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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Alkaline phosphatase activity was released up to 100% from the membrane by incubating the rat osseous plate membrane-bound enzyme with phosphatidylinositol-specific phospholipase C. The molecular weight of the released enzyme was 145,000 on Sephacryl S-300 gel filtration and 66,000 on PAGE-SDS, suggesting a dimeric structure. Solubilization of the membrane-bound enzyme with phospholipase C did not destroy its ability to hydrolyse PNPP, ATP and pyrophosphate. The hydrolysis of ATP and PNPP by phosphatidylinositol-specific phospholipase C-released enzyme exhibited 'Michaelian' kinetics with K0.5 = 70 and 979 microM, respectively. For pyrophosphate, K0.5 was 128 microM and site-site interactions were observed (n = 1.4). Magnesium ions were stimulatory (K0.5 = 1.5 mM) and zinc ions were a powerful noncompetitive inhibitor (Ki = 6.2 microM) of phosphatidylinositol-specific phospholipase C-released enzyme. Phosphatidylinositol-specific phospholipase C-released alkaline phosphatase was relatively stable at 40 degrees C. However, with increasing temperature from 40-60 degrees C, the enzyme was inactivated rapidly following first order kinetics and thermal inactivation constants varied from 5.08 x 10(-4) min-1 to 0.684 min-1. Treatment of phosphatydilinositol-specific phospholipase C-released alkaline phosphatase with Chellex 100 depleted to 5% its original PNPPase activity. Magnesium (K0.5 = 29.5 microM), manganese (K0.5 = 5 microM) and cobalt ions (K0.5 = 10.1 microM) restored the activity of Chelex-treated enzyme, demonstrating its metalloenzyme nature. The stimulation of Chelex-treated enzyme by calcium ions (K0.5 = 653 microM) was less effective (only 26%) and occurred with site-site interactions (n = 0.7). Zinc ions had no stimulatory effects. The possibility that the soluble form of the enzyme, detected during endochondral ossification, would arise by the hydrolysis of the Pl-anchored form of osseous plate alkaline phosphatase is discussed.
Mol Cell Biochem 1995 Nov 22
PMID:Characterization of the phosphatidylinositol-specific phospholipase C-released form of rat osseous plate alkaline phosphatase and its possible significance on endochondral ossification. 875 Nov 58

Phosphatidylinositol (PI) 3-kinase is a cytoplasmic signaling molecule recruited to the membrane by activated growth factor receptors. The p85 subunit of PI 3-kinase links the catalytic p110 subunit to activated growth factor receptors and is required for enzymatic activity of p110. In this report, we describe the effects of expressing novel forms of p110 that are targeted to the membrane by either N-terminal myristoylation or C-terminal farnesylation. The expression of membrane-localized p110 is sufficient to trigger downstream responses characteristic of growth factor action, including the stimulation of pp70 S6 kinase, Akt/Rac, and Jun N-terminal kinase (JNK). These responses can also be triggered by expression of a form of p110 (p110*) that is cytosolic but exhibits a high specific activity. Finally, targeting of pl10* to the membrane results in maximal activation of downstream responses. Our data demonstrate that either membrane-targeted forms of p110 or a form of p110 with high specific activity can act as constitutively active PI 3-kinases and induce PI 3-kinase-dependent responses in the absence of growth factor stimulation. The results also show that PI 3-kinase activation is sufficient to stimulate several kinases that appear to function in different signaling pathways.
Mol Cell Biol 1996 Aug
PMID:Membrane localization of phosphatidylinositol 3-kinase is sufficient to activate multiple signal-transducing kinase pathways. 875 10

