SUMMARY Chronic reactive oxygen species (ROS) production by mitochondria may contribute to the development of insulin resistance, a primary feature of type 2 diabetes. In recent years it has become apparent that ROS generation in response to physiological stimuli such as insulin may also facilitate signaling by reversibly oxidizing and inhibiting protein tyrosine phosphatases (PTPs). Here we report that mice lacking one of the key enzymes involved in the elimination of physiological ROS, glutathione peroxidase 1 (Gpx1), were protected from high fat diet-induced insulin resistance. The increased insulin sensitivity in Gpx1−/− mice was attributed to insulin-induced phosphatidylinositol-3-kinase/Akt signaling and glucose uptake in muscle and could be reversed by the anti-oxidant N-acetylcysteine. Increased insulin signaling correlated with enhanced oxidation of the PTP family member PTEN, which terminates signals generated by phosphatidylinositol-3-kinase. These studies provide causal evidence for the enhancement of insulin signaling by ROS in vivo.

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

Phosphoinositides are membrane-bound signalling molecules that regulate cell proliferation and survival, cytoskeletal reorganization and vesicular trafficking by recruiting effector proteins to cellular membranes. Growth factor or insulin stimulation induces a canonical cascade resulting in the transient phosphorylation of PtdIns(4,5)P(2) by PI3K (phosphoinositide 3-kinase) to form PtdIns(3,4,5)P(3), which is rapidly dephosphorylated either by PTEN (phosphatase and tensin homologue deleted on chromosome 10) back to PtdIns(4,5)P(2), or by the 5-ptases (inositol polyphosphate 5-phosphatases), generating PtdIns(3,4)P(2). The 5-ptases also hydrolyse PtdIns(4,5)P(2), forming PtdIns4P. Ten mammalian 5-ptases have been identified, which share a catalytic mechanism similar to that of the apurinic/apyrimidinic endonucleases. Gene-targeted deletion of 5-ptases in mice has revealed that these enzymes regulate haemopoietic cell proliferation, synaptic vesicle recycling, insulin signalling, endocytosis, vesicular trafficking and actin polymerization. Several studies have revealed that the molecular basis of Lowe's syndrome is due to mutations in the 5-ptase OCRL (oculocerebrorenal syndrome of Lowe). Futhermore, the 5-ptases SHIP [SH2 (Src homology 2)-domain-containing inositol phosphatase] 2, SKIP (skeletal muscle- and kidney-enriched inositol phosphatase) and 72-5ptase (72 kDa 5-ptase)/Type IV/Inpp5e (inositol polyphosphate 5-phosphatase E) are implicated in negatively regulating insulin signalling and glucose homoeostasis in specific tissues. SHIP2 polymorphisms are associated with a predisposition to insulin resistance. Gene profiling studies have identified changes in the expression of various 5-ptases in specific cancers. In addition, 5-ptases such as SHIP1, SHIP2 and 72-5ptase/Type IV/Inpp5e regulate macrophage phagocytosis, and SHIP1 also controls haemopoietic cell proliferation. Therefore the 5-ptases are a significant family of signal-modulating enzymes that govern a plethora of cellular functions by regulating the levels of specific phosphoinositides. Emerging studies have implicated their loss or gain of function in human disease.

SHIP-2 is a phosphoinositidylinositol 3,4,5 trisphosphate (PtdIns[3,4,5]P3) 5-phosphatase that contains an NH2-terminal SH2 domain, a central 5-phosphatase domain, and a COOH-terminal proline-rich domain. SHIP-2 negatively regulates insulin signaling. In unstimulated cells, SHIP-2 localized in a perinuclear cytosolic distribution and at the leading edge of the cell. Endogenous and recombinant SHIP-2 localized to membrane ruffles, which were mediated by the COOH-terminal proline–rich domain. To identify proteins that bind to the SHIP-2 proline–rich domain, yeast two-hybrid screening was performed, which isolated actin-binding protein filamin C. In addition, both filamin A and B specifically interacted with SHIP-2 in this assay. SHIP-2 coimmunoprecipitated with filamin from COS-7 cells, and association between these species did not change after epidermal growth factor stimulation. SHIP-2 colocalized with filamin at Z-lines and the sarcolemma in striated muscle sections and at membrane ruffles in COS-7 cells, although the membrane ruffling response was reduced in cells overexpressing SHIP-2. SHIP-2 membrane ruffle localization was dependent on filamin binding, as SHIP-2 was expressed exclusively in the cytosol of filamin-deficient cells. Recombinant SHIP-2 regulated PtdIns(3,4,5)P3 levels and submembraneous actin at membrane ruffles after growth factor stimulation, dependent on SHIP-2 catalytic activity. Collectively these studies demonstrate that filamin-dependent SHIP-2 localization critically regulates phosphatidylinositol 3 kinase signaling to the actin cytoskeleton.

