Characterization of a G Protein-activated Phosphoinositide 3-Kinase in Vascular Smooth Muscle Cell Nuclei

Recent studies highlight the existence of an autonomous nuclear polyphosphoinositide metabolism related to cellular proliferation and differentiation. However, only few data document the nuclear production of the putative second messengers, the 3-phosphorylated phosphoinositides, by the phosphoinositide 3-kinase (PI3K). In the present paper, we examine whether GTP-binding proteins can directly modulate 3-phosphorylated phosphoinositide metabolism in membrane-free nuclei isolated from pig aorta smooth muscle cells (VSMCs). In vitro PI3K assays performed without the addition of any exogenous substrates revealed that guanosine 5′-(γ-thio)triphosphate (GTPγS) specifically stimulated the nuclear synthesis of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3), whereas guanosine 5′-(β-thio)diphosphate was ineffective. PI3K inhibitors wortmannin and LY294002 prevented GTPγS-induced PtdIns(3,4,5)P3synthesis. Moreover, pertussis toxin inhibited partially PtdIns(3,4,5)P3 accumulation, suggesting that nuclear Gi/G0 proteins are involved in the activation of PI3K. Immunoblot experiments showed the presence of Gα0 proteins in VSMC nuclei. In contrast with previous reports, immunoblots and indirect immunofluorescence failed to detect the p85α subunit of the heterodimeric PI3K within VSMC nuclei. By contrast, we have detected the presence of a 117-kDa protein immunologically related to the PI3Kγ. These results indicate the existence of a G protein-activated PI3K inside VSMC nucleus that might be involved in the contol of VSMC proliferation and in the pathogenesis of vascular proliferative disorders. Recent studies highlight the existence of an autonomous nuclear polyphosphoinositide metabolism related to cellular proliferation and differentiation. However, only few data document the nuclear production of the putative second messengers, the 3-phosphorylated phosphoinositides, by the phosphoinositide 3-kinase (PI3K). In the present paper, we examine whether GTP-binding proteins can directly modulate 3-phosphorylated phosphoinositide metabolism in membrane-free nuclei isolated from pig aorta smooth muscle cells (VSMCs). In vitro PI3K assays performed without the addition of any exogenous substrates revealed that guanosine 5′-(γ-thio)triphosphate (GTPγS) specifically stimulated the nuclear synthesis of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3), whereas guanosine 5′-(β-thio)diphosphate was ineffective. PI3K inhibitors wortmannin and LY294002 prevented GTPγS-induced PtdIns(3,4,5)P3synthesis. Moreover, pertussis toxin inhibited partially PtdIns(3,4,5)P3 accumulation, suggesting that nuclear Gi/G0 proteins are involved in the activation of PI3K. Immunoblot experiments showed the presence of Gα0 proteins in VSMC nuclei. In contrast with previous reports, immunoblots and indirect immunofluorescence failed to detect the p85α subunit of the heterodimeric PI3K within VSMC nuclei. By contrast, we have detected the presence of a 117-kDa protein immunologically related to the PI3Kγ. These results indicate the existence of a G protein-activated PI3K inside VSMC nucleus that might be involved in the contol of VSMC proliferation and in the pathogenesis of vascular proliferative disorders. vascular smooth muscle cell phosphoinositides PI 3-kinase 3-phosphorylated phosphoinositides phosphatidylinositol Ptd 4-monophosphate Ptd 3-monophosphate 5)P2, Ptd 4,5-bisphosphate 4)P2, Ptd 3,4-bisphosphate 4,5)P3, Ptd 3,4,5-trisphosphate phospholipase C polyacrylamide gel electrophoresis guanosine 5′-(γ-thio)triphosphate guanosine 5′-(β-thio)diphosphate 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one pertussis toxin phosphate-buffered saline high performance liquid chromatography Vascular smooth muscle cells (VSMCs)1 play a central role in the fibroproliferative response during the development of atherosclerosis and of restenosis after angioplasty (1Ross R. Nature. 