Phosphatidylinositol 3-kinase (PI 3-kinase) plays a role in a variety of biological processes, including regulation of gene expression, cell growth, and differentiation. However, little is known about its role in the cytoplasmic events involved in epidermal growth factor (EGF)-induced transduction of signals to the transcriptional machinery of the nucleus and in EGF-induced cell transformation. In this study, we examined whether PI 3-kinase is a mediator for the activation of AP-1 and neoplastic transformation by EGF in the murine epidermal cell line JB6. The results showed the following. (i) EGF not only induced a high level of PI 3-kinase activity by itself but also enhanced insulin-induced PI 3-kinase activity in JB6 P+ cells, the EGF-induced PI-3 kinase activity could be blocked by constitutive overexpression of a dominant negative P85 subunit of PI 3-kinase (deltaP85), and insulin could markedly promote EGF-induced AP-1 activity in a dose-dependent manner in JB6 P+ cells as well as promote EGF-induced JB6 P+ cell transformation. (ii) Inhibition of PI-3 kinase with wortmannin or LY294002 markedly decreased the AP-1 activity induced by insulin, EGF, or EGF and insulin in a dose-dependent manner, while wortmannin did not block UVB-induced AP-1 activity. (iii) AP-1 activation by insulin, EGF, or EGF and insulin could be completely inhibited by overexpression of deltaP85 in all the dose and time courses studied. (iv) Inhibitors of PI 3-kinase (wortmannin and LY294002) and stable overexpression of deltaP85 inhibited EGF-induced transformation but had no significant inhibitory effect on cell proliferation induced by EGF or EGF and insulin. These results demonstrate for the first time that PI 3-kinase appears to be required for EGF- or insulin-induced AP-1 transactivation and cell transformation but not cell proliferation in JB6 cells.
Mol Cell Biol 1996 Nov
PMID:Requirement for phosphatidylinositol 3-kinase in epidermal growth factor-induced AP-1 transactivation and transformation in JB6 P+ cells. 888 71

Phosphatidylinositol 3-kinase (PI3K) activation is necessary for many insulin-induced metabolic and mitogenic responses. However, it is unclear whether PI3K activation is sufficient for any of these effects. To address this question we increased PI3K activity in differentiated 3T3-L1 adipocytes by adenovirus-mediated expression of both the inter-SH2 region of the regulatory p85 subunit of PI3K (iSH2) and the catalytic p110 alpha subunit (p110). Coexpression resulted in PI3K activity that exceeded insulin-stimulated activity by two- to fivefold in cytosol, total membranes, and the low density microsome (LDM) fraction, the site of greatest insulin stimulation. While insulin increased glucose transport 15-fold, coexpression of iSH2-p110 increased transport (5.2-) +/- 0.7-fold with a parallel increase in GLUT4 translocation to the plasma membrane. Constitutive activation of PI3K had no effect on maximally insulin-stimulated glucose transport. Neither basal nor insulin-stimulated activity of glycogen synthase or mitogen-activated protein kinase was altered by iSH2-p110 coexpression. DNA synthesis was increased twofold by insulin in control 3T3-L1 adipocytes transduced with beta-galactosidase-encoding recombinant adenovirus, while iSH2-p110 coexpression increased DNA synthesis fivefold. These data indicate that (i) increased PI3K activity is sufficient to activate some but not all metabolic responses to insulin, (ii) activation of PI3K to levels exceeding the effect of insulin in adipocyte LDM results in only a partial stimulation of glucose transport, and (iii) increased PI3K activity in the absence of growth factor or oncoprotein stimulation is a potent stimulus of DNA synthesis.
Mol Cell Biol 1997 Jan
PMID:Differential effects of constitutively active phosphatidylinositol 3-kinase on glucose transport, glycogen synthase activity, and DNA synthesis in 3T3-L1 adipocytes. 897 99

