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Inhibition of Phosphatidylinositol 3-Kinase Activity Blocks Depolarization- and Insulin-like Growth Factor I-mediated Survival of Cerebellar Granule Cells*

  • Timothy M. Miller
    Affiliations
    From the Departments of Molecular Biology and Pharmacology and of Neurology, Washington University School of Medicine, St. Louis, Missouri 63110
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  • Malú G. Tansey
    Affiliations
    From the Departments of Molecular Biology and Pharmacology and of Neurology, Washington University School of Medicine, St. Louis, Missouri 63110
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  • Eugene M. Johnson Jr.
    Affiliations
    From the Departments of Molecular Biology and Pharmacology and of Neurology, Washington University School of Medicine, St. Louis, Missouri 63110
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  • Douglas J. Creedon
    Correspondence
    To whom correspondence should be addressed:
    Affiliations
    From the Departments of Molecular Biology and Pharmacology and of Neurology, Washington University School of Medicine, St. Louis, Missouri 63110
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  • Author Footnotes
    * This work was supported by the Ataxia-Telangiectasia Children's Project, the Ronald McDonald Children's Charities, and National Institutes of Health Grant AG 12947. The calcium measurement system was supported by National Institutes of Health Grant NS 19988. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:April 11, 1997DOI:https://doi.org/10.1074/jbc.272.15.9847
      Depolarizing concentrations of potassium promote the survival of many neuronal cell types including cerebellar granule cells. To begin to understand the intracellular mediators of neuronal survival, we have tested whether the survival-promoting effect of potassium depolarization on cerebellar granule cells is dependent on either mitogen-activated protein (MAP) kinase or phosphatidylinositol 3-kinase (PI-3-K) activity. In 7-day cerebellar granule cell cultures, potassium depolarization activated both MAP kinase and PI-3-K. Preventing the activation of MAP kinase with the MEK1 inhibitor PD98059 did not affect potassium saving. In contrast, the survival-promoting effect of 25 mM potassium was negated by the addition of 30 μM LY 294002 or 1 μM wortmannin, two distinct inhibitors of PI-3-K. The cell death induced by PI-3-K inhibition was indistinguishable from the cell death caused by potassium deprivation; LY 294002-induced death included nuclear condensation, was blocked by cycloheximide, and had the same time course as potassium deprivation-induced cell death. Cerebellar granule cells can also be maintained in serum-free medium containing either 100 ng/ml insulin-like growth factor I (IGF-I) or 800 μM cAMP. PI-3-K inhibition completely blocked the survival-promoting activity of IGF-I, but had no effect on cAMP-mediated survival. These data indicate that the survival-promoting effects of depolarization and IGF-I, but not cAMP, require PI-3-K activity.

      INTRODUCTION

      Cell death is a widespread event during development that, in the nervous system, is thought to match the size of neuronal populations with their targets (
      • Oppenheim R.W.
      ). One mechanism for determining which neurons survive and which undergo programmed cell death is limiting the amount of neurotrophic factors (
      • Oppenheim R.W.
      ). Another important mechanism for determining survival is electrical activity; removal of afferent input or pharmacological blockade of electrical activity causes the death of some types of developing neurons (
      • Lipton S.A.
      ,
      • Maderdrut J.L.
      • Oppenheim R.W.
      • Prevette D.
      ,
      • Ruijter J.M.
      • Baker R.E.
      • De J.B.
      • Romijn H.J.
      ,
      • Catsicas M.
      • Pequignot Y.
      • Clarke P.G.
      ,
      • Galli R.L.
      • Ensini M.
      • Fusco E.
      • Gravina A.
      • Margheritti B.
      ). In vitro, mimicking electrical activity with depolarizing concentrations of extracellular potassium promotes the survival of many types of neurons (for review, see Ref.
      • Franklin J.L.
      • Johnson Jr., E.M.
      ). Depolarization leads to the sustained activation of voltage-gated calcium channels and subsequent elevation of intracellular calcium levels. The increase in intracellular calcium is important for survival because blockade of L-type calcium channels blocks K+-mediated survival (
      • Collins F.
