Scribd Logo
Upload

Welcome to Scribd!

  • Burgering 1995

    Uploaded by

    Hector Javier Burgos
    5 views4 pages

    Original Title

    burgering1995

    Copyright

    © © All Rights Reserved

    Available Formats

    PDF or read online from Scribd

    Did you find this document useful?

    Is this content inappropriate?

    Report this Document

    Copyright:

    © All Rights Reserved
    Download as PDF or read online from Scribd
    Flag for inappropriate content
    5 views4 pages

    Burgering 1995

    Uploaded by

    Hector Javier Burgos

    Copyright:

    © All Rights Reserved
    Download as PDF or read online from Scribd
    Flag for inappropriate content
    of 4
    LETTERS TO NATURE by proteolysis”. However, genes other than Jee may also mediate the effets of IRF-1 on apoptosis. “The possible involvement of IRF-I as a tumour suppressor had previously been reported in the context of the development ‘of myelodysplastic syndrome (MDS) and leukaemia’, In view of the results reported here, it may be interesting to investigate the susceptibility of JRFJ-’~ mice to. radiation-induced tumours, as well as the status of the FRF-T gene in other, particu larly Iymphocyte-derived, forms of human neoplasms.) 2 tot Seth an 8 ae a an HS ar ot ature 362, 849-052 1963, 5: Mmm a at 4209-519 (20 6 frac wat Scene a, 971-978 0093) 9: Torna et alot 77, 829-290 A000, 4 Cone 6 Fea Saas aay 37-100 102 5 Imomtey, AA 9 Nau 968, 768-774 (952, Yr hy SE Bo WC 82.8 sp tee et ee ce ean 114 Harada, 6 at Oneagene 8, 3313-3320 (1994) IESE Ar ee com waouon Peo eee 20. Kumar Vet ak EMBO 2251-7236 (1986) Fe A mena ceacamn See ree eee ho 2 EEE nm sn age 18st, oe She chert ie oe Sethi ara hanan for eaee, Te worn spp nat by ee gare ‘be nan Sty tr the Mansion of teree andthe Reseach vconrent Caran Protein kinase B (c-Akt) in phosphatidylinositol-3-OH kinase signal transduction Boudewijn M. Th. Burgering* & Paul J. Cotfert * Laboratory for Physiological Chemistry, Utrecht University, Universitetsweg 100, 3584 CG Utrecht, The Netherlands + Hubrecht Laboratory, Uppsalalaan 8, 3884 CT Utrecht, ‘The Netherlands A serine/threonine kinase, named protein kinase B (PKB)! f Sequence homology to both protein kinase A and C, has previously ‘een isolated. PKB, which is identical to the kinase Rac’, was later found to be the cellular homologue of the transforming v-Akt’. Here we show that PKB is activated by stimuli such as insulin, platelet-derived growth factor (PDGF), epidermal growth factor (EGF) and basic fibroblast growth factor (BEGF). Activa- tion of PKB was inhibited by the phosphatidlinositol-3-OH kinase (PIG)K) inhibitor wortmannin and by coexpression ofa dominant- negative mutant of PIG)K. PDGF receptor mutants that lack ce PKB a y Phosphorylation of PKB on serine. Finally, we show that a constructed Gag-PKB. fusion protein, homologous tothe -akr oncogene, displays signit- cantly increased ligand-independent kinase activity. Furthermore, this activity is sufficient to activate the p70 Sé-kinase (p70). These results suggest a role for PKB in PI(3)K-mediated signal transduction. Rat-l fibroblasts or insulin receptor overexpressing NIH3T3 cells (A14), transiently expressing an epitope-tagged version of PKB (HA-PKB), were treated with diferent stimuli, and HA- PKB was isolated. Increased HA-PKB kinase activity was cobsorved after treatment with PDGE, EGE, insulin and BFGF (Fig. 1a). Activation of a G-protcin-coupled receptor by Iyso- phosphatidic acid (LPA) of activation of PKC by 12-O-tetra- eeanoylphorbol 13-acetate (TPA) didnot enhance HA-PKB, activity in these cells. Activation of HIA-PKB by PDGF occurred at a half-maximal dose of 2ng ml ' and was rapid (maximal activation within 2-5 min) but transient (return to basal within 30-40 min). ‘We analysed the involvement of the p2I"* (Ras) signalling pathway in PDGF-indueed PKB activation. HA-PKB was coex- NATURE - VOL 376 - 17 AUGUST 1995 pressed with either the Ras dominant-negative mutant Ras or the raf-1 dominant-negative mutant NAraf. Expression of Ras‘""” and NAraf inhibited activation of HA-MAPK, as expected** (Fig. 16), but litte or no inhibition of PDGF- induced HA-PKB activation was observed. Also Ras" expres- sion did not inhibit insulin- and EGF-induced HA-PKB activity in AI cells (not shown). Activation of Ras and/or Raf-l is apparently not essential to PDGF-induced PKB activation. We then analysed whether PKB activation might be part of a PI(3)K-dependent signalling pathway. First, we tested the effect of the PI(3)K inhibitor wortmannin’ on HA-PKB activa- tion (Fig. 22). Pretreatment with wortmannin inbibited HA- PKB activation by PDGF, with only a small effect on HA- MAPK activation at higher doses of wortmannin. Both the 50% inhibitory concentration (ICs) for PKB