Phosphatidylinositol (PI) 3-kinase is a cytoplasmic signaling molecule that is recruited to activated growth factor receptors after growth factor stimulation of cells. Activation of PI 3-kinase results in increased intracellular levels of 3' phosphorylated inositol phospholipids and the induction of signaling responses, including the activation of the protein kinase Akt, which is also known as RAC-PK or PKB. We tested the possibility that the phospholipid products of PI 3-kinase directly mediate the activation of Akt. We have previously described a constitutively active PI 3-kinase, p110, which can stimulate Akt activity. We used purified p110 protein to generate a series of 3' phosphorylated inositol phospholipids and tested whether any of these lipids could activate Akt in vitro. Phospholipid vesicles containing PI3,4 bisphosphate (P2) specifically activated Akt in vitro. By contrast, the presence of phospholipid vesicles containing PI3P or PI3,4,5P3 failed to increase the kinase activity of Akt. Akt could also be activated by synthetic dipalmitoylated PI3,4P2 or after enzymatic conversion of PI3,4,5P3 into PI3,4P2 with the signaling inositol polyphosphate 5' phosphatase SIP. We show that PI3,4P2-mediated activation is dependent on a functional pleckstrin homology domain in Akt, since a point mutation in the pleckstrin homology domain abrogated the response to PI3,4P2. Our findings show that a phospholipid product of PI 3-kinase can directly stimulate an enzyme known to be an important mediator of PI 3-kinase signaling.
Mol Cell Biol 1997 Jan
PMID:A specific product of phosphatidylinositol 3-kinase directly activates the protein kinase Akt through its pleckstrin homology domain. 897 14

SIP (signaling inositol phosphatase) or SHIP (SH2-containing inositol phosphatase) is a recently identified SH2 domain-containing protein which has been implicated as an important signaling molecule. SIP/SHIP becomes tyrosine phosphorylated and binds the phosphotyrosine-binding domain of SHC in response to activation of hematopoietic cells. The signaling pathways and biological responses that may be regulated by SIP have not been demonstrated. SIP is a phosphatidylinositol- and inositol-polyphosphate 5-phosphatase with specificity in vitro for substrates phosphorylated at the 3' position. Phosphatidylinositol 3'-kinase (PI 3-kinase) is an enzyme which is involved in mitogenic signaling and whose phosphorylated lipid products are predicted to be substrates for SIP. We tested the hypothesis that SIP can modulate signaling by PI 3-kinase in vivo by injecting SIP cRNAs into Xenopus oocytes. SIP inhibited germinal vesicle breakdown (GVBD) induced by expression of a constitutively activated form of PI 3-kinase (p110*) and blocked GVBD induced by insulin. SIP had no effect on progesterone-induced GVBD. Catalytically inactive SIP had little effect on insulin- or PI 3-kinase-induced GVBD. Expression of SIP, but not catalytically inactive SIP, also blocked insulin-induced mitogen-activated protein kinase phosphorylation in oocytes. SIP specifically and markedly reduced the level of phosphatidylinositol (3,4,5) triphosphate [PtdIns(3,4,5)P3] generated in oocytes in response to insulin. These results demonstrate that a member of the phosphatidylinositol polyphosphate 5-phosphatase family can inhibit signaling in vivo. Further, our data suggest that the generation of PtdIns(3,4,5)P3 by PI 3-kinase is necessary for insulin-induced GVBD in Xenopus oocytes.
Mol Cell Biol 1997 May
PMID:SIP/SHIP inhibits Xenopus oocyte maturation induced by insulin and phosphatidylinositol 3-kinase. 911 25