      • Lile J.D.
      ,
      • Collins F.
      • Schmidt M.F.
      • Guthrie P.B.
      • Kater S.B.
      ,
      • Franklin J.L.
      • Sanz-Rodriguez C.
      • Juhasz A.
      • Deckwerth T.L.
      • Johnson Jr., E.M.
      ,
      • Galli C.
      • Meucci O.
      • Scorziello A.
      • Werge T.M.
      • Calissano P.
      • Schettini G.
      ,
      • Koike T.
      • Tanaka S.
      ,
      • Koike T.
      • Martin D.P.
      • Johnson Jr., E.M.
      ,
      • Nishi R.
      • Berg D.K.
      ), yet the intracellular pathways that mediate this survival are unknown.
      Dissociated cerebellar granule cells from early postnatal rats can be maintained in serum-containing medium by raising the extracellular potassium concentration to 25 mM (
      • Gallo V.
      • Kingsbury A.
      • Balazs R.
      • Jorgensen O.S.
      ). Dissociated granule cells develop characteristics of mature cerebellar granule cells in vivo including an extensive neuritic network, expression of excitatory amino acid receptors, and production and release of L-glutamate (
      • Burgoyne R.D.
      • Graham M.E.
      • Cambray D.M.
      ). Removal of both potassium and serum from the culture medium triggers a cell death that is morphologically apoptotic, accompanied by DNA fragmentation, and dependent on macromolecular synthesis (
      • D'Mello S.R.
      • Galli C.
      • Ciotti T.
      • Calissano P.
      ). This programmed cell death (PCD)
      The abbreviations used are: PCD
      programmed cell death
      MAP
      mitogen-activated protein
      PI-3-K
      phosphatidylinositol 3-kinase
      NGF
      nerve growth factor
      IGF-I
      insulin-like growth factor I
      CPT-cAMP
      chlorophenylthio-cAMP.
      presumably mimics the naturally occurring death of 20-30% of granule cells (
      • Caddy K.W.
      • Biscoe T.J.
      ), thought to be important for matching the number of granule cells with Purkinje cells, that occurs during the third through fifth postnatal weeks (
      • Williams R.W.
      • Herrup K.
      ,
      • Wetts R.
      • Herrup K.
      ).
      One possible mechanism by which depolarization could promote survival is by activating survival-promoting signaling pathways similar to those activated by neurotrophic factors. Tyrosine kinase growth factor receptors activate several intracellular signaling pathways. Of these, the MAP kinase and phosphatidylinositol 3-kinase (PI-3-K) pathways have been implicated in survival. In PC12 cells, overexpression of a constitutively active form of MEK1 (AP/xtracellular signal-regulated kinase inase), an activator of MAP kinase, promotes survival in the absence of NGF (
      • Xia Z.
      • Dickens M.
      • Raingeaud J.
      • Davis R.J.
      • Greenberg M.E.
      ). However, MAP kinase is not required for the survival of NGF-maintained sympathetic neurons (
      • Virdee K.
      • Talkovsky A.
      ,
      • Creedon D.J.
      • Johnson Jr., E.M.
      • Lawrence Jr., J.C.
      ) or PC12 cells (
      • Yao R.
      • Cooper G.M.
      ). In contrast, inhibition of PI-3-K blocks the survival-promoting effects of NGF in PC12 cells (
      • Yao R.
      • Cooper G.M.
      ). PI-3-K is a lipid kinase that mediates the mitogenic signal of the platelet-derived growth factor receptor (
      • Cantley L.C.
      • Auger K.R.
      • Carpenter C.
      • Duckworth B.
      • Graziani A.
      • Kapeller R.
      • Soltoff S.
      ). PI-3-K products are involved in regulating mitogenesis, membrane ruffling, glucose uptake, receptor sorting, and receptor down-regulation in response to growth factors (for review, see Ref.
      • Kapeller R.
      • Cantley L.C.
      ). In this report, we present evidence that PI-3-K activity is required for survival promotion by K+ depolarization and insulin-like growth factor I (IGF-I).

