A selective role for phosphatidylinositol 3,4,5-trisphosphate in the Gi-dependent activation of platelet Rap1B

The small GTP-binding protein Rap1B is activated in human platelets upon stimulation of a G(i)-dependent signaling pathway. In this work, we found that inhibition of platelet adenylyl cyclase by dideoxyadenosine or SQ22536 did not cause activation of Rap1B and did not restore Rap1B activation in platelets stimulated by cross-linking of Fcgamma receptor IIA (FcgammaRIIA) in the presence of ADP scavengers. Moreover, elevation of the intracellular cAMP concentration did not impair the G(i)-dependent activation of Rap1B. Two unrelated inhibitors of phosphatidylinositol 3-kinase (PI3K), wortmannin and LY294002, totally prevented Rap1B activation in platelets stimulated by cross-linking of FcgammaRIIA, by stimulation of the P2Y(12) receptor for ADP, or by epinephrine. However, in platelets from PI3Kgamma-deficient mice, both ADP and epinephrine were still able to normally stimulate Rap1B activation through a PI3K-dependent mechanism, suggesting the involvement of a different isoform of the enzyme. Moreover, the lack of PI3Kgamma did not prevent the ability of epinephrine to potentiate platelet aggregation through a G(i)-dependent pathway. The inhibitory effect of wortmannin on Rap1B activation was overcome by addition of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)), but not PtdIns(3,4)P(2), although both lipids were found to support phosphorylation of Akt. Moreover, PtdIns(3,4,5)P(3) was able to relieve the inhibitory effect of apyrase on FcgammaRIIA-mediated platelet aggregation. We conclude that stimulation of a G(i)-dependent signaling pathway causes activation of the small GTPase Rap1B through the action of the PI3K product PtdIns(3,4,5)P(3), but not PtdIns(3,4)P(2), and that this process may contribute to potentiation of platelet aggregation.     

Regulation of P-Rex1 by phosphatidylinositol (3,4,5)-trisphosphate and Gbetagamma subunits

P-Rex1 is a guanine-nucleotide exchange factor (GEF) for the small GTPase Rac. We have investigated here the mechanisms of stimulation of P-Rex1 Rac-GEF activity by the lipid second messenger phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) and the Gbetagamma subunits of heterotrimeric G proteins. We show that a P-Rex1 mutant lacking the PH domain (DeltaPH) cannot be stimulated by PtdIns(3,4,5)P3, which implies that the PH domain confers PtdIns(3,4,5)P3 regulation of P-Rex1 Rac-GEF activity. Consistent with this, we found that PtdIns(3,4,5)P3 binds to the PH domain of P-Rex1 and that the DH/PH domain tandem is sufficient for PtdIns(3,4,5)P3-stimulated P-Rex1 activity. The Rac-GEF activities of the DeltaPH mutant and the DH/PH domain tandem can both be stimulated by Gbetagamma subunits, which infers that Gbetagamma subunits regulate P-Rex1 activity by binding to the catalytic DH domain. Deletion of the DEP, PDZ, or inositol polyphosphate 4-phosphatase homology domains has no major consequences on the abilities of either PtdIns(3,4,5)P3 or Gbetagamma subunits to stimulate P-Rex1 Rac-GEF activity. However, the presence of any of these domains impacts on the levels of basal and/or stimulated P-Rex1 Rac-GEF activity, suggesting that there are important functional interactions between the DH/PH domain tandem and the DEP, PDZ, and inositol polyphosphate 4-phosphatase homology domains of P-Rex1.     

Regulation of phosphatidylinositol 3-kinase activity and phosphatidylinositol 3,4,5-trisphosphate accumulation by neutrophil priming agents