1993; 362: 801-809Crossref PubMed Scopus (10004) Google Scholar, 2Ross R. N. Engl. J. Med. 1999; 340: 115-126Crossref PubMed Scopus (19370) Google Scholar). Recently, we have demonstrated that phosphoinositide 3-kinase (PI3K) was essential for the progression of VSMCs throughout the G1phase of the cell cycle (3Bacqueville D. Casagrande F. Perret B. Chap H. Darbon J.M. Breton-Douillon M. Biochem. Biophys. Res. Commun. 1998; 244: 630-636Crossref PubMed Scopus (40) Google Scholar), implying that a better understanding of the PI3K signaling pathway might be of pathophysiological relevance. PI3K phosphorylates the D3 position of the inositol ring in phosphoinositides (PI) to generate the putative second messengers PtdIns(3,4)P2 and PtdIns(3,4,5)P3 (4Wymann M.P. Pirola L. Biochim. Biophys. Acta. 1998; 1436: 127-150Crossref PubMed Scopus (580) Google Scholar). In addition to proliferation, the PI3K products have also been involved in cell transformation, apoptosis, vesicle trafficking, and cytoskeleton organization (5Toker A. Cantley L.C. Nature. 1997; 387: 673-676Crossref PubMed Scopus (1229) Google Scholar, 6Rameh L.E. Cantley L.C. J. Biol. Chem. 1999; 274: 8347-8350Abstract Full Text Full Text PDF PubMed Scopus (852) Google Scholar). Three distinct classes of PI3Ks have now been identified on the basis of their in vitro substrate specificity, structure, and mode of regulation (7Domin J. Waterfield M.D. FEBS Lett. 1997; 410: 91-95Crossref PubMed Scopus (209) Google Scholar, 8Vanhaesebroeck B. Leevers S.J. Panayotou G. Waterfield M.D. Trends Biochem. Sci. 1997; 22: 267-272Abstract Full Text PDF PubMed Scopus (835) Google Scholar). The most studied are class I PI3Ks, whichin vitro phosphorylate PtdIns, PtdIns(4)P, and PtdIns(4,5)P2, and display a preference for PtdIns(4,5)P2 in vivo. Class I PI3Ks form a heterodimeric complex and are subdivided according to the adaptor protein associated with the catalytic subunit. Class IA PI3Ks consist of a 110-kDa catalytic subunit (p110 α, β, δ) (9Hiles I.D. Otsu M. Volinia S. Fry M.J. Gout I. Dhand R. Panayotou G. Ruiz-Larrea F. Thompson A. Totty N.F. Hsuan J.J. Courtneidge S.A. Parker P.J. Waterfield M.D. Cell. 1992; 70: 419-429Abstract Full Text PDF PubMed Scopus (541) Google Scholar) and a 85-kDa adaptor protein (p85 α, β) (10Skolnik E.Y. Margolis B. Mohammadi M. Lowenstein E. Fischer R. Drepps A. Ullrich A. Schlessinger J. Cell. 1991; 65: 83-90Abstract Full Text PDF PubMed Scopus (439) Google Scholar) containing Src homology 2 (SH2) domains that link them to tyrosine kinase signaling. In contrast, class IB PI3K or PI3Kγ defines a G protein-coupled receptor-regulated PI3K (11Stephens L. Smrcka A. Cooke F.T. Jackson T.R. Sternweis P.C. Hawkins P.T. Cell. 1994; 77: 83-93Abstract Full Text PDF PubMed Scopus (520) Google Scholar). It is made of a p110γ catalytic subunit and a p101 regulatory subunit unrelated to p85 (12Stephens L.R. Eguinoa A. Erdjument-Bromage H. Lui M. Cooke F. Coadwell J. Smrcka A.S. Thelen M. Cadwallader K. Tempst P. Hawkins P.T. Cell. 1997; 89: 105-114Abstract Full Text Full Text PDF PubMed Scopus (494) Google Scholar). The p110γ can be activated in vitro by both the α and βγ subunits of heterotrimeric G proteins (11Stephens L. Smrcka A. Cooke F.T. Jackson T.R. Sternweis P.C. Hawkins P.T. Cell. 1994; 77: 83-93Abstract Full Text PDF PubMed Scopus (520) Google Scholar, 12Stephens L.R. Eguinoa A. Erdjument-Bromage H. Lui M. Cooke F. Coadwell J. Smrcka A.S. Thelen M. Cadwallader K. Tempst P. Hawkins P.T. Cell. 1997; 89: 105-114Abstract Full Text Full Text PDF PubMed Scopus (494) Google Scholar, 13Stoyanov B. Volinia S. Hanck T. Rubio I. Loubtchenkov M. Malek D. Stoyanova S. Vanhaesebroeck B. Dhand R. Nürnberg B. Gierschik P. Seedorf K. Hsuan J.J. Waterfield M.D. Wetzker R. Science. 