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Requirement of Phosphatidylinositol 3-Kinase Activity for Translocation of Exogenous aFGF to the Cytosol and Nucleus*

  • Olav Klingenberg
    Footnotes
    Affiliations
    Department of Biochemistry at The Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, 0310 Oslo, Norway
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  • Antoni Wi IJ dłocha
    Affiliations
    Department of Biochemistry at The Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, 0310 Oslo, Norway
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  • Lucı́a Citores
    Footnotes
    Affiliations
    Department of Biochemistry at The Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, 0310 Oslo, Norway
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  • Sjur Olsnes
    Correspondence
    To whom correspondence should be addressed. Tel.: 47 22935640; Fax: 47 22508692
    Affiliations
    Department of Biochemistry at The Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, 0310 Oslo, Norway
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  • Author Footnotes
    * This work was supported by the Norwegian Cancer Society, Novo Nordisk Foundation, the Norwegian Research Council for Science and Humanities, Blix Legat, Rachel and Otto Kr. Bruun's Legat, and by The Jahre Foundation.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.
    ‡ Postdoctoral fellow of The Norwegian Cancer Society.
Open AccessPublished:April 21, 2000DOI:https://doi.org/10.1074/jbc.275.16.11972
      Acidic fibroblast growth factor (aFGF) is a potent mitogen for many cells. Exogenous aFGF is able to enter the cytosol and nucleus of sensitive cells. There are indications that both activation of the receptor tyrosine kinase and translocation of aFGF to the nucleus are of importance for mitogenesis. However, the mechanism of transport of aFGF from the cell surface to the nucleus is poorly understood. In this work we demonstrate that inhibition of phosphatidylinositol (PI) 3-kinase by chemical inhibitors and by expression of a dominant negative mutant of PI 3-kinase blocks translocation of aFGF to the cytosol and nucleus. Translocation to the cytosol and nucleus was monitored by cell fractionation, by farnesylation of aFGF modified to contain a farnesylation signal, and by phosphorylation by protein kinase C of aFGF added externally to cells. If aFGF is fused to diphtheria toxin A-fragment, it can be artificially translocated from the cell surface to the cytoplasm by the diphtheria toxin pathway. Upon further incubation, the fusion protein enters the nucleus due to a nuclear localization sequence in aFGF. We demonstrate here that upon inhibition of PI 3-kinase the fusion protein remains in the cytosol. We also provide evidence that the phosphorylation status of the fusion protein does not regulate its nucleocytoplasmic distribution.
      aFGF
      acidic fibroblast growth factor
      FGFR
      FGF receptor
      DT
      diphtheria toxin
      DT-A
      DT A-fragment
      DT-B
      DT B-fragment
      NLS
      nuclear localization sequence
      PI
      phosphatidylinositol
      SH2-/SH3-domain
      src homology 2/3 domain
      DMEM
      Dulbecco's modified essential medium
      PAGE
      polyacrylamide gel electrophoresis
      PBS
      phosphate-buffered saline
      Acidic fibroblast growth factor (aFGF)1 is considered to be involved in several important physiological and pathological processes, such as angiogenesis, wound healing (
      • Burgess W.H.
      • Maciag T.
      ), and atheromatosis (
      • Ananyeva N.M.
      • Tjurmin A.V.
      • Berliner J.A.
      • Chisolm G.M.
      • Liau G.
      • Winkles J.A.
      • Haudenschild C.C.
      ,
      • Hughes S.E.
      ,
      • Liau G.
      • Winkles J.A.
      • Cannon M.S.
      • Kuo L.
      • Chilian W.M.
      ,
      • Brogi E.
      • Winkles J.A.
      • Underwood R.
      • Clinton S.K.
      • Alberts G.F.
      • Libby P.
      ,
      • Hughes S.E.
      • Crossman D.
      • Hall P.A.
      ). In cell cultures it can stimulate growth, cell migration, and differentiation. As the growth factor lacks a classical signal sequence, its mechanism of secretion is still unclear, but it appears to involve a pathway different from the classical secretion pathway through the endoplasmic reticulum and Golgi apparatus (
      • Carreira C.M.
      • LaVallee T.M.
      • Tarantini F.
      • Jackson A.
      • Lathrop J.T.
      • Hampton B.
      • Burgess W.H.
      • Maciag T.
      ,
      • LaVallee T.M.
      • Tarantini F.
      • Gamble S.
      • Carreira C.M.
      • Jackson A.
      • Maciag T.
      ,
      • Tarantini F.
      • LaVallee T.
      • Jackson A.
      • Gamble S.
      • Carreira C.M.
      • Garfinkel S.
      • Burgess W.H.
      • Maciag T.
      ). At the cell surface, aFGF binds with high affinity to transmembrane FGF-receptors (FGFR) containing a cytoplasmic split tyrosine kinase domain. There are four different genes encoding FGFR (FGFR1–4) with several different splicing variants. aFGF also binds to heparan sulfate proteoglycans at the cell surface, although with lower affinity. Upon activation, the FGFR is phosphorylated on tyrosine residues, and it activates downstream effectors such as phosholipase Cγ and the mitogen-activated protein kinase pathway. There are some conflicting data as to whether phosphatidylinositol (PI) 3-kinase is also activated (
      • Kanda S.
      • Hodgkin M.N.
      • Woodfield R.J.
      • Wakelam M.J.
      • Thomas G.
      • Claesson-Welsh L.
      ,
      • Wennstrom S.
      • Siegbahn A.
      • Yokote K.
      • Arvidsson A.K.
      • Heldin C.H.
      • Mori S.
      • Claesson-Welsh L.
      ,
      • Raffioni S.
      • Bradshaw R.A.
      ,
      • Jackson T.R.
      • Stephens L.R.
      • Hawkins P.T.
      ). After binding to high affinity cell surface receptors, aFGF is translocated across cellular membranes and transported to the nucleus (
      • Munoz R.
      • Klingenberg O.
      • Wiedlocha A.
      • Rapak A.
      • Falnes P.O.