To determine the effects of the aging process on the regulation of phosphoinositide signal transduction pathway, inositol 1,4,5-trisphosphate and inositol 1,4,5-trisphosphate receptor-associated parameters were examined in the striatum of brains removed from young (3 months), adult (12 months) and senescent (25 months) male Fischer 344 rats. Inositol 1,4,5-trisphosphate content was significantly increased (P < or = 0.01) at 25 months of age compared to 3 and 12 months. No age-related differences in phosphatidylinositol 4,5-bisphosphate hydrolysis were found in striatal slices after stimulation with trans-(1S,3R)-1-aminocyclopentane-1,3-dicarboxylate, a metabotropic glutamatergic receptor agonist. Phosphatidylinositol 4,5-bisphosphate hydrolysis following stimulation with (R,S)-alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionic acid, a glutamatergic/quisqualate agonist, showed a significantly increased accumulation of net [3H]inositol 1,4,5-trisphosphate in senescent striatum whereas the muscarinic cholinergic agonist carbachol induced highest response in the young striatum. In each case, agonist-stimulated response was significantly reduced in the presence of the receptor-associated antagonist. The density of inositol 1,4,5-trisphosphate receptor in the particulate membranes derived from 12- and 25-month-old rats was decreased (P < 0.01) compared to that from young rats. Binding affinity of inositol 1,4,5-trisphosphate receptor for [3H]inositol 1,4,5-trisphosphate was increased (P = 0.05) only at 25 months of age when compared with 3 months of age. Incubation of partially purified inositol 1,4,5-trisphosphate receptor with striatal cytosol in the presence of Ca2+ showed an age-dependent susceptibility to proteolytic degradation of this receptor that was completely inhibited by calpain I inhibitor peptide. Paradoxically, the quantity of inositol 1,4,5-trisphosphate receptor mRNA-encoding transcripts was increased (P < or = 0.01) at 25 months of age, suggesting an age-dependent change in either transcriptional rate, stability or processing of inositol 1,4,5-trisphosphate receptor mRNAs in the striatum. The activity of inositol 1,4,5-trisphosphate3-kinase decreased (P < or = 0.01) with age whereas the activity of soluble inositol 1,4,5-trisphosphate 5-phosphatase was highest at 3 months but significantly decreased at 12 months of age. However, the activity of inositol 1,4,5-trisphosphate 5-phosphatase remained unchanged between 12 and 25 months of age, suggesting possible developmental modulation of the activity of the enzyme. Taken together with the established 'cross-talk' between signal transduction systems, the present data suggest that molecular/cellular changes in striatal inositol 1,4,5-trisphosphate/Ca2+ signal transduction pathway along with neuronal cell loss may contribute to aging-related decrease in striatal functioning.
Brain Res Mol Brain Res 1997 Jun
PMID:Aging-related regulation of myo-inositol 1,4,5-trisphosphate signal transduction pathway in the rat striatum. 919 Oct 77

Phosphatidylinositol 4,5-bisphosphate (PIP2) reorganizes actin filaments by modulating the functions of a variety of actin-regulatory proteins. Until now, it was thought that bound PIP2 is hydrolyzed only by tyrosine-phosphorylated phospholipase Cgamma (PLCgamma) after the activation of tyrosine kinases. Here, we show a new mechanism for the hydrolysis of bound PIP2 and the regulation of actin filaments by PIP2 phosphatase (synaptojanin). We isolated a 150-kDa protein (p150) from brains that binds the SH3 domains of Ash/Grb2. The sequence of this protein was found to be homologous to that of synaptojanin. The expression of p150 in COS 7 cells produces a decrease in the number of actin stress fibers in the center of the cells and causes the cells to become multinuclear. On the other hand, the expression of a PIP2 phosphatase-negative mutant does not disrupt actin stress fibers or produce the multinuclear phenotype. We have also shown that p150 forms the complexes with Ash/Grb2 and epidermal growth factor (EGF) receptors only when the cells are treated with EGF and that it reorganizes actin filaments in an EGF-dependent manner. Moreover, the PIP2 phosphatase activity of native p150 purified from bovine brains is not inhibited by profilin, cofilin, or alpha-actinin, although PLCdelta1 activity is markedly inhibited by these proteins. Furthermore, p150 suppresses actin gelation, which is induced by smooth muscle alpha-actinin. All these data suggest that p150 (synaptojanin) hydrolyzes PIP2 bound to actin regulatory proteins, resulting in the rearrangement of actin filaments downstream of tyrosine kinase and Ash/Grb2.
Mol Cell Biol 1997 Jul
PMID:Phosphatidylinositol 4,5-bisphosphate phosphatase regulates the rearrangement of actin filaments. 919 18