      DISCUSSION

      In this study, we provide evidence for a link between K+ depolarization and a signaling pathway that promotes the survival of neurons. Depolarizing concentrations of extracellular potassium increased PI-3-K activity; that this activity is critical for survival is indicated by the ability of inhibitors of PI-3-K to block depolarization-mediated survival. The cell death resulting from inhibition of PI-3-K was indistinguishable from the programmed cell death triggered by lowering the K+ concentration. In both cases, cell death was dependent on macromolecular synthesis and accompanied by morphologically apoptotic nuclei. In addition, the time course of cell loss was identical, and the cell death was prevented by cAMP. These data strongly suggest that PI-3-K inhibition resulted in programmed cell death by specifically blocking the survival-promoting activity of K+ depolarization.
      The survival-promoting activity of IGF-I was also blocked by inhibition of PI-3-K. K+ depolarization and growth factors may therefore converge on PI-3-K to signal survival promotion. PI-3-K activity increases in response to several growth factors including platelet-derived growth factor, insulin, colony-stimulating factor 1, nerve growth factor, hepatocyte growth factor, stem cell growth factor, and epidermal growth factor (
      • Kapeller R.
      • Cantley L.C.
      ). As previously reported in cell lines (
      • Backer J.M.
      • Myers M.J.
      • Shoelson S.E.
      • Chin D.J.
      • Sun X.J.
      • Miralpeix M.
      • Hu P.
      • Margolis B.
      • Skolnik E.Y.
      • Schlessinger J.
      ,
      • Giorgetti S.
      • Ballotti R.
      • Kowalski-Chauvel A.
      • Tartare S.
      • Van Obberghen E.
      ), we found that IGF-I increased PI-3-K activity in cerebellar granule neurons. Although both IGF-I and 25 mM potassium increased PI-3-K activity and inhibiting PI-3-K blocked their ability to promote survival, we did not directly test whether the rise in PI-3-K activity per se was critical for survival. Basal activity could augment other actions of these agents that result in survival. In the absence of 25 mM potassium or IGF-I, however, the basal PI-3-K activity was not sufficient to maintain the neurons.
      For growth factors, activation of PI-3-K is dependent on the protein-tyrosine kinase activity of the growth factor receptor. Autophosphorylation of specific tyrosine residues on the receptor recruits the Src homology 2 domain (SH2) of the p85 regulatory subunit of PI-3-K. This leads to relocalization of PI-3-K to the membrane and activation of the catalytic p110 subunit. In contrast to other known growth factors, phosphorylation of the insulin or IGF-I receptor leads to phosphorylation of insulin receptor substrate 1, which serves as an SH2-docking protein for p85 (
      • Backer J.M.
      • Myers M.J.
      • Shoelson S.E.
      • Chin D.J.
      • Sun X.J.
      • Miralpeix M.
      • Hu P.
      • Margolis B.
      • Skolnik E.Y.
      • Schlessinger J.
      ,
      • Giorgetti S.
      • Ballotti R.
      • Kowalski-Chauvel A.
      • Tartare S.
      • Van Obberghen E.
      ,
      • Backer J.M.
      • Myers Jr., M.-G.
      • Sun X.-J.
      • Chin D.J.
      • Shoelson S.E.
      • Miralpeix M.
      • White M.F.
      ,
      • Sun X.J.
      • Rothenberg P.
      • Kahn C.R.
      • Backer J.M.
      • Araki E.
      • Wilden P.A.
      • Cahill D.A.
      • Goldstein B.J.
      • White M.F.
      ).
      One mechanism by which potassium depolarization may increase PI-3-K activity is by causing phosphorylation events similar to those caused by growth factor receptors. Some evidence supports this hypothesis. For example, PYK2, a novel protein-tyrosine kinase, is rapidly phosphorylated in response to stimuli that increase intracellular calcium (
      • Lev S.
      • Moreno H.
      • Martinez R.
      • Canoll P.
      • Peles E.
      • Musacchio J.M.