      • Olsnes S.
      ,
      • Mehta V.B.
      • Connors L.
      • Wang H.C.
      • Chiu I.M.
      ,
      • Wiedlocha A.
      • Falnes P.Ø.
      • Madshus I.H.
      • Sandvig K.
      • Olsnes S.
      ,
      • Wiedlocha A.
      • Falnes P.Ø.
      • Rapak A.
      • Klingenberg O.
      • Munoz R.
      • Olsnes S.
      ,
      • Klingenberg O.
      • Wiedlocha A.
      • Rapak A.
      • Munoz R.
      • Falnes P.Ø.
      • Olsnes S.
      ,
      • Zhan X.
      • Hu X.
      • Friedman S.
      • Maciag T.
      ,
      • Imamura T.
      • Engleka K.
      • Zhan X.
      • Tokita Y.
      • Forough R.
      • Roeder D.
      • Jackson A.
      • Maier J.A.
      • Hla T.
      • Maciag T.
      ,
      • Prudovsky I.A.
      • Savion N.
      • LaVallee T.M.
      • Maciag T.
      ,
      • Zhan X.
      • Hu X.
      • Friesel R.
      • Maciag T.
      ). The mechanism of this process is not well defined. It depends on FGFR and, at least in the case of FGFR4, also on the intactness of the C terminus of its cytosolic part.
      Klingenberg, O., Wiedtocha, A., Rapak, A., Khnykin, D., Citores, L., and Olsnes, S. (2000) J. Cell Sci.,in press.
      2Klingenberg, O., Wiedtocha, A., Rapak, A., Khnykin, D., Citores, L., and Olsnes, S. (2000) J. Cell Sci.,in press.
      Human PI 3-kinases phosphorylate PI in the 3′ position of the inositol ring and can be divided into three main classes on the basis ofin vitro substrate specificity (
      • Vanhaesebroeck B.
      • Leevers S.J.
      • Panayotou G.
      • Waterfield M.D.
      ). Class I phosphorylates PI, PI 4-phosphate and PI 4,5-diphosphate and includes the p85/p110 heterodimeric PI 3-kinases and the G-protein βγ subunit-activated PI 3-kinases. Class II phosphorylates PI and PI 4-phosphate, whereas class III phosphorylates only PI and includes the human homologue of the yeast vps34 protein (
      • Vanhaesebroeck B.
      • Leevers S.J.
      • Panayotou G.
      • Waterfield M.D.
      ,
      • Lemmon M.A.
      • Ferguson K.M.
      • Schlessinger J.
      ,
      • Ward S.G.
      • June C.H.
      • Olive D.
      ,
      • Shepherd P.R.
      • Reaves B.J.
      • Davidson H.W.
      ,
      • Carpenter C.L.
      • Cantley L.C.
      ). PI 3-kinase has been reported to be involved in the regulation of diverse cellular processes, such as intracellular vesicle transport (
      • Shepherd P.R.
      • Reaves B.J.
      • Davidson H.W.
      ,
      • Li G.
      • D'Souza-Schorey C.
      • Barbieri M.A.
      • Roberts R.L.
      • Klippel A.
      • Williams L.T.
      • Stahl P.D.
      ,
      • Joly M.
      • Kazlauskas A.
      • Fay F.S.
      • Corvera S.
      ,
      • De Camilli P.
      • Emr S.D.
      • McPherson P.S.
      • Novick P.
      ,
      • Martys J.L.
      • Wjasow C.
      • Gangi D.M.
      • Kielian M.C.
      • McGraw T.E.
      • Backer J.M.
      ,
      • Hansen S.H.
      • Olsson A.
      • Casanova J.E.
      ,
      • Shpetner H.
      • Joly M.
      • Hartley D.
      • Corvera S.
      ,
      • Gaullier J.M.
      • Simonsen A.
      • D'Arrigo A.
      • Bremnes B.
      • Stenmark H.
      • Aasland R.
      ,
      • Patki V.
      • Lawe D.C.
      • Corvera S.
      • Virbasius J.V.
      • Chawla A.
      ), mitogenesis (
      • Varticovski L.
      • Harrison-Findik D.
      • Keeler M.L.
      • Susa M.
      ), cell motility and invasiveness (
      • Keely P.J.
      • Westwick J.K.
      • Whitehead I.P.
      • Der C.J.
      • Parise L.V.
      ), insulin-regulated glucose uptake (
      • Carpenter C.L.
      • Cantley L.C.
      ,
      • Okada T.
      • Kawano Y.
      • Sakakibara T.
      • Hazeki O.
      • Ui M.
      ,
      • Gould G.W.
      • Jess T.J.
      • Andrews G.C.
      • Herbst J.J.
      • Plevin R.J.
      • Gibbs E.M.
      ,
      • Shepherd P.R.
      • Nave B.T.
      • Siddle K.
      ), and apoptosis (
      • Franke T.F.
      • Kaplan D.R.
      • Cantley L.C.
      ,
      • Franke T.F.
      • Cantley L.C.
      ,
      • Yao R.
      • Cooper G.M.
      ), to mention some.
      Best studied is the heterodimeric PI 3-kinase consisting of a 110-kDa catalytic subunit noncovalently bound to an 85-kDa regulatory subunit (
      • Ward S.G.
      • June C.H.
      • Olive D.
      ,
      • Varticovski L.
      • Harrison-Findik D.
      • Keeler M.L.
      • Susa M.
      ). PI 4,5-diphosphate is likely its preferred physiological substrate (
      • Vanhaesebroeck B.
      • Leevers S.J.
      • Panayotou G.
      • Waterfield M.D.
      ,
      • Shepherd P.R.
      • Reaves B.J.
      • Davidson H.W.
      ). The regulatory 85-kDa subunit contains several domains, among which are two SH2 (src homology 2) domains and one SH3 domain. The SH2 domains can bind specifically to phosphorylated tyrosine residues in the consensus sequence pYXXM (
      • Ward S.G.
      • June C.H.
      • Olive D.