1995; 269: 690-693Crossref PubMed Scopus (641) Google Scholar). This stimulation is considerably enhanced by the p101 adaptor (12Stephens L.R. Eguinoa A. Erdjument-Bromage H. Lui M. Cooke F. Coadwell J. Smrcka A.S. Thelen M. Cadwallader K. Tempst P. Hawkins P.T. Cell. 1997; 89: 105-114Abstract Full Text Full Text PDF PubMed Scopus (494) Google Scholar). Moreover, there is now considerable evidence that a nuclear PI cycle, apart from that occurring in the plasma membrane, is involved in the regulation of nuclear functions (14D'Santos C.S. Clarke J.H. Divecha N. Biochim. Biophys. Acta. 1998; 1436: 201-232Crossref PubMed Scopus (159) Google Scholar). Indeed, it has been demonstrated that nuclei contain almost all the enzymes involved in the classical PI cycle, including kinases required for the synthesis of PtdIns(4,5)P2, phospholipases C, and diacylglycerol kinase (15Payrastre B. Nievers M. Boonstra J. Breton M. Verkleij A.J. Van Bergen en Henegouwen P.M.P. J. Biol. Chem. 1992; 267: 5078-5084Abstract Full Text PDF PubMed Google Scholar, 16Martelli A.M. Gilmour R.S. Bertagnolo V. Neri L.M. Manzoli L. Cocco L. Nature. 1992; 358: 242-245Crossref PubMed Scopus (309) Google Scholar). 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Biol. 1994; 40: 619-626PubMed Google Scholar, 22Zini N. Ognibene A. Bavelloni A. Santi S. Sabatelli P. Baldini N. Scotlandi K. Serra M. Maraldi N.M. Histochem. Cell Biol. 1996; 106: 457-464Crossref PubMed Scopus (38) Google Scholar) and the growth factor-dependent nuclear translocation of the p110β catalytic subunit in osteoblast-like cells (23Martelli A.M. Borgatti P. Bortul R. Manfredini M. Massari L. Capitani S. Neri L.M. J. Bone Miner. Res. 2000; 15: 1716-1730Crossref PubMed Scopus (41) Google Scholar), suggesting that class IA PI3Ks exist in the nucleus. Recently, a study based on the immunolocalization of epitope-tagged p110γ in HepG2 cells reported that PI3Kγ translocates to the nucleus after serum stimulation (24Metjian A. Roll R.L. Ma A.D. Abrams C.S. J. Biol. Chem. 1999; 274: 27943-27947Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Since a nuclear G protein-regulated PI3K activity has not yet been demonstrated, we investigated whether GTP-binding proteins directly modulate 3-phosphorylated phosphoinositide (3-PI) metabolism in membrane-free nuclei isolated from pig aorta VSMCs. Our data provide the first evidence for the existence of a pertussis toxin (PTX)-sensitive PI3K inside VSMC nucleus. This enzyme, which might be related to PI3Kγ, phosphorylates an intranuclear pool of PtdIns(4,5)P2, suggesting that nuclear PtdIns(3,4,5)P3 may regulate protein kinases involved in vascular proliferative disorders. All culture reagents were obtained from Life Technologies Inc. U73122, wortmannin, and LY294002 were obtained from Biomol (Plymouth Meeting, PA). GTPγS, GDPβS, and RNase-free DNase I were from Roche Molecular Biochemicals. [γ-32P]ATP (3000 Ci/mmol) was purchased from PerkinElmer Life and the enhanced were purchased from Inc. was by S. the of the of the and G or the of the α subunit of were obtained and B. N. J. R. J. V. Mol. 1992; Google Scholar). and from Inc. and Inc. was from and and all reagents were obtained from cells human p110γ or were a of A. and P. VSMCs were from pig aorta the R. J. Cell Biol. PubMed Scopus Google Scholar) and in containing M. E. E. J. Cell Res. PubMed Scopus Google Scholar). all the VSMCs were from the to the VSMCs were with and and with a containing All were was to with and for and VSMCs were by the addition of and were to for and were by a The cell was a and was for in with a in a to nuclei nuclei were the of and with the containing serum 2 2 and to the of the nuclear an of nuclei were obtained from cells and nuclei and of were by for an cell and for with nuclei RNase-free DNase I. All assays were in a of of containing and C experiments in the presence of PI3K inhibitors and a Gi/G0 nuclei were with wortmannin or and pertussis for on or was for The assays were by the addition of containing of and the was for exogenous of containing and were the were by the addition of of of 2 and of were after the of and J. Biochem. 