      • Plowman G.D.
      • Rudy B.
      • Schlessinger J.
      ). The p85 subunit of PI-3-K may associate with this calcium-responsive protein-tyrosine kinase. Alternatively, PYK2 may activate PI-3-K by first activating Ras, which can be an upstream activator of PI-3-K (
      • Kodaki T.
      • Woscholski R.
      • Hallberg B.
      • Rodriguez V.P.
      • Downward J.
      • Parker P.J.
      ). Potassium depolarization of PC12 cells results in phosphorylation of the adapter protein Shc and leads to the association of Shc with Grb2, causing Ras and MAP kinase activation (
      • Rusanescu G.
      • Qi H.
      • Thomas S.M.
      • Brugge J.S.
      • Halegoua S.
      ,
      • Rosen L.B.
      • Ginty D.D.
      • Weber M.J.
      • Greenberg M.E.
      ,
      • Rosen L.B.
      • Greenberg M.E.
      ) and phosphorylation of the epidermal growth factor receptor (
      • Rosen L.B.
      • Greenberg M.E.
      ). At this point, which of these phosphorylation events occurs in depolarized granule cells is unclear.
      In cultures of cortical neurons, depolarization promotes survival by stimulating the release of the neurotrophin brain-derived neurotrophic factor that serves as an autocrine or paracrine trophic agent (
      • Ghosh A.
      • Carnahan J.
      • Greenberg M.E.
      ). This contrasts with sympathetic neurons, however, in which potassium depolarization fails to induce the phosphorylation of growth factor receptors (
      • Franklin J.L.
      • Sanz-Rodriguez C.
      • Juhasz A.
      • Deckwerth T.L.
      • Johnson Jr., E.M.
      ). In granule cell cultures, antibodies to IGF-I, neurotrophin-3, NGF, or brain-derived neurotrophic factor do not affect granule cell survival in depolarizing medium,2 suggesting that granule cells behave similarly to sympathetic neurons in this regard. Another mechanism whereby calcium may influence survival is by increasing calcium/calmodulin kinase activity. Inhibitors of calmodulin- or Ca2+/calmodulin-dependent protein kinase II activity block K+-mediated survival (
      • Gallo V.
      • Kingsbury A.
      • Balazs R.
      • Jorgensen O.S.
      ,
      • Hack N.
      • Hidaka H.
      • Wakefield M.J.
      • Balazs R.
      ). However, these compounds may affect survival by blocking the sustained rise in intracellular calcium (
      • Franklin J.L.
      • Sanz-Rodriguez C.
      • Juhasz A.
      • Deckwerth T.L.
      • Johnson Jr., E.M.
      ).
      We found that LY 294002 did not interfere with increases in intracellular calcium after 1 h (Fig. 6), a time when inhibitors of calcium channels, such as nifedipine, will block calcium channels completely (
      • Franklin J.L.
      • Sanz-Rodriguez C.
      • Juhasz A.
      • Deckwerth T.L.
      • Johnson Jr., E.M.
      ). It is possible that LY 294002 affected calcium at later time points, but measurement of calcium at later time points would be difficult to interpret because the cells begin to degenerate within 6 h, and changes in metabolic parameters such as glucose uptake and protein synthesis occur within 2 h of the induction of apoptosis (
      • Miller T.M.
      • Johnson Jr., E.M.
      ). In addition to our direct measurement of calcium at 1 h, two other pieces of evidence argue against LY 294002 affecting calcium. First, since the time course of death triggered by K5−S medium or by K25−S medium plus LY 294002 overlaps (Fig. 5), an LY 294002 effect on calcium would have to occur rapidly. If LY 294002 caused a delayed decrease in intracellular calcium that initiated PCD, we would expect to see a corresponding delay in the cell death time course, but this was not the case (Fig. 5). Second, LY 294002 blocked the survival-promoting effect of IGF-I, which does not elevate intracellular calcium (
      • Galli C.
      • Meucci O.
      • Scorziello A.
      • Werge T.M.
      • Calissano P.
      • Schettini G.
      ).