Neutrophil priming by agents such as TNF-alpha and GM-CSF causes a dramatic increase in the response of these cells to secretagogue agonists and affects the capacity of neutrophils to induce tissue injury. In view of the central role of phosphatidylinositol 3-kinase (PI3-kinase) in regulating NADPH oxidase activity we examined the influence of priming agents on agonist-stimulated phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) accumulation in human neutrophils. Pretreatment of neutrophils with TNF-alpha or GM-CSF, while not influencing fMLP-stimulated PtdIns(3,4,5)P3 accumulation at 5 s, caused a major increase in PtdIns(3,4,5)P3 at later times (10-60 s), which paralleled the augmented superoxide anion (O2-) response. The intimate relationship between PtdIns(3,4,5)P3 accumulation and O2- release was confirmed using platelet-activating factor, which caused full but transient priming of both responses. Likewise, LY294002, a PI3-kinase inhibitor, and genistein, a tyrosine kinase inhibitor, caused parallel inhibition of O2- generation and PtdIns(3,4,5)P3 accumulation; in contrast, radicicol, which inhibits receptor-mediated activation of p85 PI3-kinase, had no effect on either response. Despite major increases in PI3-kinase activity observed in p85 and anti-phosphotyrosine immunoprecipitates in growth factor-stimulated smooth muscle cells, no such increase was observed in primed/stimulated neutrophils. In contrast, both fMLP and TNF-alpha alone caused a 3-fold increase in PI3-kinase activity in p110gamma PI3-kinase immunoprecipitates. p21(ras) activation (an upstream regulator of PI3-kinase) was unaffected by priming. These data demonstrate that timing and magnitude of PtdIns(3,4,5)P3 accumulation in neutrophils correlate closely with O2- generation, that PI3-kinase-gamma is responsible for the enhanced PtdIns(3,4,5)P3 production seen in primed cells, and that factors other than activation of p21(ras) underlie this response.     

抑癌基因D L C-1 的研究进展

DLC-1（frequently deletedin liver cancer）基因是新发现的一个肿瘤抑制基因。它的失活有可能参与肿瘤的发生和发展。本文拟就DLC-1基因的结构功能及其在遗传和表遗传方面的失活机制作一综述。     

Identification of a functional phosphatidylinositol 3,4,5-trisphosphate binding site in the epithelial Na+ channel

Membrane phospholipids, such as phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P(3)), are signaling molecules that can directly modulate the activity of ion channels, including the epithelial Na(+) channel (ENaC). Whereas PI(3,4,5)P(3) directly activates ENaC, its binding site within the channel has not been identified. We identify here a region of gamma-mENaC just following the second trans-membrane domain (residues 569-583) important to PI(3,4,5)P(3) binding and regulation. Deletion of this track decreases activity of ENaC heterologously expressed in Chinese hamster ovary cells. K-Ras and its first effector phosphoinositide 3-OH kinase (PI3-K), as well as RhoA and its effector phosphatidylinositol 4-phosphate 5-kinase increase ENaC activity. Whereas the former, via generation of PI(3,4,5)P(3), increases ENaC open probability, the latter increases activity by increasing membrane levels of the channel. Deletion of the region just distal to the second trans-membrane domain disrupted regulation by K-Ras and PI3-K but not RhoA and phosphatidylinositol 4-phosphate 5-kinase. Moreover, PI(3,4,5)P(3) binds ENaC with deletion of the region following the second transmembrane domain disrupting this interaction and disrupting direct activation of the channel by PI(3,4,5)P(3). Mutation analysis revealed the importance of conserved positive and negative charged residues as well as bulky amino acids within this region to modulation of ENaC by PI3-K. The current results identify the region just distal to the second trans-membrane domain within gamma-mENaC as being part of a functional PI(3,4,5)P(3) binding site that directly impacts ENaC activity. Phospholipid binding to this site is probably mediated by the positively charged amino acids within this track, with negatively charged and bulky residues also influencing specificity of interactions.     

Nuclear phosphatidylinositol 3,4,5-trisphosphate, phosphatidylinositol 3-kinase, Akt, and PTen: emerging key regulators of anti-apoptotic signaling and carcinogenesis

Inositol lipid-derived second messengers have long been known to have an important regulatory role in cell physiology. Phosphatidylinositol 3-kinase (PI3K) synthesizes the second messenger 3,4,5'-phosphatidylinositol trisphosphate (Ptdlns 3,4,5P3) which controls a multitude of cell functions. Down-stream of PI3K/PtdIns 3,4,5P3 is the serine/threonine protein kinase Akt (protein kinase B, PKB). Since the PI3K/ PtdIns 3,4,5P3 /Akt pathway stimulates cell proliferation and suppresses apoptosis, it has been implicated in carcinogenesis. The lipid phosphatase PTEN is a negative regulator of this signaling network. Until recently, it was thought that this signal transduction cascade would promote its anti-apoptotic effects when activated in the cytoplasm. Several lines of evidence gathered over the past 20 years, have highlighted the existence of an autonomous nuclear inositol lipid cycle, strongly suggesting that lipids are important components of signaling pathways operating at the nuclear level. PI3K, PtdIns(3,4,5)P3, Akt, and PTEN have been identified within the nucleus and recent findings suggest that they are involved in cell survival also by operating in this organelle, through a block of caspase-activated DNase and inhibition of chromatin condensation. Here, we shall summarize the most updated and intriguing findings about nuclear PI3K/ PtdIns(3,4,5)P3/Akt/PTEN in relationship with carcinogenesis and suppression of apoptosis.     