The binding of the spermatozoon to the oocyte zona pellucida (ZP) occurs via specific receptors localized over the anterior head region of the spermatozoon. Zona pellucida binding stimulates the spermatozoa to undergo the acrosome reaction resulting in the release of hydrolytic enzymes and in the exposure of new membrane domains, both of which are essential for fertilization. We suggest that ZP binds to at least two different receptors in the plasma membrane. One (R) is a Gi-coupled receptor that activates phospholipase C (PLC) beta 1. The other (TK) is a tyrosine kinase receptor coupled to PLC gamma. Binding to R would regulate adenylyl cyclase (AC) leading to elevation of cAMP and protein kinase (PKA) activation. The PKA activates a voltage-dependent Ca2+ channel in the outer acrosomal membrane which releases Ca2+ from the interior of the acrosome to the cytosol. This is the first, relatively small, rise in [Ca2+]i (I) which leads to activation of the PLC gamma. The products of phosphatidyl-inositol bisphosphate (PIP2) hydrolysis by PLC diacylglycerol (DAG) and inositol-trisphosphate (IP3) will lead to PKC translocation to the plasma membrane and its activation. PKC opens a voltage-dependent Ca2+ channel (L) in the plasma membrane, leading to the second (II) higher increase in [Ca2+]i. The Gi or TK can also activate an Na+/H+ exchanger leading to alkalization of the cytosol. PKC also activates phospholipase A2 (PLA2) to generate arachidonic acid (AA) from membrane phospholipids. AA will be converted to prostaglandins (PG) and leukotriens (LT) by the enzymes cyclooxygenase (COX) and lipoxygenase (LOX) respectively. The increase in [Ca2+]i and pH leads to membrane fusion and acrosomal exocytosis.
Mol Hum Reprod 1997 Mar
PMID:The biochemistry of the acrosome reaction. 923 45

Phosphatidylinositol 3 (PI 3)-kinases are potently inhibited by two structurally unrelated membrane-permeant reagents: wortmannin and LY294002. By using these two inhibitors we first suggested the involvement of a PI 3-kinase activity in muscle cell differentiation. However, several reports have described that these compounds are not as selective for PI 3-kinase activity as assumed. Here we show that LY294002 blocks the myogenic pathway elicited by insulin-like growth factors (IGFs), and we confirm the specific involvement of PI 3-kinase in IGF-induced myogenesis by overexpressing in L6E9 myoblasts a dominant negative p85 PI 3-kinase-regulatory subunit (L6E9-delta p85). IGF-I, des(1-3)IGF-I, or IGF-II induced L6E9 skeletal muscle cell differentiation as measured by myotube formation, myogenin gene expression, and GLUT4 glucose carrier induction. The addition of LY294002 to the differentiation medium totally inhibited these IGF-induced myogenic events without altering the expression of a non-muscle-specific protein, beta1-integrin. Independent clones of L6E9 myoblasts expressing a dominant negative mutant of the p85-regulatory subunit (delta p85) showed markedly impaired glucose transport activity and formation of p85/p110 complexes in response to insulin, consistent with the inhibition of PI 3-kinase activity. IGF-induced myogenic parameters in L6E9-delta p85 cells, ie. cell fusion and myogenin gene and GLUT4 expression, were severely impaired compared with parental cells or L6E9 cells expressing wild-type p85. In all, data presented here indicate that PI 3-kinase is essential for IGF-induced muscle differentiation and that the specific PI 3-kinase subclass involved in myogenesis is the heterodimeric p85-p110 enzyme.
Mol Endocrinol 1998 Jan
PMID:Insulin-like growth factors require phosphatidylinositol 3-kinase to signal myogenesis: dominant negative p85 expression blocks differentiation of L6E9 muscle cells. 944 Aug 11


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