      An indication that Ras is activated by depolarization is that depolarization activates the MAP kinase pathway (Fig. 6) (
      • Rusanescu G.
      • Qi H.
      • Thomas S.M.
      • Brugge J.S.
      • Halegoua S.
      ,
      • Rosen L.B.
      • Ginty D.D.
      • Weber M.J.
      • Greenberg M.E.
      ,
      • Rosen L.B.
      • Greenberg M.E.
      ,
      • Finkbeiner S.
      • Greenberg M.E.
      ). In PC12 cells, overexpression of a constitutively active form of MEK1, an activator of MAP kinase, promotes survival in the absence of NGF (
      • Xia Z.
      • Dickens M.
      • Raingeaud J.
      • Davis R.J.
      • Greenberg M.E.
      ). However, MAP kinase is not required for the survival of NGF-maintained sympathetic neurons (
      • Virdee K.
      • Talkovsky A.
      ,
      • Creedon D.J.
      • Johnson Jr., E.M.
      • Lawrence Jr., J.C.
      ) or NGF-maintained PC12 cells (
      • Yao R.
      • Cooper G.M.
      ). Consistent with this, our data demonstrate that MAP kinase was not required for survival because the MEK inhibitor, which blocks activation of MAP kinase, did not affect survival. The fact that PI-3-K inhibitors do not prevent the activation of MAP kinase2 yet lead to PCD (Fig. 3) indicates that MAP kinase activity was also not sufficient to promote granule neuron survival.
      Several possible downstream targets of PI-3-K may be involved in neuronal survival. Protein kinase C isozymes (
      • Zhang J.
      • Falck J.R.
      • Reddy K.K.
      • Abrams C.S.
      • Zhao W.
      • Rittenhouse S.E.
      ,
      • Toker A.
      • Meyer M.
      • Reddy K.K.
      • Falck J.R.
      • Aneja R.
      • Aneja S.
      • Parra A.
      • Burns D.J.
      • Ballas L.M.
      • Cantley L.C.
      ,
      • Toker A.
      • Bachelot C.
      • Chen C.-S.
      • Falck J.R.
      • Hartwig J.H.
      • Cantley L.C.
      • Kovacsovics T.J.
      ,
      • Palmer R.H.
      • Dekker L.V.
      • Woscholski R.
      • Good J.A.L.
      • Gigg R.
      • Parker P.J.
      ), Rac (
      • Parker P.J.
      ,
      • Wennstrom S.
      • Hawkins P.
      • Cooke F.
      • Hara K.
      • Yonezawa K.
      • Kasuga M.
      • Jackson T.
      • Claesson W.L.
      • Stephens L.
      ), p70S6K (
      • Petritsch C.
      • Woscholski R.
      • Edelmann H.M.
      • Parker P.J.
      • Ballou L.M.
      ,
      • Chung J.
      • Grammer T.
      • Lemon K.
      • Kazlauskas A.
      • Blenis J.
      ,
      • Cheatham B.
      • Vlahos C.
      • Cheatham L.
      • Wang L.
      • Blenis J.
      ), and AKT/protein kinase B serine/threonine kinase (
      • Bos J.L.
      ,
      • Franke T.F.
      • Yang S.I.
      • Chan T.O.
      • Datta K.
      • Kazlauskas A.
      • Morrison D.K.
      • Kaplan D.R.
      • Tsichlis P.N.
      ) are all effectors of PI-3-K. PI-3-K also affects glucose transporters (
      • Conricode K.M.
      ,
      • Kaliman P.
      • Vinals F.
      • Testar X.
      • Palacin M.
      • Zorzano A.
      ,
      • Martin S.S.
      • Haruta T.
      • Morris A.J.
      • Klippel A.
      • Williams L.T.
      • Olefsky J.M.
      ) and, through this pathway, may be critical for maintaining the metabolic functions of cells. We have found that glucose uptake, protein synthesis, and RNA synthesis decrease dramatically as granule cells (
      • Miller T.M.
      • Johnson Jr., E.M.