37: PubMed Scopus Google Scholar). were on chromatography in or by on a after Breton M. G. P. M. Chap H. Biochem. J. 269: PubMed Scopus Google Scholar). The synthesis of and PtdIns(3,4,5)P3 was performed from specific in B. D. M. G. Breton M. Chap H. FEBS Lett. 1994; PubMed Scopus Google Scholar). from cells or nuclei were in electrophoresis for with cells RNase-free DNase for by and polyacrylamide and B. D. M. G. Breton M. Chap H. FEBS Lett. 1994; PubMed Scopus Google Scholar). was the for and and and and were of and proteins were with reagents according to the VSMCs were on stimulated for with and in for were and the was for in a of 2 in were with and were for in containing serum and were with the with in and a to a The nuclei were in in in in and in were on with and and in a nuclei from pig aorta VSMCs were isolated by with The of nuclear was by biochemical and and for and plasma to be and of the activity in the Furthermore, showed the of to the proteins in the nuclei In the nuclear was with a protein of nuclear complex that the nuclei of high of the nuclei was during the the was and the the nuclear to the and nuclear C, studies the existence of a nuclear PI3K pathway (20Lu P.J. Hsu A, L. Wang D, S. Yan H.Y. Yin H.L. Chen C.S. Biochemistry. 1998; 37: 5738-5745Crossref PubMed Scopus (73) Google Scholar, 21Neri L.M. Milani D. Bertolaso L. Stroscio M. Bertagnolo V. Capitani S. Cell. Mol. Biol. 1994; 40: 619-626PubMed Google Scholar, 22Zini N. Ognibene A. Bavelloni A. Santi S. Sabatelli P. Baldini N. Scotlandi K. Serra M. Maraldi N.M. Histochem. Cell Biol. 1996; 106: 457-464Crossref PubMed Scopus (38) Google Scholar, A.M. Borgatti P. Bortul R. Manfredini M. Massari L. Capitani S. Neri L.M. J. Bone Miner. Res. 2000; 15: 1716-1730Crossref PubMed Scopus (41) Google Scholar, A. Roll R.L. Ma A.D. Abrams C.S. J. Biol. Chem. 1999; 274: 27943-27947Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar), the intranuclear and regulation of a PI3K activity has not been we first investigated whether membrane-free nuclei were to from nuclear PI3K activity in vitro by from [γ-32P]ATP inositol All assays were in the presence of 2 and a phospholipase C that nuclear activity not with PI3K in 2 analyses revealed a of suggesting a we detected To for the presence of a G protein-regulated PI3K (11Stephens L. Smrcka A. Cooke F.T. Jackson T.R. Sternweis P.C. Hawkins P.T. 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Science. 1995; 269: 690-693Crossref PubMed Scopus (641) Google Scholar), we whether GTPγS, a the production of in isolated nuclei we detected the synthesis of only PI3K the and the was nuclei This with the of a 2 In the was and we the production of any inositol and stimulation of PtdIns(3,4,5)P3 was with not Moreover, was to PtdIns(3,4,5)P3 in 2 These results were by the specific activation of only PtdIns(3,4,5)P3 synthesis by in isolated nuclei and production of in the of and in the PI3K activity was to be These data that a nuclear PI3K phosphorylates a intranuclear pool of PtdIns(4,5)P2 to investigated the of specific PI3K inhibitors on GTPγS-induced nuclear PI3K and studies showed that of PtdIns(3,4,5)P3 was by after of nuclei with or that the nuclear PI3K is to PI3K inhibitors in a classical of of PI3K inhibitors on GTPγS-induced nuclear nuclei were and nuclear kinase activity was in were for with wortmannin or LY294002 with A, were