The inositol-polyphosphate 5-phosphatase enzyme family removes the 5-position phosphate from both inositol phosphate and phosphoinositide signaling molecules. We have cloned and characterized a novel 5-phosphatase, which demonstrates a restricted substrate specificity and tissue expression. The 3.9-kb cDNA predicts for a 72-kDa protein with an N-terminal proline rich domain, a central 5-phosphatase domain, and a Cterminal CAAX motif. The 3.9-kilobase mRNA showed a restricted expression but was abundant in testis and brain. Antibodies against the sequence detected a 72-kDa protein in the testis in the detergent-insoluble fraction. Indirect immunofluorescence of the Tera-1 cell line using anti-peptide antibodies to the 72-kDa 5-phosphatase demonstrated that the enzyme is predominantly located to the Golgi. Expression of green fluorescent protein-tagged 72-kDa 5-phosphatase in COS-7 cells revealed that the enzyme localized predominantly to the Golgi, mediated by the N-terminal proline-rich domain, but not the C-terminal CAAX motif. In vitro, the protein inserted into microsomal membranes on the cytoplasmic face of the membrane. Immunoprecipitated recombinant 72-kDa 5-phosphatase hydrolyzed phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,5-bisphosphate, forming phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3-phosphate, respectively. We propose that the novel 5-phosphatase hydrolyzes phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,5-bisphosphate on the cytoplasmic Golgi membrane and thereby may regulate Golgi-vesicular trafficking.The inositol-polyphosphate 5-phosphatases (5-phosphatases) 1 are a large family of enzymes that remove the 5-position phosphate from the inositol ring of phosphatidylinositols including phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ), phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P 3 ), and the inositol phosphates, inositol 1,4,5-trisphosphate (Ins(1,4,5)-P 3 ) and inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P 4 ). Nine distinct mammalian 5-phosphatases have been identified and characterized, while four 5-phosphatases have been described in Saccharomyces cerevisiae. All 5-phosphatases are defined by the presence of a conserved 300-amino acid domain, which contains two signature motifs, proposed to mediate substrate binding and catalysis (1, 2). Although all 5-phosphatase enzymes contain these signature motifs, there is considerable diversity in the substrate specificity of 5-phosphatase isoforms. The enzyme family has been subclassified on this basis into four types, I-IV. The type I enzymes, characterized by the 43-kDa 5-phosphatase (also called 5-phosphatase I), hydrolyze Ins(1,4,5)P 3 and Ins(1,3,4,5)P 4 but not any of the 5-position phosphoinositide substrates; the type II 5-phosphatases, including synaptojanin, 5-phosphatase II (originally designated the 75-kDa 5-phosphatase), and the protein product of the oculocerebrorenal syndrome (OCRL) gene, hydrolyze PtdIns-(4,5)P 2 , PtdIns(3,4,5)P 3 , Ins(1,3,4,5)P 4 ...