      ) and sympathetic neurons (
      • Deckwerth T.L.
      • Johnson Jr., E.M.
      ) undergo PCD and have suggested that this dramatic decrease in metabolic parameters is part of PCD in neurons. Inhibiting PI-3-K may specifically decrease metabolic functions maintained by either K+ depolarization or growth factors and thus trigger PCD.
      Although peripheral neurons are particularly dependent on growth factors for survival, neurons from the central nervous system may depend on a combination of trophic factors and electrical activity (
      • Meyer F.A.
      • Kaplan M.R.
      • Pfrieger F.W.
      • Barres B.A.
      ). Our results highlight PI-3-K as an important intracellular mediator of survival promotion by either activity or trophic factors. PI-3-K is one of the pathways upon which neurons critically depend for survival in vitro and, perhaps, in vivo

      Acknowledgments

      We thank Monsanto Co. for the generous gift of IGF-I; S. Handran and Dr. S. Rothman for help with calcium measurements and for the use of the calcium measurement system; Dr. L. Pike for preparing the radiolabeled phosphatidylinositol 4-phosphate; M. Deshmukh, J. L. Franklin, and P. A. Osborne for critical evaluation of this manuscript; and N. Girgis for help with cell counts.

      REFERENCES

        • Oppenheim R.W.
        Annu. Rev. Neurosci. 1991; 14: 453-501
        • Lipton S.A.
        Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 9774-9778
        • Maderdrut J.L.
        • Oppenheim R.W.
        • Prevette D.
        Brain Res. 1988; 444: 189-194
        • Ruijter J.M.
        • Baker R.E.
        • De J.B.
        • Romijn H.J.
        Int. J. Dev. Neurosci. 1991; 9: 331-338
        • Catsicas M.
        • Pequignot Y.
        • Clarke P.G.
        J. Neurosci. 1992; 12: 4642-4650
        • Galli R.L.
        • Ensini M.
        • Fusco E.
        • Gravina A.
        • Margheritti B.
        J. Neurosci. 1993; 13: 243-250
        • Franklin J.L.
        • Johnson Jr., E.M.
        Trends Neurosci. 1992; 15: 501-508
        • Collins F.
        • Lile J.D.
        Brain Res. 1989; 502: 99-108
        • Collins F.
        • Schmidt M.F.
        • Guthrie P.B.
        • Kater S.B.
        J. Neurosci. 1991; 11: 2582-2587
        • Franklin J.L.
        • Sanz-Rodriguez C.
        • Juhasz A.
        • Deckwerth T.L.
        • Johnson Jr., E.M.
        J. Neurosci. 1995; 15: 643-664
        • Galli C.
        • Meucci O.
        • Scorziello A.
        • Werge T.M.
        • Calissano P.
        • Schettini G.
        J. Neurosci. 1995; 15: 1172-1179
        • Koike T.
        • Tanaka S.
        Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3892-3896
        • Koike T.
        • Martin D.P.
        • Johnson Jr., E.M.
        Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6421-6425
        • Nishi R.
        • Berg D.K.
        Dev. Biol. 1981; 87: 301-307
        • Gallo V.
        • Kingsbury A.
        • Balazs R.
        • Jorgensen O.S.
        J. Neurosci. 1987; 7: 2203-2213
        • Burgoyne R.D.
        • Graham M.E.
        • Cambray D.M.
        J. Neurocytol. 1993; 22: 689-695
        • D'Mello S.R.
        • Galli C.
        • Ciotti T.
        • Calissano P.
        Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10989-10993
        • Caddy K.W.
        • Biscoe T.J.
        Philos. Trans. R. Soc. Lond. B Biol. Sci. 1979; 287: 167-201
        • Williams R.W.
        • Herrup K.
        Annu. Rev. Neurosci. 1988; 11: 423-453
        • Wetts R.
        • Herrup K.
        Dev. Brain Res. 1983; 10: 41-47
        • Xia Z.
        • Dickens M.
        • Raingeaud J.
        • Davis R.J.
        • Greenberg M.E.
