Phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P 3 ) is a key molecule involved in cell growth signaling. We demonstrated that overexpression of PTEN, a putative tumor suppressor, reduced insulininduced PtdIns(3,4,5)P 3 production in human 293 cells without effecting insulin-induced phosphoinositide 3-kinase activation. Further, transfection of the catalytically inactive mutant of PTEN (C124S) caused PtdIns(3,4,5)P 3 accumulation in the absence of insulin stimulation. Purified recombinant PTEN catalyzed dephosphorylation of PtdIns(3,4,5)P 3 , specifically at position 3 on the inositol ring. PTEN also exhibited 3-phosphatase activity toward inositol 1,3,4,5-tetrakisphosphate. Our results raise the possibility that PTEN acts in vivo as a phosphoinositide 3-phosphatase by regulating PtdIns(3,4,5)P 3 levels. As expected, the C124S mutant of PTEN was incapable of catalyzing dephosphorylation of PtdIns(3,4,5)P 3 consistent with the mechanism observed in protein-tyrosine phosphatase-catalyzed reactions.A recently identified candidate tumor suppressor gene, PTEN/MMAC1, shares sequence identity with the family of protein-tyrosine phosphatases (PTPases) 1 (1). Deletions and mutations within the PTEN gene have been observed in several cancer cell types and tumor cell lines (2, 3). Additional evidence that PTEN functions as a tumor suppressor was obtained by Furnari et al. (4), who showed that PTEN had a growth suppressor activity in glioma cells. PTEN encodes the active site consensus motif HCXXGXXR(S/T) found in all PTPases. In contrast, the recombinant protein is a poor catalyst toward both phosphoproteins and peptide substrates with the highest activity of PTEN observed toward the highly negatively charged, multiply phosphorylated polymer of (Glu-Tyr) n (5, 6). Based on these observations we thought it possible that PTEN could catalyze the dephosphorylation of acidic nonproteinaceous substrate. Identification of possible in vivo substrates would not only suggest a possible physiological function of PTEN, but they might also provide insight into how PTEN functions as a tumor suppressor.PtdIns(3,4,5)P 3 is an important second messenger involved in cell growth signaling (7). PtdIns(3,4,5)P 3 is specifically produced from PtdIns(4,5)P 2 by PI 3-kinase upon stimulation by a variety of ligands (7). Recent studies have identified that PtdIns(3,4,5)P 3 can directly activate Akt, which in turn activates p70 S6 kinase and inhibits glycogen synthase kinase-3 (8, 9). Although there are several phosphoinositide 5-phosphatases, the mechanism of regulation and particularly the degradation pathway of PtdIns(3,4,5)P 3 in vivo is still unclear (10,11). In the present study we demonstrate that recombinant PTEN has PtdIns(3,4,5)P 3 3-phosphatase activity. In addition, we provide evidence that PTEN may act in vivo as a regulator of PtdIns(3,4,5)P 3 , which produces a substrate that can be recycled by PI 3-kinase. EXPERIMENTAL PROCEDURESDetermination of PtdIns(3,4,5)P 3 in Vivo-The coding sequence of human PTEN and the C124S mutant o...

The PTEN tumor suppressor is mutated in diverse human cancers and in hereditary cancer predisposition syndromes. PTEN is a phosphatase that can act on both polypeptide and phosphoinositide substrates in vitro. The PTEN structure reveals a phosphatase domain that is similar to protein phosphatases but has an enlarged active site important for the accommodation of the phosphoinositide substrate. The structure also reveals that PTEN has a C2 domain. The PTEN C2 domain binds phospholipid membranes in vitro, and mutation of basic residues that could mediate this reduces PTEN's membrane affinity and its ability to suppress the growth of glioblastoma tumor cells. The phosphatase and C2 domains associate across an extensive interface, suggesting that the C2 domain may serve to productively position the catalytic domain on the membrane.

We demonstrate the efficacy of double-stranded RNA-mediated interference (RNAi) of gene expression in generating ''knock-out'' phenotypes for specific proteins in several Drosophila cell lines. We prove the applicability of this technique for studying signaling cascades by dissecting the well-characterized insulin signal transduction pathway. Specifically, we demonstrate that inhibiting the expression of the DSOR1 (mitogen-activated protein kinase kinase, MAPKK) prevents the activation of the downstream ERK-A (MAPK).In contrast, blocking ERK-A expression results in increased activation of DSOR1. We also show that Drosophila AKT (DAKT) activation depends on the insulin receptor substrate, CHICO (IRS1-4). Finally, we demonstrate that blocking the expression of Drosophila PTEN results in the activation of DAKT. In all cases, the interference of the biochemical cascade by RNAi is consistent with the known steps in the pathway. We extend this powerful technique to study two proteins, DSH3PX1 and Drosophila ACK (DACK). DSH3PX1 is an SH3, phox homology domain-containing protein, and DACK is homologous to the mammalian activated Cdc42 tyrosine kinase, ACK. Using RNAi, we demonstrate that DACK is upstream of DSH3PX1 phosphorylation, making DSH3PX1 an identified downstream target͞substrate of ACK-like tyrosine kinases. These experiments highlight the usefulness of RNAi in dissecting complex biochemical signaling cascades and provide a highly effective method for determining the function of the identified genes arising from the Drosophila genome sequencing project.

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