(3,4), and two SH2-containing adapter proteins, Grb2 and Shc, have been implicated in its activation. Specifically, these proteins have been shown to bind directly to tyrosine-phosphorylated receptors (5-7) or SH2 docking proteins (such as the insulin receptor substrate 1) (8). Grb2, a 25-kDa protein with two SH3 domains flanking one SH2 domain, shuttles the Ras guanine nucleotide exchange factor, Sosl, to activated receptors (or to insulin receptor substrate 1) (5, 7-11) so that Sosl can activate Ras by catalyzing the exchange of GDP for GTP (5, 9-11). Shc, another widely expressed protein that contains an N-terminal phosphotyrosine binding (PTB) domain (12-16) and a C-terminal SH2 domain (17), can associate, in its tyrosine-phosphorylated form, with Grb2-Sosl complexes and may increase Grb2-Sosl interactions after growth factor stimulation (9,18,19 MATERIALS AND METHODS Reagents. COS-cell-derived murine IL-3 and GM-CSF were produced as described (29). Rabbit antiserum to the Shcassociated p145 protein was generated by immunizing rabbits with a 15-mer obtained from amino acid sequencing (VPAE-GVSSLNEMINP) and crosslinked to keyhole limpet hemocyanin with glutaraldehyde (30). The glutathione S-transferase (GST) fusion proteins, consisting of the 27-kDa N-terminal and of GST linked to the Grb2 N-terminal (amino acid residues 5-56) or C-terminal (residues 163-215) SH3 domains of Grb2, were expressed in Escherichia coli in pGEX-2T plasmids (Pharmacia/LKB) and the fusion proteins were recovered from clarified lysates with glutathione (GSH)-agarose beads as described (31 Abbreviations: GM-CSF, granulocyte-macrophage colony-stimulating factor; GSH, glutathione; GST, glutathione S-transferase; IL-3, interleukin 3; NRS, normal rabbit serum; 5-ptase, inositol polyphosphate 5-phosphatase; Ptd1ns(3,4,5)P3, phosphatidylinositol 3,4,5-trisphosphate; Ins(1,3,4,5)P4, inositol 1,3,4,5-tetrakisphosphate; SH2, src homology 2 domain; PTB, phosphotyrosine binding; p145, the Shc-associated 145-kDa protein.

Phosphotidylinositol (PtdIns) signaling is tightly regulated, both spatially and temporally, by subcellularly localized PtdIns kinases and phosphatases that dynamically alter downstream signaling events 1. Joubert Syndrome (JS) characterized by a specific midbrain-hindbrain malformation (“molar tooth sign”) and variably associated retinal dystrophy, nephronophthisis, liver fibrosis and polydactyly 2, and is included in the newly emerging group of “ciliopathies”. In patients linking to JBTS1, we identified mutations in the INPP5E gene, encoding inositol polyphosphate-5-phosphatase E, which hydrolyzes the 5-phosphate of PtdIns(3,4,5)P3 and PtdIns(4,5)P2. Mutations clustered in the phosphatase domain and impaired 5-phosphatase activity, resulting in altered cellular PtdIns ratios. INPP5E localized to cilia in major organs affected in JS, and mutations promoted premature destabilization of cilia in response to stimulation. Thus, these data links PtdIns signaling to the primary cilium, a cellular structure that is becoming increasingly appreciated for its role in mediating cell signals and neuronal function.

Several proteins secreted by enteric bacteria are thought to contribute to virulence by disturbing the signal transduction of infected cells. Here, we report that SopB, a protein secreted by Salmonella dublin, has sequence homology to mammalian inositol polyphosphate 4-phosphatases and that recombinant SopB has inositol phosphate phosphatase activity in vitro.