        Science. 1995; 270: 1326-1331
        • Virdee K.
        • Talkovsky A.
        J. Neurochem. 1996; 67: 1801-1805
        • Creedon D.J.
        • Johnson Jr., E.M.
        • Lawrence Jr., J.C.
        J. Biol. Chem. 1996; 271: 20713-20718
        • Yao R.
        • Cooper G.M.
        Science. 1995; 267: 2003-2006
        • Cantley L.C.
        • Auger K.R.
        • Carpenter C.
        • Duckworth B.
        • Graziani A.
        • Kapeller R.
        • Soltoff S.
        Cell. 1991; 64: 281-302
        • Kapeller R.
        • Cantley L.C.
        Bioessays. 1994; 16: 565-576
        • Miller T.M.
        • Johnson Jr., E.M.
        J. Neurosci. 1996; 16: 7487-7495
        • Thangnipon W.
        • Kingsbury A.
        • Webb M.
        • Balazs R.
        Dev. Brain Res. 1983; 11: 177-189
        • Kingsbury A.E.
        • Gallo V.
        • Woodhams P.L.
        • Balazs R.
        Dev. Brain Res. 1985; 17: 17-25
        • Bozyczko C.D.
        • McKenna B.W.
        • Connors T.J.
        • Neff N.T.
        J. Neurosci. Methods. 1993; 50: 205-216
        • Newberry E.P.
        • Pike L.J.
        Biochem. Biophys. Res. Commun. 1995; 208: 253-259
        • Auger K.R.
        • Serunian L.A.
        • Cantley L.C.
        Irvine R.F. Methods in Inositide Research. Raven Press, Ltd., New York1990: 159-166
        • Hope H.M.
        • Pike L.J.
        J. Biol. Chem. 1994; 269: 23648-23654
        • Grynkiewicz G.
        • Poenie M.
        • Tsien R.Y.
        J. Biol. Chem. 1985; 260: 3440-3450
        • Vlahos C.J.
        • Matter W.F.
        • Hui K.Y.
        • Brown F.B.
        J. Biol. Chem. 1994; 269: 5241-5248
        • Dudley D.T.
        • Pang L.
        • Decker S.J.
        • Bridges A.J.
        • Saltiel A.R.
        Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7686-7689
        • Alessi D.R.
        • Cuenda A.
        • Cohen P.
        • Dudley D.T.
        • Saltiel A.R.
        J. Biol. Chem. 1995; 270: 27489-27494
        • Kimura K.
        • Hattori S.
        • Kabuyama Y.
        • Shizawa Y.
        • Takayanagi J.
        • Nakamura S.
        • Toki S.
        • Matsuda Y.
        • Onodera K.
        • Fukui Y.
        J. Biol. Chem. 1994; 269: 18961-18967
        • Backer J.M.
        • Myers M.J.
        • Shoelson S.E.
        • Chin D.J.
        • Sun X.J.
        • Miralpeix M.
        • Hu P.
        • Margolis B.
        • Skolnik E.Y.
        • Schlessinger J.
        EMBO J. 1992; 11: 3469-3479
        • Giorgetti S.
        • Ballotti R.
        • Kowalski-Chauvel A.
        • Tartare S.
        • Van Obberghen E.
        J. Biol. Chem. 1993; 268: 7358-7364
        • Backer J.M.
        • Myers Jr., M.-G.
        • Sun X.-J.
        • Chin D.J.
        • Shoelson S.E.
        • Miralpeix M.
        • White M.F.
        J. Biol. Chem. 1993; 268: 8204-8212
        • Sun X.J.
        • Rothenberg P.
        • Kahn C.R.
        • Backer J.M.
        • Araki E.
        • Wilden P.A.
        • Cahill D.A.
        • Goldstein B.J.
        • White M.F.
        Nature. 1991; 352: 73-77
        • Lev S.
        • Moreno H.
        • Martinez R.
        • Canoll P.
        • Peles E.
        • Musacchio J.M.
        • Plowman G.D.
        • Rudy B.
        • Schlessinger J.