      ,
      • Varticovski L.
      • Harrison-Findik D.
      • Keeler M.L.
      • Susa M.
      ), contained in transmembrane receptors such as the platelet-derived growth factor receptor and the colony stimulating factor-1 receptor. Binding of PI 3-kinase by its SH2 domains to such phosphorylated tyrosine residues activates the enzyme. The same consensus sequence is conserved in all four FGF receptors (
      • Jaye M.
      • Schlessinger J.
      • Dionne C.A.
      ), and the tyrosine in this sequence of FGFR 1 was recently shown to be phosphorylated in vivo upon receptor activation (
      • Mohammadi M.
      • Dikic I.
      • Sorokin A.
      • Burgess W.H.
      • Jaye M.
      • Schlessinger J.
      ). At high concentrations, a tyrosine-phosphorylated pentapeptide derived from this FGFR1 sequence was demonstrated to compete with platelet-derived growth factor receptor and v-fms for binding to PI 3-kinase in vitro (
      • Fantl W.J.
      • Escobedo J.A.
      • Martin G.A.
      • Turck C.W.
      • Del Rosario M.
      • McCormick F.
      • Williams L.T.
      ). However, a direct interaction of FGFR and PI 3-kinase has been difficult to demonstrate (
      • Vainikka S.
      • Joukov V.
      • Wennstrom S.
      • Bergman M.
      • Pelicci P.G.
      • Alitalo K.
      ,
      • Wennstrom S.
      • Landgren E.
      • Blume-Jensen P.
      • Claesson-Welsh L.
      ). There is one recent report that in extracts from Xenopus blastulae, p85 of PI 3-kinase co-precipitates with FGFR1 (
      • Ryan P.J.
      • Paterno G.D.
      • Gillespie L.L.
      ).
      PI 3-kinase is also regulated by other means. Ras can bind to the catalytic subunit and thereby activate it (
      • Kodaki T.
      • Woscholski R.
      • Hallberg B.
      • Rodriguez-Viciana P.
      • Downward J.
      • Parker P.J.
      ,
      • Rodriguez-Viciana P.
      • Warne P.H.
      • Dhand R.
      • Vanhaesebroeck B.
      • Gout I.
      • Fry M.J.
      • Waterfield M.D.
      • Downward J.
      ,
      • Rodriguez-Viciana P.
      • Warne P.H.
      • Vanhaesebroeck B.
      • Waterfield M.D.
      • Downward J.
      ,
      • Rodriguez-Viciana P.
      • Warne P.H.
      • Khwaja A.
      • Marte B.M.
      • Pappin D.
      • Das P.
      • Waterfield M.D.
      • Ridley A.
      • Downward J.
      ). CDC42 and Rac are reported to activate the enzyme by binding to the regulatory subunit (
      • Keely P.J.
      • Westwick J.K.
      • Whitehead I.P.
      • Der C.J.
      • Parise L.V.
      ,
      • Zheng Y.
      • Bagrodia S.
      • Cerione R.A.
      ,
      • Tolias K.F.
      • Cantley L.C.
      • Carpenter C.L.
      ), and tyrosine phosphorylation of this subunit also has a stimulatory effect (
      • Varticovski L.
      • Harrison-Findik D.
      • Keeler M.L.
      • Susa M.
      ,
      • Soltoff S.P.
      • Rabin S.L.
      • Cantley L.C.
      • Kaplan D.R.
      ). Serine autophosphorylation down-regulates the enzyme (
      • Varticovski L.
      • Harrison-Findik D.
      • Keeler M.L.
      • Susa M.
      ).
      Diphtheria toxin (DT) is synthesized as a single polypeptide chain that can be cleaved at a protease-sensitive site into two fragments, A and B (or DT-A and DT-B), held together by a disulfide bond. DT-B binds to diphtheria toxin receptors, and the complex is endocytosed. Triggered by the low pH in the endosomes, DT-A translocates across the endosomal membrane and reaches the cytosol, where it exerts its toxic effect by ADP-ribosylating elongation factor 2, thereby inhibiting protein synthesis (
      • London E.
      ). Experimentally, the translocation can be rapidly induced at the level of the surface membrane if cells with receptor-bound toxin are exposed to low pH, resembling the conditions in the endosomes (
      • Sandvig K.
      • Olsnes S.
      ,
      • Sandvig K.
      • Olsnes S.
      ). Several proteins, including aFGF, are capable of being translocated into cells as fusion proteins with DT-A (
      • Madshus I.H.
      • Olsnes S.
      • Stenmark H.
      ,
      • Wiedlocha A.
      • Madshus I.H.
      • Mach H.
      • Middaugh C.R.
      • Olsnes S.
      ,
      • Klingenberg O.
      • Olsnes S.
      ). We have previously used this as a tool to study the intracellular role of aFGF (
      • Wiedlocha A.
      • Falnes P.Ø.
      • Madshus I.H.
      • Sandvig K.
      • Olsnes S.
      ,
      • Klingenberg O.
      • Wiedlocha A.
      • Rapak A.
      • Munoz R.
      • Falnes P.Ø.
      • Olsnes S.
      ,
      • Wiedlocha A.
      • Falnes P.Ø.
      • Rapak A.
      • Munoz R.
      • Klingenberg O.
      • Olsnes S.
      ).
      In this work we demonstrate that PI 3-kinase activity is required both for transport of exogenously added aFGF to the cytosol and to the nuclear fraction and for accumulation in the nucleus of aFGF that has been translocated to the cytosol as a fusion protein with diphtheria toxin.

      DISCUSSION

      The data here presented demonstrate that two inhibitors of PI3-kinase, wortmannin and LY294002, inhibited transport to the cytosol and nuclear fraction of aFGF added externally to cells. Also, the drugs inhibited accumulation in the nucleus of the fusion protein aFGF-DTA that had been translocated into the cytosol by the diphtheria toxin pathway. The concentrations required to inhibit these processes suggest that in both cases, inhibition of PI 3-kinase was responsible for the effect, since most other targets for these inhibitors require higher concentrations of the drugs (
      • Ward S.G.