AZU-1: a candidate breast tumor suppressor and biomarker for tumor progression

To identify genes misregulated in the final stages of breast carcinogenesis, we performed differential display to compare the gene expression patterns of the human tumorigenic mammary epithelial cells, HMT-3522-T4-2, with those of their immediate premalignant progenitors, HMT-3522-S2. We identified a novel gene, called anti-zuai-1 (AZU-1), that was abundantly expressed in non- and premalignant cells and tissues but was appreciably reduced in breast tumor cell types and in primary tumors. The AZU-1 gene encodes an acidic 571-amino-acid protein containing at least two structurally distinct domains with potential protein-binding functions: an N-terminal serine and proline-rich domain with a predicted immunoglobulin-like fold and a C-terminal coiled-coil domain. In HMT-3522 cells, the bulk of AZU-1 protein resided in a detergent-extractable cytoplasmic pool and was present at much lower levels in tumorigenic T4-2 cells than in their nonmalignant counterparts. Reversion of the tumorigenic phenotype of T4-2 cells, by means described previously, was accompanied by the up-regulation of AZU-1. In addition, reexpression of AZU-1 in T4-2 cells, using viral vectors, was sufficient to reduce their malignant phenotype substantially, both in culture and in vivo. These results indicate that AZU-1 is a candidate breast tumor suppressor that may exert its effects by promoting correct tissue morphogenesis.     

Production of phosphatidylinositol 3,4,5-trisphosphate and phosphatidic acid in platelet rafts: evidence for a critical role of cholesterol-enriched domains in human platelet activation.

Glycosphingolipid- and cholesterol-enriched membrane microdomains, called rafts, can be isolated from several mammalian cells, including platelets. These microdomains appear to play a critical role in signal transduction in several hematopoietic cells, but their function in blood platelets remains unknown. Herein, we first characterized the lipid composition, including the fatty acid composition of phospholipids, of human platelet rafts. Then their role in platelet activation process was investigated. Interestingly, thrombin stimulation led to morphological changes of rafts correlating with the production of lipid second messengers in these microdomains. Indeed, we could demonstrate for the first time that a large part of the stimulation-dependent production of phosphatidic acid and phosphoinositide 3-kinase products was concentrated in rafts. Moreover, cholesterol depletion with methyl-beta-cyclodextrin disrupted platelet rafts, dramatically decreased the agonist-dependent production of these lipid signaling molecules, and impaired platelet secretion and aggregation. Cholesterol repletion restored the physiological platelet responses. Altogether our data indicate that rafts are highly dynamic platelet membrane structures involved in critical signaling mechanisms linked to the production of lipid second messengers. The demonstration of phosphatidylinositol 3,4,5-trisphosphate production in rafts may have general implications for the understanding of the role of this key second messenger found ubiquitously in higher eucaryotic cells.     

LL5beta is a phosphatidylinositol (3,4,5)-trisphosphate sensor that can bind the cytoskeletal adaptor,gamma-filamin

We identified a potential phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P(3)) binding pleckstrin homology domain in the data bases and have cloned and expressed its full coding sequence (LL5beta). The protein bound PtdIns(3,4,5)P(3) selectively in vitro. Strikingly, a substantial proportion of LL5beta became associated with an unidentified intracellular vesicle population in the context of low PtdIns(3,4,5)P(3) levels produced by the addition of wortmannin or LY294002. In addition, expression of platelet-derived growth factor-receptor mutants unable to activate type 1A phosphoinositide 3-kinase (PI3K) or serum starvation in porcine aortic endothelial cells lead to redistribution of LL5beta to this vesicle population. Importantly, pleckstrin homology domain mutants of LL5beta that could not bind PtdIns(3,4,5)P(3) were constitutively localized to this vesicle population. At increased PtdIns(3,4,5)P(3) levels, LL5beta was redirected to a predominantly cytoplasmic distribution, presumably through a PI3K-dependent block on its targeting to the vesicular compartment. Furthermore, at high, hormone-stimulated PtdIns(3,4,5)P(3) levels, it became significantly plasma-membrane localized. The distribution of LL5beta is thus dramatically and uniquely sensitive to low levels of PtdIns(3,4,5)P(3) indicating it can act as a sensor of both low and hormone-stimulated levels of PtdIns(3,4,5)P(3). In addition, LL5beta bound to the cytoskeletal adaptor, gamma-filamin, tightly and in a PI3K-independent fashion, both in vitro and in vivo. This interaction could co-localize heterologously expressed gamma-filamin with GFP-LL5beta in the unidentified vesicles.     

