also blocked SGK1 and Akt phosphorylation. In contrast to insulin-stimulated glucose uptake in muscle cells, p110-β inhibition did not enhance sensitivity to p110-α inhibition. These data support the conclusion that ENaC-dependent Na+ current is controlled exclusively by p110-α, the same isoform that is the principal mediator of insulin effects on glucose metabolism, and lacks any dependence on p110-13. These findings further underscore the extent to which Na+ and glucose regulation are intertwined and provide additional insight into the interconnections between diabetes and hypertension. [ABSTRACT FROM AUTHOR]