The influence of inositol phosphates and phosphoinositides on the ␣ isoform of the RAC-protein kinase B (RAC/PKB) was studied using purified wild type and mutant kinase preparations and a recombinant pleckstrin homology (PH) domain. Binding of inositol phosphates and phosphoinositides to the PH domain was measured as the quenching of intrinsic tryptophan fluorescence. Inositol phosphates and D3-phosphorylated phosphoinositides bound with affinities of 1-10 M and 0.5 M, respectively. Similar values were obtained using RAC/PKB expressed and purified from baculovirus-infected Sf9 cells in the fluorescence assay. The influence of synthetic dioctanoyl derivatives of phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate on the activity of RAC/PKB purified from transfected COS-1 cells was studied. Phosphatidylinositol 3,4,5-trisphosphate was found to inhibit the RAC/ PKB kinase activity with half-maximal inhibition at 2.5 M. In contrast, phosphatidylinositol 3,4-bisphosphate stimulated kinase activity (half-maximal stimulation at 2.5 M). A mutant RAC/PKB protein lacking the PH domain was not affected by D3-phosphorylated phosphoinositides. These results demonstrate that the PH domain of RAC/PKB binds inositol phosphates and phosphoinositides with high affinity, and suggest that the products of the phosphatidylinositide 3-kinase can act as both a membrane anchor and modulator of RAC/ PKB activity. The data also provide further evidence for a link between phosphatidylinositide 3-kinase and RAC/ PKB regulation. The stimulation of receptor tyrosine kinases (RTK)1 by agonists leads to immediate activation of intracellular signal transduction pathways. The assembly of multiprotein complexes at the plasma membrane is one important feature of RTK signal transduction mechanisms (reviewed in Refs. 1 and 2). Numerous studies suggest that the activation of phosphatidylinositide 3-kinase (PI3-K) by growth factors is also involved (3, 4), leading to the accumulation of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P 3 ) and phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P 2 ); these metabolites are assumed to act as second messengers (5). Recently RAC-protein kinase B (RAC/PKB) has emerged as a key player in the PI3-K-stimulated signaling pathway, based on the inhibition of its activation by wortmannin (6 -11).RAC/PKB is a subfamily of the second messenger-regulated serine/threonine kinases (12). Three isoforms (␣, ␤, ␥) have been identified, each consisting of an amino-terminal pleckstrin homology (PH) domain, a central kinase domain, and a serine/threonine-rich carboxyl-terminal region (13-17). Various stimuli, such as insulin, PDGF, epidermal growth factor, basic fibroblast growth factor, serum (6 -10), and protein phosphatase inhibitors (9), lead to activation of RAC/PKB kinase activity. The activation is promoted by signals emanating from RTK-regulated PI3-K and is accompanied by an increase in serine/threonine phosphorylation of RAC/PKB (6, 9). The phosphorylation of two sites has been ...

N~tnc oxide (NO) Inhibits a variety of heme-contammg enzymes, mcludmg NO synthase and cytochrome P4501Al and 2Bl The present study exammed whether NO mhlblts the production of 20-hydroxyelcosatetraenolc acid (20-HETE) by cytochrome P4504A enzymes and whether blockade of the production of this substance contributes to the vascular effects of NO Sodium mtroprusslde (SNP, lo-', lo-", and 10m3 mol/L) reduced the productlon of 20-HETE by renal mlcrosomes incubated with arachldomc acid to 71+5%, 29t4%, and 4+2% of control, respectlvely (n=S) Slmllar results were obtained with the use of 1-propanamme, 3-(2-hydroxy-2-mtroso-1-propylhydrazmo) (n=3) To determme whether mhlbitlon of 20-HETE contributes to the vasodllatory effects of NO, the effects of dlbromo-dodecenylmethylsulfinude (DDMS), a selective mhlbltor of the formatlon of 20-HETE, on the response to SNP (lo-' to 10m3 mol/L) were examined m rat renal artenoles preconstructed with phenylephrme (n=.5) SNP increased vascular diameter m a concentrafion-dependent manner to 82t4% of control After DDMS (25 ymol/L), SNP (lo-' mol/L) increased vascular diameter by only 17+3% The effects of DDMS on the mean arterial pressure (MAP) and renal blood flow (RBF) responses to mfuslon of an NO donor and a synthase mhibltor were also examined m thlobutabarbltal-anesthetrzed, Sprague-Dawley rats Infusion of MAHMA NONOate at 1, 3, 5, and 10 nmol/mm reduced MAP by 16+2, 3023,40t5, and 48?5 mm Hg and lowered renal vascular resistance (RVR) by 15+3%, 26?2%, 30?3%, and 34?4% of control After DDMS (10 mg/kg, n=7 rats), the MAP and RVR responses to I-hexamme, 6-(2-hydroxy-1 -methyl-2-mtrohydrazmo)N-methyl (MAHMA NONOate) averaged only 20% of those seen durmg control In other expenments, MAP Increased by 3224% and RBF fell to 56+5% of control after admmlstratlon of N-mtro-L-argmme (L-NArg) (10 mg/kg IV) After DDMS (10 mg/kg, n=7 rats), MAP Increased by only 19+4% and RBF fell by only 724% after L-NArg These results indicate that NO mhlblts cytochrome P4504A enzymes and that mhlbltlon of the production of 20-HETE contributes to the vasodllatory effects of NO (Hypertension. 1997;29[part 2]:320-325.)Key Words l mtnc oxide l vasculature l enzymes R ecent studies have mdlcated that the effects of many renal vasodllators are dependent on the release of NO from the endothehum Blockade of NO synthesis increases arterial pressure, decreases RBF, and potentiates tubuloglomerular feedback responses. 1 These results indicate that tonic release of NO plays an important modulatory role m the regulation of both renal and penpheral vascular tone. It 1s generally assumed that the vasodllatory effects of NO are mediated by cGMP secondary to shmulatlon of guanylyl cyclase 2.3 This concluslon 1s based on the observations that endothehum-dependent vasodilators and NO donors increase cGMP m vascular tissue and that methylene blue and other mhlbltors of guanylyl cyclase m many vessels can elm-nnate the vasodilatory response However, this generalized scheme for NO-induced vasodllatlon has been questioned ...