      • June C.H.
      • Olive D.
      ,
      • Brunn G.J.
      • Williams J.
      • Sabers C.
      • Wiederrecht G.
      • Lawrence Jr., J.C.
      • Abraham R.T.
      ,
      • Wymann M.P.
      • Bulgarelli-Leva G.
      • Zvelebil M.J.
      • Pirola L.
      • Vanhaesebroeck B.
      • Waterfield M.D.
      • Panayotou G.
      ,
      • Nakanishi S.
      • Kakita S.
      • Takahashi I.
      • Kawahara K.
      • Tsukuda E.
      • Sano T.
      • Yamada K.
      • Yoshida M.
      • Kase H.
      • Matsuda Y.
      ). This notion is also substantiated by the findings that the dominant negative PI 3-kinase mutant, Δp85α, inhibited transport to the cytosol and nucleus of aFGF, as measured by phosphorylation as well as the nuclear accumulation of aFGF-DT-A.
      When cells with bound aFGF-DT-A fusion protein reconstituted with DT-B are exposed to low pH, the fusion protein is immediately located in the cytosol (
      • Wiedlocha A.
      • Madshus I.H.
      • Mach H.
      • Middaugh C.R.
      • Olsnes S.
      ,
      • Moskaug J.O.
      • Sandvig K.
      • Olsnes S.
      ). Complete accumulation of the fusion protein in the nuclear fraction takes, however, several hours. The reason for the slow kinetics of nuclear accumulation is not clear, but the possibility that aFGF-DT-A is actually shuttling between the cytosol and nucleus is presently under investigation. If this is the case, the slow nuclear accumulation could be the result of competition between nuclear export and binding to nuclear structures retaining the fusion protein in the nucleus. In any event, nuclear accumulation of aFGF-DT-A appears to depend on the NLS of aFGF since ΔaFGF-DT-A, where this sequence has been deleted, remained in the cytosol. The NLS of aFGF has been shown to be able to direct nuclear translocation of a chimeric protein when fused to the N terminus of β-galactosidase and expressed in NIH 3T3 cells (
      • Zhan X.
      • Hu X.
      • Friedman S.
      • Maciag T.
      ). However, when full-length aFGF was fused to β-galactosidase, the construct remained in the cytosol, as was the case when aFGF alone was expressed (
      • Zhan X.
      • Hu X.
      • Friedman S.
      • Maciag T.
      ). Exogenously added aFGF, on the other hand, was transported to the nucleus (
      • Munoz R.
      • Klingenberg O.
      • Wiedlocha A.
      • Rapak A.
      • Falnes P.O.
      • Olsnes S.
      ,
      • Mehta V.B.
      • Connors L.
      • Wang H.C.
      • Chiu I.M.
      ,
      • Wiedlocha A.
      • Falnes P.Ø.
      • Madshus I.H.
      • Sandvig K.
      • Olsnes S.
      ,
      • Wiedlocha A.
      • Falnes P.Ø.
      • Rapak A.
      • Klingenberg O.
      • Munoz R.
      • Olsnes S.
      ,
      • Klingenberg O.
      • Wiedlocha A.
      • Rapak A.
      • Munoz R.
      • Falnes P.Ø.
      • Olsnes S.
      ,
      • Zhan X.
      • Hu X.
      • Friedman S.
      • Maciag T.
      ,
      • Imamura T.
      • Engleka K.
      • Zhan X.
      • Tokita Y.
      • Forough R.
      • Roeder D.
      • Jackson A.
      • Maier J.A.
      • Hla T.
      • Maciag T.
      ,
      • Prudovsky I.A.
      • Savion N.
      • LaVallee T.M.
      • Maciag T.
      ,
      • Zhan X.
      • Hu X.
      • Friesel R.
      • Maciag T.
      ).
      With aFGF as such, the transport pathway from the cell surface into the nucleus is not clear. It is not known whether aFGF is first translocated to the cytosol and then to the nucleus or vice versa. For the NLS of aFGF to be functional (
      • Imamura T.
      • Engleka K.
      • Zhan X.
      • Tokita Y.
      • Forough R.
      • Roeder D.
      • Jackson A.
      • Maier J.A.
      • Hla T.
      • Maciag T.
      ), interaction with cytoplasmic transport proteins appears to be required, which favors the first alternative. The NLS could also function to counteract diffusion from the nucleus into the cytosol. We incubated calf pulmonary artery endothelial cells in the presence of 125I-labeled aFGF for different periods of time, fractionated the cells in membrane, cytosol, and nuclear fractions, and analyzed the fractions by SDS-PAGE. At the earliest time points, aFGF was contained in the membrane fraction. Later, it was also obtained from the nuclear fraction, and finally aFGF was found also in the cytosol fraction.
      A. Wiedlocha, unpublished results.
      These data argue for a transport mechanism in which aFGF is first translocated into the nucleus and then later exported to the cytosol. However, the data can also be explained by a model in which aFGF is first translocated to the cytosol and then rapidly transported from the cytosol to the nucleus, so that the concentration in the cytosol is below the detection limit in our assay. This issue requires further work.
      PI 3-kinase inhibition seems to interfere with nuclear localization at different steps for aFGF and aFGF-DT-A. In the case of aFGF, it appears that a transport step prior to or involved in membrane translocation is blocked by PI 3-kinase inhibition since labeled aFGF was only found in the membrane fraction in the presence of PI 3-kinase inhibitors and since, upon PI 3-kinase inhibition, aFGF-CAAX was not farnesylated, and aFGF was not phosphorylated. It could therefore not have reached even the cytosol.
      PI 3-kinase has a well established role in intracellular vesicle transport, from yeast to man. In Saccharomyces cerevisiaethe VPS34 gene product, which is the only PI 3-kinase in yeast, is involved in protein sorting to the vacuole (
      • De Camilli P.
      • Emr S.D.
      • McPherson P.S.
      • Novick P.