Serine proteinase inhibitors (serpins) are classically regulators of extracellular proteolysis, however, recent evidence suggests that some function intracellularly. Such "ovalbumin" serpins include the human proteinase inhibitors 6 (PI-6), 8 (PI-8), and 9 (PI-9), plasminogen activator inhibitor 2, and the monocyte/neutrophil elastase inhibitor. PI-9 is a potent granzyme B (graB) inhibitor that has an unusual P 1 Glu and is present primarily in lymphocytes. In a search for the murine equivalent of PI-9 we screened cDNA libraries, and performed reverse transcriptase-polymerase chain reaction on RNA isolated from leukocyte cell lines and from lymph nodes and spleens of allo-immunized mice. We identified 10 new ovalbumin serpin sequences: two resemble PI-8, two resemble PI-9, and the remaining six have no obvious human counterparts. By RNA analysis only one of the two sequences resembling PI-9 (designated SPI6) is present in mouse lymphocytes while the other (a partial clone designated mBM2A) is predominantly in testis. SPI6 comprises a 1.8-kilobase cDNA encoding a 374-amino acid polypeptide that is 68% identical to PI-9. mBM2A is 65% identical to PI-9 and over 80% identical to SPI6. Although the reactive loops of SPI6 and mBM2A differ from PI-9, both contain a Glu in a region likely to contain the P 1 -P 1 bond. SPI6 produced in vitro using a coupled transcription/translation system formed an SDS-stable complex with human graB and did not interact with trypsin, chymotrypsin, leukocyte elastase, pancreatic elastase, thrombin, or cathepsin G. Recombinant SPI6 produced in a yeast expression system was used to examine the interaction with human graB in more detail. The second-order rate constant for the interaction was estimated as 8 ؋ 10 4 M ؊1 s ؊1 , and inhibition depended on the Glu in the SPI6 reactive center. The SPI6 gene was mapped to the same region on mouse chromosome 13 as Spi3, which encodes the murine homolog of PI-6. We conclude that even though their reactive centers are not highly conserved, SPI6 is a functional homolog of PI-9, and that the regulation of graB in the mouse may involve a second serpin encoded by mBM2A. Our identification of multiple sequence homologs of PI-8 and PI-9, and six new ovalbumin serpins, is consonant with the idea that the larger set of granule and other proteinases known to exist in the mouse (compared with human) is balanced by a larger array of serpins.

Metastasis is the major cause of breast cancer mortality. Phosphoinositide 3-kinase (PI3K) generated PtdIns(3,4,5)P3 activates AKT, which promotes breast cancer cell proliferation and regulates migration. To date, none of the inositol polyphosphate 5-phosphatases that inhibit PI3K/AKT signaling have been reported as tumor suppressors in breast cancer. Here, we show depletion of the inositol polyphosphate 5-phosphatase PIPP (INPP5J) increases breast cancer cell transformation, but reduces cell migration and invasion. Pipp ablation accelerates oncogene-driven breast cancer tumor growth in vivo, but paradoxically reduces metastasis by regulating AKT1-dependent tumor cell migration. PIPP mRNA expression is reduced in human ER-negative breast cancers associated with reduced long-term outcome. Collectively, our findings identify PIPP as a suppressor of oncogenic PI3K/AKT signaling in breast cancer.

The spatial activation of phosphoinositide 3-kinase (PI3-kinase) signaling at the axon growth cone generates phosphatidylinositol 3,4,5 trisphosphate (PtdIns(3,4,5)P3), which localizes and facilitates Akt activation and stimulates GSK-3beta inactivation, promoting microtubule polymerization and axon elongation. However, the molecular mechanisms that govern the spatial down-regulation of PtdIns(3,4,5)P3 signaling at the growth cone remain undetermined. The inositol polyphosphate 5-phosphatases (5-phosphatase) hydrolyze the 5-position phosphate from phosphatidylinositol 4,5 bisphosphate (PtdIns(4,5)P2) and/or PtdIns(3,4,5)P3. We demonstrate here that PIPP, an uncharacterized 5-phosphatase, hydrolyzes PtdIns(3,4,5)P3 forming PtdIns(3,4)P2, decreasing Ser473-Akt phosphorylation. PIPP is expressed in PC12 cells, localizing to the plasma membrane of undifferentiated cells and the neurite shaft and growth cone of NGF-differentiated neurites. Overexpression of wild-type, but not catalytically inactive PIPP, in PC12 cells inhibited neurite elongation. Targeted depletion of PIPP using RNA interference (RNAi) resulted in enhanced neurite differentiation, associated with neurite hyperelongation. Inhibition of PI3-kinase activity prevented neurite hyperelongation in PIPP-deficient cells. PIPP targeted-depletion resulted in increased phospho-Ser473-Akt and phospho-Ser9-GSK-3beta, specifically at the neurite growth cone, and accumulation of PtdIns(3,4,5)P3 at this site, associated with enhanced microtubule polymerization in the neurite shaft. PIPP therefore inhibits PI3-kinase-dependent neurite elongation in PC12 cells, via regulation of the spatial distribution of phospho-Ser473-Akt and phospho-Ser9-GSK-3beta signaling.