and by of synthesis is nuclei. for are the from for PI3K inhibitors the of were and by The data are of The position of is the The data that the PI3K is for nuclear PtdIns(4,5)P2 or that and present in the nuclear were during nuclei To containing were to nuclear PI3K and the of from was by of nuclei with exogenous in the of in the synthesis of (15Payrastre B. Nievers M. Boonstra J. Breton M. Verkleij A.J. Van Bergen en Henegouwen P.M.P. J. Biol. Chem. 1992; 267: 5078-5084Abstract Full Text PDF PubMed Google Scholar), and also of not of any A, and This that membrane-free VSMC nuclei also contain a PI3K in vitro exogenous to to phosphorylate the intranuclear pool of In the presence of GTPγS, synthesis was by and that we also PtdIns(3,4,5)P3 synthesis A, These results indicate that the nuclear PI3K can phosphorylate both nuclear PtdIns(4,5)P2 and exogenous performed experiments to the PI3K in isolated nuclei and The existence of a nuclear PI3K that phosphorylates both and PtdIns(4,5)P2 in a that it be a class IB PI3K. Indeed, we detected the presence of the catalytic subunit of the G protein-regulated PI3K by The with a the of human p110γ revealed a 117-kDa protein in both the cell A, and the nuclear A, in in of nuclei with human p110γ in cells also showed a A, Moreover, the p110γ was the not with human in and was detected in VSMC nuclei the was and The also the presence of a 117-kDa protein in VSMC nuclei in HepG2 cells (24Metjian A. Roll R.L. Ma A.D. Abrams C.S. J. Biol. Chem. 1999; 274: 27943-27947Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar), all were to detect PI3Kγ by immunofluorescence in of p85α in VSMCs. A, of of proteins from cells and nuclei were on and with The data are from a of p85α by were on and with the of that a nuclear PI3K phosphorylates exogenous in the of and that p85α has been reported in rat nuclei (20Lu P.J. Hsu A, L. Wang D, S. Yan H.Y. Yin H.L. Chen C.S. Biochemistry. 1998; 37: 5738-5745Crossref PubMed Scopus (73) Google Scholar), we whether class IA PI3Ks also be present in VSMC nuclei an to detect adaptor protein and the of proteins from cell or from nuclei In contrast to previous reports, p85α was in VSMC nuclei with cell nuclei were The of nuclear p85α was by indirect immunofluorescence in the p85α was a ring the and in the and the plasma membrane, whereas the nuclear These results that the heterodimeric PI3K is from nuclear be present in the nuclear whereas a 117-kDa PI3K kinase inside VSMC nuclei. to whether nuclear heterotrimeric Gi/G0 proteins be for the nuclear PI3K activation by of isolated nuclei with inhibited of the GTPγS-induced PI3K activity Moreover, to G proteins inside VSMC we performed immunoblots specific both subunits or the α subunit of in a protein of is by both in nuclear These results that G proteins are present in VSMC nuclei and be involved in nuclear PI3K In the present we provide the first evidence that a PI3K the second PtdIns(3,4,5)P3 from a nuclear pool of PtdIns(4,5)P2, directly within VSMC nucleus. Furthermore, we showed that PI3K which be related to a PI3Kγ, is to nuclear G VSMC nuclei of their nuclear Our data demonstrated that the PI3K and the PtdIns(4,5)P2 with suggesting that and substrate are associated with nuclear In with PtdIns(4,5)P2 was in in rat G. N. E. R. A. Ognibene A. Santi S. A. A.M. Maraldi N.M. J. Histochem. 1995; PubMed Scopus Google Scholar), and assays have revealed that of nuclear PtdIns(4,5)P2 of in rat nuclei with L.R. Irvine R.F. Divecha N. Biochem. J. 1997; PubMed Scopus Google Scholar). In data from M. Mol. Biol. Cell. 1998; PubMed Scopus Google Scholar) provide the of PtdIns(4,5)P2 within the nucleus. demonstrated that PtdIns(4,5)P2 is to in nuclear are from and contain and their is to the of These that might for in nuclei. 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