      ). In mammals, PI 3-kinase is not required for the initial endocytic process but for later sorting events (
      • Shepherd P.R.
      • Reaves B.J.
      • Davidson H.W.
      ). A mutant platelet-derived growth factor receptor that is deficient in PI 3-kinase binding and activation did not traffic normally to the juxtanuclear region but stayed at the cell periphery (
      • Joly M.
      • Kazlauskas A.
      • Fay F.S.
      • Corvera S.
      ). It was recently shown that the Rab5 binding human early-endosomal autoantigen, EEA1, binds directly to PI 3-phosphate (
      • Gaullier J.M.
      • Simonsen A.
      • D'Arrigo A.
      • Bremnes B.
      • Stenmark H.
      • Aasland R.
      ,
      • Patki V.
      • Lawe D.C.
      • Corvera S.
      • Virbasius J.V.
      • Chawla A.
      ), providing a molecular link between PI 3-kinase and endosome fusion (
      • Simonsen A.
      • Lippe R.
      • Christoforidis S.
      • Gaullier J.M.
      • Brech A.
      • Callaghan J.
      • Toh B.H.
      • Murphy C.
      • Zerial M.
      • Stenmark H.
      ). It is therefore quite possible that PI 3-kinase inhibition interferes with intracellular transport of aFGF at a sorting step after endocytosis. If this is the case, it can be invoked that endocytosis is required for membrane translocation of aFGF to take place. For other proteins that translocate across membranes, this is often the case as for e.g. diphtheria toxin (
      • Sandvig K.
      • Olsnes S.
      ,
      • Sandvig K.
      • Olsnes S.
      ) and ricin (
      • Rapak A.
      • Falnes P.O.
      • Olsnes S.
      ). There are, however, also examples of proteins that most likely are able to translocate across the cell surface membrane such as the invasive adenylate cyclase from Bordetella pertussis (
      • Rogel A.
      • Hanski E.
      ), the VP22 protein of herpesvirus (
      • Elliott G.
      • O'Hare P.
      ,
      • Cleves A.E.
      ) and the Antennapedia homeodomain fromDrosophila (
      • Prochiantz A.
      ). It should be kept in mind that the data here presented do not rule out the possibility that the effect of PI 3-kinase inhibition is not on intracellular transport but rather on the membrane translocation process as such.
      The cytoplasmic retention of aFGF-DT-A observed upon PI 3-kinase inhibition could not be caused by a block in vesicle sorting since the fusion protein was translocated into the cytosol during a brief low pH treatment in the absence of wortmannin and LY294002. In the case of the fusion protein, either transport from the cytosol to the nucleus (
      • Schlenstedt G.
      ,
      • Nigg E.A.
      ,
      • Gorlich D.
      • Mattaj I.W.
      ), intranuclear binding (
      • Schmidt-Zachmann M.S.
      • Dargemont C.
      • Kuhn L.C.
      • Nigg E.A.
      ,
      • Paine P.L.
      ), or exit from the nucleus must have been affected by PI 3-kinase inhibition. Wortmannin blocked the nuclear translocation of protein kinase Cζ (PKCζ) during ischemia, but this result was interpreted as inhibition of protein kinase Cζ activation, since this isotype of protein kinase C is stimulated by PI 3-kinase lipid products (
      • Mizukami Y.
      • Hirata T.
      • Yoshida K.
      ). Wortmannin also prevented nuclear translocation of mitogen-activated protein kinase in polyomavirus middle-T-transformed cells, but also in this case inhibition of nuclear localization of mitogen-activated protein kinase was coupled with inhibition of its activation (
      • Urich M.
      • el Shemerly M.Y.
      • Besser D.
      • Nagamine Y.
      • Ballmer-Hofer K.
      ). Protein kinase Bβ, a signal transducer downstream of PI 3-kinase, was reported to undergo nuclear translocation within 20–30 min after stimulation (
      • Meier R.
      • Alessi D.R.
      • Cron P.
      • Andjelkovic M.
      • Hemmings B.A.
      ). Even PI 3-kinase itself was reported to translocate to the nucleus after treatment of PC 12 cells with nerve growth factor (
      • Neri L.M.
      • Milani D.
      • Bertolaso L.
      • Stroscio M.
      • Bertagnolo V.
      • Capitani S.
      ). However, in all these cases nuclear translocation was coupled to activation of the proteins.
      Protein kinase activity has been involved in regulation of transport of proteins to the nucleus. In many cases a phosphorylation site close to a nuclear localization sequence regulates nucleocytoplasmic distribution (
      • Nigg E.A.
      ,
      • Tagawa T.
      • Kuroki T.
      • Vogt P.K.
      • Chida K.
      ,
      • Moll T.
      • Tebb G.
      • Surana U.
      • Robitsch H.
      • Nasmyth K.
      ). aFGF is phosphorylated in living cells (
      • Klingenberg O.
      • Wiedlocha A.
      • Rapak A.
      • Munoz R.
      • Falnes P.Ø.
      • Olsnes S.
      ,
      • Mascarelli F.
      • Raulais D.
      • Courtois Y.
      ), and both aFGF and aFGF-DT-A are phosphorylated in vitro in a cell lysate (
      • Klingenberg O.
      • Wiedlocha A.
      • Rapak A.
      • Munoz R.
      • Falnes P.Ø.
      • Olsnes S.
      ). However, the nucleocytoplasmic distribution of aFGF(K132E)-DT-A was apparently regulated by PI 3-kinase in the same manner as aFGF-DT-A. aFGF(K132E)-DT-A was not phosphorylated in vitro because the Lys-132Glu mutation destroys a consensus site for phosphorylation by protein kinase C, which appears to be the major phosphorylation site in the growth factor (
      • Klingenberg O.
      • Wiedlocha A.
      • Rapak A.
      • Munoz R.
      • Falnes P.Ø.
      • Olsnes S.
      ,
      • Klingenberg O.
      • Wiedlocha A.
      • Olsnes S.