        Nature. 1995; 376: 737-745
        • Kodaki T.
        • Woscholski R.
        • Hallberg B.
        • Rodriguez V.P.
        • Downward J.
        • Parker P.J.
        Curr. Biol. 1994; 4: 798-806
        • Rusanescu G.
        • Qi H.
        • Thomas S.M.
        • Brugge J.S.
        • Halegoua S.
        Neuron. 1995; 15: 1415-1425
        • Rosen L.B.
        • Ginty D.D.
        • Weber M.J.
        • Greenberg M.E.
        Neuron. 1994; 12: 1207-1221
        • Rosen L.B.
        • Greenberg M.E.
        Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1113-1118
        • Ghosh A.
        • Carnahan J.
        • Greenberg M.E.
        Science. 1994; 263: 1618-1623
        • Hack N.
        • Hidaka H.
        • Wakefield M.J.
        • Balazs R.
        Neuroscience. 1993; 57: 9-20
        • Finkbeiner S.
        • Greenberg M.E.
        Neuron. 1996; 16: 233-236
        • Zhang J.
        • Falck J.R.
        • Reddy K.K.
        • Abrams C.S.
        • Zhao W.
        • Rittenhouse S.E.
        J. Biol. Chem. 1995; 270: 22807-22810
        • Toker A.
        • Meyer M.
        • Reddy K.K.
        • Falck J.R.
        • Aneja R.
        • Aneja S.
        • Parra A.
        • Burns D.J.
        • Ballas L.M.
        • Cantley L.C.
        J. Biol. Chem. 1994; 269: 32358-32367
        • Toker A.
        • Bachelot C.
        • Chen C.-S.
        • Falck J.R.
        • Hartwig J.H.
        • Cantley L.C.
        • Kovacsovics T.J.
        J. Biol. Chem. 1995; 270: 29525-29531
        • Palmer R.H.
        • Dekker L.V.
        • Woscholski R.
        • Good J.A.L.
        • Gigg R.
        • Parker P.J.
        J. Biol. Chem. 1995; 270: 22412-22416
        • Parker P.J.
        Curr. Biol. 1995; 5: 577-579
        • Wennstrom S.
        • Hawkins P.
        • Cooke F.
        • Hara K.
        • Yonezawa K.
        • Kasuga M.
        • Jackson T.
        • Claesson W.L.
        • Stephens L.
        Curr. Biol. 1994; 4: 385-393
        • Petritsch C.
        • Woscholski R.
        • Edelmann H.M.
        • Parker P.J.
        • Ballou L.M.
        Eur. J. Biochem. 1995; 230: 431-438
        • Chung J.
        • Grammer T.
        • Lemon K.
        • Kazlauskas A.
        • Blenis J.
        Nature. 1994; 370: 71-75
        • Cheatham B.
        • Vlahos C.
        • Cheatham L.
        • Wang L.
        • Blenis J.
        Mol. Cell. Biol. 1994; 14: 4902-4911
        • Bos J.L.
        Trends Biochem. Sci. 1995; 20: 441-442
        • Franke T.F.
        • Yang S.I.
        • Chan T.O.
        • Datta K.
        • Kazlauskas A.
        • Morrison D.K.
        • Kaplan D.R.
        • Tsichlis P.N.
        Cell. 1995; 81: 727-736
        • Conricode K.M.
        Biochem. Mol. Biol. Int. 1995; 36: 835-843
        • Kaliman P.
        • Vinals F.
        • Testar X.
        • Palacin M.
        • Zorzano A.
        Biochem. J. 1995; 312: 471-477
        • Martin S.S.
        • Haruta T.
        • Morris A.J.
        • Klippel A.
        • Williams L.T.
        • Olefsky J.M.
        J. Biol. Chem. 1996; 271: 17605-17608
        • Deckwerth T.L.
        • Johnson Jr., E.M.
        J. Cell Biol. 1993; 123: 1207-1222
        • Meyer F.A.
        • Kaplan M.R.
        • Pfrieger F.W.
        • Barres B.A.
        Neuron. 1995; 15: 805-819