When microsomes of sheep or bovine semi-nal vesicles are incubated with [acetyl-3H]aspirin (acetyl sali-cylic acid), 200 Ci/mol, we observe acetylation of a single protein, as measured by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The protein has a molecular weight of 85,000 and correspnds to a similar acetylated protein found in the particulate fraction of aspirin-treated human platelets. The aspirin-mediated acetylation reaction proceeds with the same time course and at the same concentration as does the inhibition of prostaglandin synthase (cyclo-oxygenase) (EC 1.14.99.1;8,11, Prostaglandin biosynthesis involves the initial conversion of arachidonic acid to cyclic endoperoxide intermediates designated PGG2 and PGH2 (1, 2). These relatively unstable compounds are then metabolized by other enzymes to form stable prostaglandins, or the endoperoxide intermediates are broken down to form malonaldehyde and other products (1,3). The initial conversion is catalyzed by a microsomal enzyme (or enzymes) contained in numerous tissues, including bovine and sheep seminal vesicles and human platelets (2-4). The microsomal enzyme that metabolize's arachidonic acid has been termed prostaglandin synthase or cyclo-oxygenase (EC 1.14.99.1; 8,11,14-eicosatrienoate, hydrogendonor:oxygen oxidoreductase). While the enzyme has been solubilized by neutral detergents (5-7), it has not been purified or characterized as to molecular weight or subunit structure.The anti-inflammatory effect (8) as well as the anti-platelet effect of aspirin (9) may be mediated through inhibition of prostaglandin synthesis. Recent experiments indicate that aspirin blocks conversion of arachidonic acid to cyclic endoperoxides (3, 10). Conversely, arachidonic acid prevents the inhibitory effect of aspirin on the enzyme, and the concentration of arachidonic acid that protects the enzyme correlates with the Michaelis constant of the fatty acid as cyclooxygenase substrate (11). Therefore, Rome and Lands have postulated that aspirin inhibits cyclo-oxygenase by competing with substrate for the active site (11). The inhibition by aspirin is not strictly competitive since it irreversibly inactivates cyclo-oxygenase in vitro.The mechanism by which aspirin inhibits cyclo-oxygenase is undefined. The drug is known to acetylate proteins such as albumin, immunoglobulins, and fibrinogen (12, 13). However, these reactions proceed slowly over hours and require 1-20 mM concentrations of aspirin. Aspirin inhibits cyclooxygenase under clearly different conditions; namely, within minutes at concentrations in the micromolar range (3,14).We have recently examined the relationship between protein acetylation and the inhibition of platelet function by aspirin (15). We demonstrated that aspirin acetylates a single 85,000 molecular weight protein of the particulate fraction of human platelets. The time course and aspirin concentration dependence of the acetylation reaction correlated well with the same parameters of aspirin's effect on platelet function (16)....

Inositol polyphosphate 4-phosphatase-II (INPP4B) is a regulator of the phosphoinositide 3-kinase (PI3K) signaling pathway and is implicated as a tumor suppressor in epithelial carcinomas. INPP4B loss of heterozygosity (LOH) is detected in some human breast cancers; however, the expression of INPP4B protein in breast cancer subtypes and the normal breast is unknown. We report here that INPP4B is expressed in nonproliferative estrogen receptor (ER)-positive cells in the normal breast, and in ER-positive, but not negative, breast cancer cell lines. INPP4B knockdown in ER-positive breast cancer cells increased Akt activation, cell proliferation, and xenograft tumor growth. Conversely, reconstitution of INPP4B expression in ER-negative, INPP4B-null human breast cancer cells reduced Akt activation and anchorage-independent growth. INPP4B protein expression was frequently lost in primary human breast carcinomas, associated with high clinical grade and tumor size and loss of hormone receptors and was lost most commonly in aggressive basal-like breast carcinomas. INPP4B protein loss was also frequently observed in phosphatase and tensin homolog (PTEN)-null tumors. These studies provide evidence that INPP4B functions as a tumor suppressor by negatively regulating normal and malignant mammary epithelial cell proliferation through regulation of the PI3K/Akt signaling pathway, and that loss of INPP4B protein is a marker of aggressive basal-like breast carcinomas. phosphatidylinositol 3,4-bisphosphate