      ). Therefore, it is unlikely that the effect of PI 3-kinase inhibition on nucleocytoplasmic distribution is mediated by phosphorylation of aFGF-DT-A.
      In conclusion, we have demonstrated the requirement of PI 3-kinase activity for transport of externally added aFGF to the cytosol and nucleus. We have also demonstrated that nuclear accumulation of aFGF-DT-A, translocated to the cytosol by the diphtheria toxin pathway, depends on PI 3-kinase activity. The molecular mechanisms of these observations remain to be elucidated.

      Acknowledgments

      The expert work with the cell cultures by Jorunn Jacobsen and the skillful technical assistance by Mette Sværen is gratefully acknowledged.

      REFERENCES

        • Burgess W.H.
        • Maciag T.
        Annu. Rev. Biochem. 1989; 58: 575-606
        • Ananyeva N.M.
        • Tjurmin A.V.
        • Berliner J.A.
        • Chisolm G.M.
        • Liau G.
        • Winkles J.A.
        • Haudenschild C.C.
        Arterioscler. Thromb. Vasc. Biol. 1997; 17: 445-453
        • Hughes S.E.
        Cardiovasc. Res. 1996; 32: 557-569
        • Liau G.
        • Winkles J.A.
        • Cannon M.S.
        • Kuo L.
        • Chilian W.M.
        J. Vasc. Res. 1993; 30: 327-332
        • Brogi E.
        • Winkles J.A.
        • Underwood R.
        • Clinton S.K.
        • Alberts G.F.
        • Libby P.
        J. Clin. Invest. 1993; 92: 2408-2418
        • Hughes S.E.
        • Crossman D.
        • Hall P.A.
        Cardiovasc. Res. 1993; 27: 1214-1219
        • Carreira C.M.
        • LaVallee T.M.
        • Tarantini F.
        • Jackson A.
        • Lathrop J.T.
        • Hampton B.
        • Burgess W.H.
        • Maciag T.
        J. Biol. Chem. 1998; 273: 22224-22231
        • LaVallee T.M.
        • Tarantini F.
        • Gamble S.
        • Carreira C.M.
        • Jackson A.
        • Maciag T.
        J. Biol. Chem. 1998; 273: 22217-22223
        • Tarantini F.
        • LaVallee T.
        • Jackson A.
        • Gamble S.
        • Carreira C.M.
        • Garfinkel S.
        • Burgess W.H.
        • Maciag T.
        J. Biol. Chem. 1998; 273: 22209-22216
        • Kanda S.
        • Hodgkin M.N.
        • Woodfield R.J.
        • Wakelam M.J.
        • Thomas G.
        • Claesson-Welsh L.
        J. Biol. Chem. 1997; 272: 23347-23353
        • Wennstrom S.
        • Siegbahn A.
        • Yokote K.
        • Arvidsson A.K.
        • Heldin C.H.
        • Mori S.
        • Claesson-Welsh L.
        Oncogene. 1994; 9: 651-660
        • Raffioni S.
        • Bradshaw R.A.
        Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9121-9125
        • Jackson T.R.
        • Stephens L.R.
        • Hawkins P.T.
        J. Biol. Chem. 1992; 267: 16627-16636
        • Munoz R.
        • Klingenberg O.
        • Wiedlocha A.
        • Rapak A.
        • Falnes P.O.
        • Olsnes S.
        Oncogene. 1997; 15: 525-536
        • Mehta V.B.
        • Connors L.
        • Wang H.C.
        • Chiu I.M.
        J. Biol. Chem. 1998; 273: 4197-4205
        • Wiedlocha A.
        • Falnes P.Ø.
        • Madshus I.H.
        • Sandvig K.
        • Olsnes S.
        Cell. 1994; 76: 1039-1051
        • Wiedlocha A.
        • Falnes P.Ø.
        • Rapak A.
        • Klingenberg O.
        • Munoz R.
        • Olsnes S.
        J. Biol. Chem. 1995; 270: 30680-30685
        • Klingenberg O.
        • Wiedlocha A.
        • Rapak A.
        • Munoz R.
        • Falnes P.Ø.
        • Olsnes S.
        J. Biol. Chem. 1998; 273: 11164-11172
        • Zhan X.
        • Hu X.
        • Friedman S.
        • Maciag T.
        Biochem. Biophys. Res. Commun. 1992; 188: 982-991
        • Imamura T.
        • Engleka K.
        • Zhan X.
        • Tokita Y.
        • Forough R.
        • Roeder D.
        • Jackson A.
        • Maier J.A.
        • Hla T.
        • Maciag T.
        Science. 1990; 249: 1567-1570
        • Prudovsky I.A.
        • Savion N.
        • LaVallee T.M.
        • Maciag T.
        J. Biol. Chem. 1996; 271: 14198-14205
        • Zhan X.
        • Hu X.
        • Friesel R.
        • Maciag T.
        J. Biol. Chem. 1993; 268: 9611-9620
        • Vanhaesebroeck B.
        • Leevers S.J.
        • Panayotou G.
        • Waterfield M.D.
        Trends Biochem. Sci. 1997; 22: 267-272
        • Lemmon M.A.
        • Ferguson K.M.
        • Schlessinger J.
        Cell. 1996; 85: 621-624
        • Ward S.G.
        • June C.H.
        • Olive D.
        Immunol. Today. 1996; 17: 187-197
        • Shepherd P.R.
        • Reaves B.J.
        • Davidson H.W.
        Trends Cell Biol. 1996; 6: 92-97
        • Carpenter C.L.
        • Cantley L.C.
        Curr. Opin. Cell Biol. 1996; 8: 153-158
        • Li G.
        • D'Souza-Schorey C.
        • Barbieri M.A.
        • Roberts R.L.
        • Klippel A.
        • Williams L.T.
        • Stahl P.D.
        Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10207-10211
        • Joly M.
        • Kazlauskas A.
        • Fay F.S.
        • Corvera S.
        Science. 1994; 263: 684-687
        • De Camilli P.
        • Emr S.D.
