Philip W. Majerus

Inhibition of platelet prostaglandin synthetase by oral aspirin.

Aspirin inhibits platelet function by permanently acetylating the cyclooxygenase that forms prostaglandins. We determined the sensitivity of platelets to aspirin in normal subjects by measuring [3H-acetyl]aspirin-susceptible cyclooxygenase in washed platelets obtained at various times after aspirin ingestion. A single 325-mg aspirin dose inactivated 89% of platelet cyclooxygenase. The inhibition persisted for 2 days suggesting that oral aspirin also inactivated megakaryocyte cyclooxygenase. Thereafter, active enzyme returned with a time-course reflecting platelet turnover (life-span 8.2+/-2 days). Single doses of 20-650 mg aspirin resulted in 34- greater than 95% inhibition after 24 h. Daily doses of 20-325 mg aspirin for brief periods produced 61- greater than 95% inactivation when measured 24 h after cessation of the drug. Platelet cyclooxygenase is more sensitive to inactivation by aspirin than enzyme in sheep seminal vesicles.

Multiple forms of an inositol polyphosphate 5-phosphatase form signaling complexes with Shc and Grb2

BACKGROUND Shc and Grb2 form a complex in cells in response to growth factor stimulation and link tyrosine kinases to Ras during the resulting signaling process. Shc and Grb2 each contain domains that mediate interactions with other unidentified intracellular proteins. For example, the Shc PTB domain binds to 130 kDa and 145 kDa tyrosine-phosphorylated proteins in response to stimulation of cells by growth factors, cytokines and crosslinking of antigen receptors. The Grb2 SH3 domains bind to an unidentified 116 kDa protein in T cells. We have identified three proteins, of 110 kDa, 130 kDa and 145 kDa, as a new family of molecules encoded by the same gene. In vivo studies show that these proteins form signal transduction complexes with Shc and with Grb2. RESULTS The 130 kDa and 145 kDa tyrosine-phosphorylated proteins that associate with the Shc PTB domain were purified by conventional chromatographic methods. Partial peptide and cDNA sequences corresponding to these proteins, termed SIP-145 and SIP-130 (SIP for signaling inositol polyphosphate 5-phosphatase), identified them as SH2 domain-containing products of a single gene and as members of the inositol polyphosphate 5-phosphatase family. The SIP-130 and SIP-145 proteins and inositol polyphosphate 5-phosphatase activity associated with Shc in vivo in response to B-cell activation. By using an independent approach, expression cloning, we found that the Grb2 SH3 domains bind specifically to SIP-110, a 110 kDa splice variant of SIP-145 and SIP-130, which lacks the SH2 domain. The SIP proteins hydrolyzed phosphatidylinositol (3,4,5)-trisphosphate (PtdIns (3,4,5)-P3) and Ins (1,3,4,5)-P4, but not PtdIns (4,5)-P2 or Ins (1,4,5)-P3. CONCLUSIONS These findings strongly implicate the inositol polyphosphate 5-phosphatases in Shc- and Grb2-mediated signal transduction. Furthermore, SIP-110, SIP-130 and SIP-145 prefer 3-phosphorylated substrates, suggesting a link to the phosphatidylinositol 3-kinase signaling pathway.

Prevention of Thrombosis in Patients on Hemodialysis by Low-Dose Aspirin

Since platelet cyclo-oxygenase is much more sensitive to inactivation by aspirin than is the enzyme in the arterial wall and low doses of aspirin may prevent thrombosis by blocking thromboxane synthesis, we conducted a randomized, double-blind trial of aspirin (160 mg per day) vs. placebo in 44 patients on chronic hemodialysis. The study was continued until there were 24 patients with thrombi and both groups had been under observation for a mean of nearly five months. Thrombi occurred in 18 of 25 (72 per cent) of patients given placebo and 16 of 19 (32 per cent) of those given aspirin (P less than 0.01). The incidence of thrombosis was reduced from 0.46 thrombi per patient month in the placebo group to 0.16 thrombi per patient month in the aspirin group (p less than 0.005). A dose of 160 mg of aspirin per day is an effective, nontoxic antithrombotic regimen in patients on hemodialysis.

The role of phosphatases in inositol signaling reactions.

The phosphatidylinositol signaling pathway employs a host of kinases and phosphatases that form and degrade the many signaling molecules that act in this system. The complexity and robustness of the system are indicated by the fact that there are six inositol phospholipids and more than 20 soluble inositol phosphates that have been found in mammalian cells (1). The system is present in all eukaryotic cells in one form or another. This review will focus on a few of the phosphatases as they exist in mammalian cells. In general, the inositol phosphatases are analogous to protein phosphatases in that they both tend to inhibit or terminate signaling reactions. There are specific exceptions to this general rule in both systems. Numerous inositol phosphatases have been discovered and characterized in the past 15 years. Surprisingly, in many cases the same enzymes hydrolyze phosphate from both water-soluble inositol phosphates and the corresponding lipids with the same arrangement of phosphate groups.

Recent insights in phosphatidylinositol signaling

Studies of phosphatidylinositol signaling pathways are entering a new phase in which molecular genetic techniques are providing powerful tools to dissect the functions of various metabolites and pathways. Studies with phospholipase C are most advanced and clearly indicate that phosphatidylinositol turnover is critical for vision in Drosophila and cell proliferation in various cultured cells. Expression of cDNA constructs and microinjection of PLC or antibodies against it clearly establish a role for PtdIns signaling distinct from its role in calcium mobilization and protein kinase C activation. The importance of inositol cyclic phosphates is also beginning to be realized from the study of cyclic hydrolase using similar techniques. Elucidation of the function of the 3-phosphate inositol phospholipid pathway awaits similar studies. The recent cDNA cloning of inositol monophosphatase (Diehl et al., 1990), Ins(1,4,5)P3 3-kinase (Choi et al., 1990), and inositol polyphosphate 1-phosphatase (York and Majerus, 1991) should provide tools to define further the cell biology of the phosphatidylinositol signaling pathway.