Identification of protein kinase B (PKB) as a phosphatidylinositol 3,4,5-trisphosphate binding protein in Dictyostelium discoideum

We have searched for phosphatidylinositol (PI)-3,4,5-trisphosphate (PIP3) binding proteins in Dictyostelium discoideum using beads bearing a PIP3 analogue, PIP3-APB. One of the binding proteins with a molecular mass of 55 kDa was purified and its amino acid sequence was partially analyzed. Database searches showed that the analyzed sequence was identical to that of protein kinase B (PKB) of D. discoideum. The specific activity of D. discoideum PKB, when expressed together with constitutively active PI-3 kinase in mammalian cells, was elevated by about three-fold, suggesting that PKB could also act downstream of PI-3 kinase in Dictyostelium cells.     

MicroRNA-1 is a candidate tumor suppressor and prognostic marker in human prostate cancer

We previously reported that miR-1 is among the most consistently down-regulated miRs in primary human prostate tumors. In this follow-up study, we further corroborated this finding in an independent data set and made the novel observation that miR-1 expression is further reduced in distant metastasis and is a candidate predictor of disease recurrence. Moreover, we performed in vitro experiments to explore the tumor suppressor function of miR-1. Cell-based assays showed that miR-1 is epigenetically silenced in human prostate cancer. Overexpression of miR-1 in these cells led to growth inhibition and down-regulation of genes in pathways regulating cell cycle progression, mitosis, DNA replication/repair and actin dynamics. This observation was further corroborated with protein expression analysis and 3'-UTR-based reporter assays, indicating that genes in these pathways are either direct or indirect targets of miR-1. A gene set enrichment analysis revealed that the miR-1-mediated tumor suppressor effects are globally similar to those of histone deacetylase inhibitors. Lastly, we obtained preliminary evidence that miR-1 alters the cellular organization of F-actin and inhibits tumor cell invasion and filipodia formation. In conclusion, our findings indicate that miR-1 acts as a tumor suppressor in prostate cancer by influencing multiple cancer-related processes and by inhibiting cell proliferation and motility.     

The role of phosphatidylinositol 3'-kinase and its downstream signals in erbB-2-mediated transformation

We previously demonstrated that erbB-2-overexpressing human mammary epithelial (HME) cells exhibit several transformed phenotypes including growth factor independence, anchorage-independent growth, motility, and invasiveness. Because phosphatidylinositol 3'-kinase (PI3K) is a major target of erbB-2 activation, we tested the contribution that PI3K and its downstream signaling pathways make to these phenotypes. Utilizing a constitutively active form of PI3K, p110CAAX, we show that PI3K can mediate most phenotypes observed in erbB-2-overexpressing cells. To identify pathways leading from PI3K to specific phenotypes, we expressed constitutively active AKT or PTEN in erbB-2-overexpressing cells or in HME cells. HME cells expressing constitutively active AKT were growth factor independent, anchorage independent and motile, but not invasive. PTEN expression blocked erbB-2-mediated invasion but none of the other phenotypes. Rottlerin blocked invasion induced by p110CAAX and erbB-2, suggesting that protein kinase C delta (PKC-delta) is the downstream effector of PI3K responsible for the invasive capacity of the cells. Consistent with these observations, phospho-AKT remained detectable in erbB-2 cells treated with LY294002 or expressing exogenous PTEN, but was abolished by treatment with the p38MAP kinase inhibitor SB202190. Thus, both PI3K-dependent and p38MAP kinase-dependent pathways lead to activation of AKT, and activation of PKC-delta, via PI3K, mediates invasion.     

Synthesis of phosphatidylinositol 3,4-bisphosphate but not phosphatidylinositol 3,4,5-trisphosphate is closely correlated with protein-tyrosine phosphorylation in thrombin-activated human platelets.

Synthesis of D-3-phosphorylated phosphoinositides and its correlation with protein-tyrosine phosphorylation were examined using human platelets. Thrombin stimulation of platelets resulted in time- and dose-dependent production of phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2), which is absent from resting platelets. In contrast, phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) was detected in resting platelets, but remained unaffected by thrombin treatment. The production of PtdIns(3,4)P2 but not PtdIns(3,4,5)P3 was inhibited by pretreatment with staurosporine or dibutyryl cyclic adenosine monophosphate (dbcAMP). Protein-tyrosine phosphorylation, which is reportedly involved in generation of 3-phosphorylated phosphoinositides, was elicited in thrombin-activated platelets. The tyrosine phosphorylation was suppressed by pretreatment with staurosporine or dbcAMP. These observations suggest that synthesis of PtdIns(3,4)P2 but not PtdIns(3,4,5) P3 is closely correlated with protein-tyrosine phosphorylation in human platelets.     