        • McPherson P.S.
        • Novick P.
        Science. 1996; 271: 1533-1539
        • Martys J.L.
        • Wjasow C.
        • Gangi D.M.
        • Kielian M.C.
        • McGraw T.E.
        • Backer J.M.
        J. Biol. Chem. 1996; 271: 10953-10962
        • Hansen S.H.
        • Olsson A.
        • Casanova J.E.
        J. Biol. Chem. 1995; 270: 28425-28432
        • Shpetner H.
        • Joly M.
        • Hartley D.
        • Corvera S.
        J. Cell Biol. 1996; 132: 595-605
        • Gaullier J.M.
        • Simonsen A.
        • D'Arrigo A.
        • Bremnes B.
        • Stenmark H.
        • Aasland R.
        Nature. 1998; 394: 432-433
        • Patki V.
        • Lawe D.C.
        • Corvera S.
        • Virbasius J.V.
        • Chawla A.
        Nature. 1998; 394: 433-434
        • Varticovski L.
        • Harrison-Findik D.
        • Keeler M.L.
        • Susa M.
        Biochim. Biophys. Acta. 1994; 1226: 1-11
        • Keely P.J.
        • Westwick J.K.
        • Whitehead I.P.
        • Der C.J.
        • Parise L.V.
        Nature. 1997; 390: 632-636
        • Okada T.
        • Kawano Y.
        • Sakakibara T.
        • Hazeki O.
        • Ui M.
        J. Biol. Chem. 1994; 269: 3568-3573
        • Gould G.W.
        • Jess T.J.
        • Andrews G.C.
        • Herbst J.J.
        • Plevin R.J.
        • Gibbs E.M.
        J. Biol. Chem. 1994; 269: 26622-26625
        • Shepherd P.R.
        • Nave B.T.
        • Siddle K.
        Biochem. J. 1995; 305: 25-28
        • Franke T.F.
        • Kaplan D.R.
        • Cantley L.C.
        Cell. 1997; 88: 435-437
        • Franke T.F.
        • Cantley L.C.
        Nature. 1997; 390: 116-117
        • Yao R.
        • Cooper G.M.
        Science. 1995; 267: 2003-2006
        • Jaye M.
        • Schlessinger J.
        • Dionne C.A.
        Biochim. Biophys. Acta. 1992; 1135: 185-199
        • Mohammadi M.
        • Dikic I.
        • Sorokin A.
        • Burgess W.H.
        • Jaye M.
        • Schlessinger J.
        Mol. Cell. Biol. 1996; 16: 977-989
        • Fantl W.J.
        • Escobedo J.A.
        • Martin G.A.
        • Turck C.W.
        • Del Rosario M.
        • McCormick F.
        • Williams L.T.
        Cell. 1992; 69: 413-423
        • Vainikka S.
        • Joukov V.
        • Wennstrom S.
        • Bergman M.
        • Pelicci P.G.
        • Alitalo K.
        J. Biol. Chem. 1994; 269: 18320-18326
        • Wennstrom S.
        • Landgren E.
        • Blume-Jensen P.
        • Claesson-Welsh L.
        J. Biol. Chem. 1992; 267: 13749-13756
        • Ryan P.J.
        • Paterno G.D.
        • Gillespie L.L.
        Biochem. Biophys. Res. Commun. 1998; 244: 763-767
        • Kodaki T.
        • Woscholski R.
        • Hallberg B.
        • Rodriguez-Viciana P.
        • Downward J.
        • Parker P.J.
        Curr. Biol. 1994; 4: 798-806
        • Rodriguez-Viciana P.
        • Warne P.H.
        • Dhand R.
        • Vanhaesebroeck B.
        • Gout I.
        • Fry M.J.
        • Waterfield M.D.
        • Downward J.
        Nature. 1994; 370: 527-532
        • Rodriguez-Viciana P.
        • Warne P.H.
        • Vanhaesebroeck B.
        • Waterfield M.D.
        • Downward J.
        EMBO J. 1996; 15: 2442-2451
        • Rodriguez-Viciana P.
        • Warne P.H.
        • Khwaja A.
        • Marte B.M.
        • Pappin D.
        • Das P.
        • Waterfield M.D.
        • Ridley A.
        • Downward J.
        Cell. 1997; 89: 457-467
        • Zheng Y.
        • Bagrodia S.
        • Cerione R.A.
        J. Biol. Chem. 1994; 269: 18727-18730
        • Tolias K.F.
        • Cantley L.C.
        • Carpenter C.L.
        J. Biol. Chem. 1995; 270: 17656-17659
        • Soltoff S.P.
        • Rabin S.L.
        • Cantley L.C.
        • Kaplan D.R.
        J. Biol. Chem. 1992; 267: 17472-17477
        • London E.
        Biochim. Biophys. Acta. 1992; 1113: 25-51
        • Sandvig K.
        • Olsnes S.
        J. Cell Biol. 1980; 87: 828-832
        • Sandvig K.
        • Olsnes S.
        J. Biol. Chem. 1981; 256: 9068-9076
        • Madshus I.H.
        • Olsnes S.
        • Stenmark H.
        Infect. Immun. 1992; 60: 3296-3302
        • Wiedlocha A.
        • Madshus I.H.
        • Mach H.
        • Middaugh C.R.
        • Olsnes S.
        EMBO J. 1992; 11: 4835-4842
        • Klingenberg O.
        • Olsnes S.
        Biochem. J. 1996; 313: 647-653
        • Wiedlocha A.
        • Falnes P.Ø.
        • Rapak A.
        • Munoz R.
        • Klingenberg O.
        • Olsnes S.
        Mol. Cell. Biol. 1996; 16: 270-280
        • Laemmli U.K.
        Nature. 1970; 227: 680-685
        • Stenmark H.
        • Afanasiev B.N.
        • Ariansen S.
        • Olsnes S.
        Biochem. J. 1992; 281: 619-625
        • Wang J.K.
        • Gao G.
        • Goldfarb M.