Cultured human skin fibroblasts and arterial cells produce a labile platelet-inhibitory prostaglandin.

Abstract Human skin fibroblasts and cells cultured from human arterial smooth muscle produce a platelet-inhibitory prostaglandin in response to mechanical trauma. This prostaglandin is synthesized from an endogenous precursor rather than exogenous cyclic endoperoxides; it differs from PGE 1 and PGD 2 and resembles PGI 2 (prostacyclin) in its stability properties, being stable at pH ≥ 8.5 and labile at pH 7.4 and below. The prostaglandin synthesis pathway in these cultured cells is less sensitive to inhibition by aspirin than that in human platelets.

The Inositol 5′-Phosphatase SHIP Binds to Immunoreceptor Signaling Motifs and Responds to High Affinity IgE Receptor Aggregation

Immunoreceptors such as the high affinity IgE receptor, FcεRI, and T-cell receptor-associated proteins share a common motif, the immunoreceptor tyrosine-based activation motif (ITAM). We used the yeast tribrid system to identify downstream effectors of the phosphorylated FcεRI ITAM-containing subunits β and γ. One novel cDNA was isolated that encodes a protein that is phosphorylated on tyrosine, contains a Src-homology 2 (SH2) domain, inositolpolyphosphate 5-phosphatase activity, three NXXY motifs, several proline-rich regions, and is called SHIP. Mutation of the conserved tyrosine or leucine residues within the FcεRI β or γ ITAMs eliminates SHIP binding and indicates that the SHIP-ITAM interaction is specific. SHIP also binds to ITAMs from the CD3 complex and T cell receptor ζ chain in vitro. SHIP protein possesses both phosphatidylinositol-3,4,5-trisphosphate 5′-phosphatase and inositol-1,3,4,5-tetrakisphosphate 5′-phosphatase activity. Phosphorylation of SHIP by a protein-tyrosine kinase, Lck, results in a reduction in enzyme activity. FcεRI activation induces the association of several tyrosine phosphoproteins with SHIP. SHIP is constitutively tyrosine-phosphorylated and associated with Shc and Grb2. These data suggest that SHIP may serve as a multifunctional linker protein in receptor activation.

The Activation Loop of Phosphatidylinositol Phosphate Kinases Determines Signaling Specificity

Phosphatidylinositol-4,5-bisphosphate plays a pivotal role in the regulation of cell proliferation and survival, cytoskeletal reorganization, and membrane trafficking. However, little is known about the temporal and spatial regulation of its synthesis. Higher eukaryotic cells have the potential to use two distinct pathways for the generation of phosphatidylinositol-4,5-bisphosphate. These pathways require two classes of phosphatidylinositol phosphate kinases, termed type I and type II PIP kinases. While highly related by sequence, these kinases localize to different subcellular compartments, phosphorylate distinct substrates, and are functionally nonredundant. Here, we show that a 20- to 25-amino acid loop spanning the catalytic site, termed the activation loop, determines both enzymatic specificity and subcellular targeting of PIP kinases. Therefore, the activation loop controls signaling specificity and PIP kinase function at multiple levels.

Inositol polyphosphate 4-phosphatase II regulates PI3K/Akt signaling and is lost in human basal-like breast cancers

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.

Characterization of prostacyclin synthesis in cultured human arterial smooth muscle cells, venous endothelial cells and skin fibroblasts

Abstract The interaction of human platelets with one another and with the blood vessel wall is thought to be regulated in part by a balance between two arachidonic acid metabolites: thromboxane A 2 , synthesized by platelets, and prostacyclin (PGI 2 ), synthesized by the vessel wall. We have studied the ability of cultured human vascular cells to synthesize PGI 2 from arachidonic acid. Four strains of human arterial smooth muscle cells synthesized a mean of 1.36 ng PGI 2 per 10 5 cells, with a range of 0.2–5.3 ng PGI 2 per 10 5 cells among the different strains. Human umbilical vein endothelial cells synthesized a mean of 7.16 ng PGI 2 per 10 5 cells with a range of 2.3–14.0 ng per 10 5 cells. In contrast, cultured human diploid skin fibroblasts synthesized only 0.27 ng PGI 2 per 10 5 cells with a range of 0.05–0.6 ng per 10 5 cells. When cultured cells were mixed with platelets, PGI 2 synthesis from added arachidonate was reduced rather than stimulated. Thus the major precursor cyclic endoperoxides utilized for PGI 2 synthesis are formed within the cells and not from endoperoxides synthesized by platelet cyclooxygenase. Aspirin has been proposed as an anti-thrombotic agent. Aspirin could be ineffective, however, if it inhibited not only platelet cyclooxygenase but that of vessel wall cells as well. Measurement of the rate constant or potency for aspirin inhibition of PGI 2 synthesis in cultured cells indicates that the cyclooxygenase in both cell types of the blood vessel wall is 14–44 fold less sensitive to aspirin inactivation than that in platelets, and appropriate levels of aspirin can selectively block human platelet thromboxane A 2 synthesis without compromising the capacity of the vasculature to produce PGI 2 .