Evidence that SHIP-1 contributes to phosphatidylinositol 3,4,5-trisphosphate metabolism in T lymphocytes and can regulate novel phosphoinositide 3-kinase effectors

The leukemic T cell line Jurkat is deficient in protein expression of the lipid phosphatases Src homology 2 domain containing inositol polyphosphate phosphatase (SHIP) and phosphatase and tensin homolog deleted on chromosome ten (PTEN). We examined whether the lack of expression of SHIP-1 and PTEN is shared by other leukemic T cell lines and PBLs. Analysis of a range of cell lines and PBLs revealed that unlike Jurkat cells, two other well-characterized T cell lines, namely CEM and MOLT-4 cells, expressed the 5'-phosphatase SHIP at the protein level. However, the 3-phosphatase PTEN was not expressed by CEM or MOLT-4 cells or Jurkat cells. The HUT78 cell line and PBLs expressed both SHIP and PTEN. Jurkat cells exhibited high basal levels of phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P(3); the lipid substrate for both SHIP and PTEN) as well as saturated protein kinase B (PKB) phosphorylation. Lower levels of PI(3,4,5)P(3) and higher levels of phosphatidylinositol 3,4-bisphosphate (PI(3,4)P(2)) as well as unsaturated constitutive phosphorylation of PKB were observed in CEM and MOLT-4 cells compared with Jurkat cells. In PBLs and HUT78 cells which express both PTEN and SHIP-1, there was no constitutive PI(3,4,5)P(3) or PKB phosphorylation, and receptor stimuli were able to elicit robust phosphorylation of PKB. Expression of a constitutively active SHIP-1 protein in Jurkat cells was sufficient to reduce both constitutive PKB membrane localization and PKB phosphorylation. Together, these data indicate important differences between T leukemic cells as well as PBLs, regarding expression of key lipid phosphatases. This study provides the first evidence that SHIP-1 can influence the constitutive levels of PI(3,4,5)P(3) and the activity of downstream phosphoinositide 3-kinase effectors in T lymphocytes.     

Cloning of a novel gene (ING1L) homologous to ING1, a candidate tumor suppressor

The ING1 gene encodes p33(ING1), a putative tumor suppressor for neuroblastomas and breast cancers, which has been shown to cooperate with p53 in controlling cell proliferation. We have isolated a novel human gene, ING1L, that potentially encodes a PHD-type zinc-finger protein highly homologous to p33(ING1). Fluorescence in situ hybridization and radiation-hybrid analyses assigned ING1L to human chromosome 4. Both ING1 and ING1L are expressed in a variety of human tissues, but we found ING1L expression to be significantly more pronounced in tumors from several colon-cancer patients than in normal colon tissues excised at the same surgical sites. Although the significance of this observation with respect to carcinogenesis remains to be established, the data suggest that ING1L might be involved in colon cancers through interference with signal(s) transmitted through p53 and p33(ING1).     

Direct activation of the epithelial Na(+) channel by phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,4-bisphosphate produced by phosphoinositide 3-OH kinase

The phospholipid phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) is accepted to be a direct modulator of ion channel activity. The products of phosphoinositide 3-OH kinase (PI3K), PtdIns(3,4)P(2) and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)), in contrast, are not. We report here activation of the epithelial Na(+) channel (ENaC) reconstituted in Chinese hamster ovary cells by PI3K. Insulin-like growth factor-I also activated reconstituted ENaC and increased Na(+) reabsorption across renal A6 epithelial cell monolayers via PI3K. Neither IGF-I nor PI3K affected the levels of ENaC in the plasma membrane. The effects of PI3K and IGF-I on ENaC activity paralleled changes in the plasma membrane levels of the PI3K product phospholipids, PtdIns(3,4)P(2)/PtdIns(3,4,5)P(3), as measured by evanescent field fluorescence microscopy. Both PtdIns(3,4)P(2) and PtdIns(3,4,5)P(3) activated ENaC in excised patches. Activation of ENaC by PI3K and its phospholipid products corresponded to changes in channel open probability. We conclude that PI3K directly modulates ENaC activity via PtdIns(3,4)P(2) and PtdIns(3,4,5)P(3). This represents a novel transduction pathway whereby growth factors, such as IGF-I, rapidly modulate target proteins independent of signaling elicited by kinases downstream of PI3K.