        Mol. Cell. Biol. 1994; 14: 181-188
        • Casey P.J.
        • Thissen J.A.
        • Moomaw J.F.
        Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8631-8635
        • Reiss Y.
        • Goldstein J.L.
        • Seabra M.C.
        • Casey P.J.
        • Brown M.S.
        Cell. 1990; 62: 81-88
        • Schaber M.D.
        • O'Hara M.B.
        • Garsky V.M.
        • Mosser S.C.
        • Bergstrom J.D.
        • Moores S.L.
        • Marshall M.S.
        • Friedman P.A.
        • Dixon R.A.
        • Gibbs J.B.
        J. Biol. Chem. 1990; 265: 14701-14704
        • Lutz R.J.
        • Trujillo M.A.
        • Denham K.S.
        • Wenger L.
        • Sinensky M.
        Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3000-3004
        • Sinensky M.
        • Fantle K.
        • Trujillo M.
        • McLain T.
        • Kupfer A.
        • Dalton M.
        J. Cell Sci. 1994; 107: 61-67
        • Garcia A.M.
        • Rowell C.
        • Ackermann K.
        • Kowalczyk J.J.
        • Lewis M.D.
        J. Biol. Chem. 1993; 268: 18415-18418
        • Klingenberg O.
        • Wiedlocha A.
        • Olsnes S.
        J. Biol. Chem. 1999; 274: 18081-18086
        • Dhand R.
        • Hara K.
        • Hiles I.
        • Bax B.
        • Gout I.
        • Panayotou G.
        • Fry M.J.
        • Yonezawa K.
        • Kasuga M.
        • Waterfield M.D.
        EMBO J. 1994; 13: 511-521
        • Burgering B.M.
        • Coffer P.J.
        Nature. 1995; 376: 599-602
        • van Weering D.H.
        • de Rooij J.
        • Marte B.
        • Downward J.
        • Bos J.L.
        • Burgering B.M.
        Mol. Cell. Biol. 1998; 18: 1802-1811
        • Alessi D.R.
        • Andjelkovic M.
        • Caudwell B.
        • Cron P.
        • Morrice N.
        • Cohen P.
        • Hemmings B.A.
        EMBO J. 1996; 15: 6541-6551
        • Burgess W.H.
        • Shaheen A.M.
        • Ravera M.
        • Jaye M.
        • Donohue P.J.
        • Winkles J.A.
        J. Cell Biol. 1990; 111: 2129-2138
        • Brunn G.J.
        • Williams J.
        • Sabers C.
        • Wiederrecht G.
        • Lawrence Jr., J.C.
        • Abraham R.T.
        EMBO J. 1996; 15: 5256-5267
        • Wymann M.P.
        • Bulgarelli-Leva G.
        • Zvelebil M.J.
        • Pirola L.
        • Vanhaesebroeck B.
        • Waterfield M.D.
        • Panayotou G.
        Mol. Cell. Biol. 1996; 16: 1722-1733
        • Nakanishi S.
        • Kakita S.
        • Takahashi I.
        • Kawahara K.
        • Tsukuda E.
        • Sano T.
        • Yamada K.
        • Yoshida M.
        • Kase H.
        • Matsuda Y.
        J. Biol. Chem. 1992; 267: 2157-2163
        • Moskaug J.O.
        • Sandvig K.
        • Olsnes S.
        J. Biol. Chem. 1988; 263: 2518-2525
        • Simonsen A.
        • Lippe R.
        • Christoforidis S.
        • Gaullier J.M.
        • Brech A.
        • Callaghan J.
        • Toh B.H.
        • Murphy C.
        • Zerial M.
        • Stenmark H.
        Nature. 1998; 394: 494-498
        • Rapak A.
        • Falnes P.O.
        • Olsnes S.
        Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3783-3788
        • Rogel A.
        • Hanski E.
        J. Biol. Chem. 1992; 267: 22599-22605
        • Elliott G.
        • O'Hare P.
        Cell. 1997; 88: 223-233
        • Cleves A.E.
        Curr. Biol. 1997; 7: 318-320
        • Prochiantz A.
        Curr. Opin. Neurobiol. 1996; 6: 629-634
        • Schlenstedt G.
        FEBS Lett. 1996; 389: 75-79
        • Nigg E.A.
        Nature. 1997; 386: 779-787
        • Gorlich D.
        • Mattaj I.W.
        Science. 1996; 271: 1513-1518
        • Schmidt-Zachmann M.S.
        • Dargemont C.
        • Kuhn L.C.
        • Nigg E.A.
        Cell. 1993; 74: 493-504
        • Paine P.L.
        Trends Cell Biol. 1993; 3: 325-329
        • Mizukami Y.
        • Hirata T.
        • Yoshida K.
        FEBS Lett. 1997; 401: 247-251
        • Urich M.
        • el Shemerly M.Y.
        • Besser D.
        • Nagamine Y.
        • Ballmer-Hofer K.
        J. Biol. Chem. 1995; 270: 29286-29292
        • Meier R.
        • Alessi D.R.
        • Cron P.
        • Andjelkovic M.
        • Hemmings B.A.
        J. Biol. Chem. 1997; 272: 30491-30497
        • Neri L.M.
        • Milani D.
        • Bertolaso L.
        • Stroscio M.
        • Bertagnolo V.
        • Capitani S.
        Cell. Mol. Biol. (Noisy-le-Grand). 1994; 40: 619-626
        • Tagawa T.
        • Kuroki T.
        • Vogt P.K.
        • Chida K.
        J. Cell Biol. 1995; 130: 255-263
        • Moll T.
        • Tebb G.
        • Surana U.
        • Robitsch H.
        • Nasmyth K.
        Cell. 1991; 66: 743-758
        • Mascarelli F.
        • Raulais D.
        • Courtois Y.
        EMBO J. 1989; 8: 2265-2273
        • Citores L.
        • Wesche J.
        • Kolpakova E.
        • Olsnes S.
        Mol. Biol. Cell. 1999; 10: 3835-3848
        • 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