Previous structural analyses of diphosphoinositol polyphosphates in biological systems have relied largely on NMR analysis. For example, in Dictyostelium discoideum, diphosphoinositol pentakisphosphate was determined by NMR to be 4- and/or 6-PPInsP5, and the bisdiphosphoinositol tetrakisphosphate was found to be 4, 5-bisPPInsP4 and/or 5,6-bisPPInsP4 [Laussmann, Eujen, Weisshuhn, Thiel and Vogel (1996) Biochem. J. 315, 715-720]. We now describe three recent technical developments to aid the analysis of these compounds, not just in Dictyostelium, but also in a wider range of biological systems: (i) improved resolution and sensitivity of detection of PPInsP5 isomers by microbore metal-dye-detection HPLC; (ii) the use of the enantiomerically specific properties of a rat hepatic diphosphatase; (iii) chemical synthesis of enantiomerically pure reference standards of all six possible PPInsP5 isomers. Thus we now demonstrate that the major PPInsP5 isomer in Dictyostelium is 6-PPInsP5. Similar findings obtained using the same synthetic standards have been published [Laussmann, Reddy, Reddy, Falck and Vogel (1997) Biochem. J. 322, 31-33]. In addition, we show that 10-25% of the Dictyostelium PPInsP5 pool is comprised of 5-PPInsP5. The biological significance of this new observation was reinforced by our demonstration that 5-PPInsP5 is the predominant PPInsP5 isomer in four different mammalian cell lines (FTC human thyroid cancer cells, Swiss 3T3 fibroblasts, Jurkat T-cells and Chinese hamster ovary cells). The fact that the cellular spectrum of diphosphoinositol polyphosphates varies across phylogenetic boundaries underscores the value of our technological developments for future determinations of the structures of this class of compounds in other systems.

Two diphospho-myo-inositol phosphates from Dictyostelium were recently investigated by two-dimensional 1H/31P NMR analysis and assigned to be either D-4-diphospho-myo-inositol pentakisphosphate (D-4-PP-InsP5) and D-4,5-bisdiphospho-myo-inositol tetrakisphosphate (D-4,5-bis-PP-InsP4) or their corresponding enantiomers D-6-PP-InsP5 and D-5,6-bis-PP-InsP4. In the present study the naturally occurring enantiomers were identified by using defined synthetic PP-InsP5 isomers as substrates for a partially purified PP-InsP5 5-kinase from Dictyostelium. This enzyme specifically phosphorylates the naturally occurring PP-InsP5 and the synthetic D-6-PP-InsP5, leading to D-5,6-bis-PP-InsP4. In contrast, neither D-4-PP-InsP5 nor D-1-PP-InsP5 or D-3-PP-InsP5 are converted by the enzyme.

No abstract

The two major diphospho inositol phosphates from the axenic strain Dictyostelium discoideum AX2 were previously investigated and identified as 6-PP-InsP5 and 5,6-bis-PP-InsP4. In order to examine whether these findings are representative of Dictyostelids in general, five non-axenic wild-type species of Dictyostelium and two of Polysphondylium were studied. It was found that all of the Dictyostelium species exhibit similar patterns of diphospho inositol phosphates. By contrast, both of the Polysphondylium species contain 5-PP-InsP5 as the predominant isomer. Besides 5,6-bis-PP-InsP4, a new bis-PP-InsP4 was detected in Polysphondylium. This compound is either 1,5-bis-PP-InsP4 or its corresponding enantiomer 3,5-bis-PP-InsP5. The structures were elucidated by two-dimensional 1H-1H and 1H-31P NMR analysis. Additionally, they were confirmed using a specific 6-PP-InsP(5)-5-kinase from D. discoideum AX2 as an enantio-specific tool and enantiomerically pure reference standards.

We have reported that platelets exposed to thrombin or thrombin receptor-directed ligand activate phospholipase C and rapidly accumulate phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P 3 ) and phosphatidylinositol (3,4)-bisphosphate (PtdIns(3,4)P 2 ) as a function of the activation of phosphoinositide (PI) 3-kinases in a GTP-binding protein-dependent manner. In such platelets, serine-and threonine-directed phosphorylation of pleckstrin also occurs and has been attributed to protein kinase C activation. We now report that the phosphorylation of pleckstrin is partially dependent upon PI 3-kinase. Pleckstrin phosphorylation in response to thrombin receptor stimulation is progressively susceptible to inhibition by wortmannin, a potent and specific inhibitor of platelet PI 3-kinases. PI 3-kinase thus seems to play a gradually increasing role in promoting pleckstrin phosphorylation. The IC 50 for wortmannin in inhibiting SFLLRN-stimulated 3-phosphorylated phosphoinositide accumulation is 10 nM, and that (i.e. 50% of maximum inhibition) for inhibiting pleckstrin phosphorylation is 15 nM. Synthetic PtdIns(3,4,5)P 3 , when added to saponin-permeabilized (but not intact) platelets, causes wortmannin-insensitive phosphorylation of pleckstrin. PtdIns(3,4,5)P 3 also overcomes the inhibition by wortmannin of thrombin-or guanosine 5-3-O-(thio)trisphosphatestimulated pleckstrin phosphorylation. In contrast, PtdIns(4,5)P 2 or inositol (1,3,4,5)-tetrakisphosphate are ineffective in these respects. The pattern of phosphorylation of pleckstrin activated by PtdIns(3,4,5)P 3 is not distinguishable from that of pleckstrin phosphorylated in intact platelets exposed to protein kinase C-activating ␤-phorbol myristate acetate, mimicking diacylglycerol. Activation of protein kinase(s) by PtdIns(3,4,5)P 3 thus offers a route for pleckstrin phosphorylation in vivo that is an alternative to activation of phospholipase C 3 diacylglycerol 3 protein kinase C.It is being appreciated increasingly that the metabolism of phosphoinositides catalyzed by phosphoinositide (PI) 1 3-kinase has signal-transducing consequences that rival those of phosphoinositidase C activation in a variety of cells. PtdIns(3,4,5)P 3 and PtdIns(3,4)P 2 are both major physiological products of activated PI 3-kinase. They have been proposed to be modulators of protein kinase(s), thereby amplifying an initial limited amount of signal (1), analogously with diacylglycerol and PKC. Indeed, several members of the PKC family have been reported recently to be stimulated by PtdIns(3,4,5)P 3 , including a mixture of rat brain PKC isozymes (2), PKC (3), and PKC⑀, ␦, and (by either PtdIns(3,4,5)P 3 or PtdIns(3,4)P 2 ) (4). Other protein kinases may also be stimulated. We have described the activation of two species of PI 3-kinase by a variety of agonists, including thrombin, thromboxane A 2 analogue, GTP␥S, and thrombin receptor-directed ligand, leading to the sequential accumulation of PtdIns(3,4,5)P 3 and PtdIns(3,4)P 2 in intact or permeabilized platelets (5-7). Another re...

L-a-Phosphatidyl-D-myo-inositol3,4,5-trisphosphate (3,4,, the most prominent member of a new class of intracellular second messengers, and two ether analogs were conveniently prepared from the differentially functionalized D-myo-inositol intermediate 7 which was ultimately derived from the unique cyclitol precursor dehydroshikimic acid (1). Critical transformations included the stereoselective hydride reduction of the shikimate ketone, exclusive osmylation from the a-face to give 3, controlled enolization of 4, and dioxirane epoxidation with in situ rearrangement affording ketone 5. Dioctanoyl 3,4,5-PIP3 (9a) and its dioctyl ether analog 9b selectively activated the 6, E , and 7-isotypes of protein kinase C (PKC).