inhibition and PI(3)K activity present in antiphosphotyrosine immunoprecipitates was approximately 10nM (not shown). Second, we analysed NMuMG cells expressing the PDGF receptor (Nm), cells expressing a kinase-insert domain deletion mutant (AKi), no longer binding NCK, Grb2, pl20GAP and PIG3)K, and cells expressing a mutant defective in p120GAP binding (SW)". Treat- ment of cells with PDGF or EGF results in Ras activation and activation of MAP kinase (see refs 6, 9; and Fig. 25). PDGF failed to activate HA-PKB in the AKi mutant (Fig. 2h), owing to the mutant PDGF receptor expressed, because EGF activated HA-PKB normally in this cell line (Fig. 2b). To investigate in more detail the possible involvement of PI(3)K in PKB activa- tion we further analysed PKB activation in PAE cells expressing different PDGF receptor point mutants. These included the single PDGF receptor point mutations Y740F and Y7SIF, as well as the double-mutant Y740F /Y7S1F". These mutants show reduced ability to stimulate PI(3)K activity. The different cell lines were transfected, and PDGF -indueed PI(3)K and HA-PKB activity were compared (Fig. 26). From this we conclude that activation of HA-PKB fully correlates with the ability of these receptors to activate PI(3)K. Finally, we investigated whether HA-PKB activation could be blocked by coexpression of a dele- tion mutant of the p85 subunit of PI(3)K (Ap85), which was previously shown to inhibit receptor-induced PI(3)K activity" Coexpression of Ap85 inhibited PDGF-induced HA-PKB acti vation, but had no effect on PDGF-induced HA-MAPK activa: tion (Fig. 2c), Taken together, these data demonstrate that I(3)K activation is essential for PKB activation. To study the mechanism of PKB activation we analysed whether PKB was regulated by phosphorylation, HA-PKB 599 LETTERS TO NATURE FIG. 1. PKB is activated by receptor tyrosine kinases, and activation is not inhibited by Ras"™” or NArat coexpression. a, HA-PKB activity in transfected Rat-1 cells following stimulation with ciferent growth stimull. HAPKB activity was analysed from extracts of untreated Rat for AL4 cells (NH3T3 overexpressing the human insulin receptor), and Ratt or A14 cells treated withthe indicated stimuli (20 ng mi * POGF: 201g mi * EGF; 100 nM TPA: 20.ng mi-* BFGF: 1 uM LPA: 100 9M insulin (ins), Before stimulation, cell cultures were cotransfected with HA-PKE (2 ig per Gish) inthe presence of 3 yg ofthe vector plasmic. Al indicated stimull activate MAP kinase in these ces", so lack of ‘activation is not due to a lack of responsiveness of Rat-1 or AL cells to these stimuli 6, HA-PKB activity or HAMAP-kinase activity was ana- lysed from extracts of untreated Rat-1 cells or POGF (20 ngmi *) treated Ratt colls. Before teatment Ratt cells were cotransfected with HA- PKB (2 pg) In the presence of Ras” or NAvaf METHODS. & fulblengtn protein kinase B cDNA wos isolated from 3 ‘bovine CDNA library a8 previously described". A 2.4 EcoRI fragment containing the complete PKB cDNA was cloned into the mammalian ‘expression vector pSGS (ret. 17) generating the vector pSGS PKB. PKB ‘was Nerminally tagged witha Qamino-acld peptide (HAL) from Influenza vius haemagglutinn'® generating the plasmid pSG5-PKB™. The N-terminal tag Is recognized by the monoclonal antibody 12CA5. The use of HA-MAP kinase, Ras"™"” and NAraf have been described previously’. Approximately 2 10” Rat-t cells were seeded per 5 cm Gish in DMEM supplemented with 10% FCS and transfected using the (Ca(PO,.2 technique. For PKB activity determination, cells were washed twice In ice-cold PBS and HA-PKB was Isolated as previously described for HA-MAP kinase®. After the final wash the precipitate was incubated In 25 pl kinase butter: 50 mM Tris, pH 7.5; 10 mM MgCis; 1 mM OTT; jIM PK; 50M ‘ATP: 10 yg myelin basic protein (MBP) and 3 pCi p-yATP. Kinase reaction was for 25 min at room temperature with continuous shaking. Reaction was stopped by the adaition of 7 yl of immunoprecipitated from transfected, "P-orthophosphate- labelled cells appeared in two phosphorylated forms. Phos- phorylation of the faster migrating form was constitutive and not induced by PDGF, but phosphorylation of the slower migrating form of PKB was induced by PDGF and inhibited by pretreatment with wortmannin (Fig. 3a). Phosphoamino-acid analysis showed that phosphorylation was mainly on serine (Fig. 36), This phosphorylation appeared essential for PKB kinase > FF oF Phy F gh |+ve° atl oe Fah ae |< ars > 44 8 NaF 224 snl? te ek ter ? ---ee-” PKB NAPE | ser 5x Laemmli sample buffer. The reaction was separated by electro: Dhoresis on 15% SDS-PAGE. MBP phosphonjation was detected by ‘autoradiography. Determination of HA-MAP kinase activity has been escrbed previously activity, because treatment of active PKB immune complexes ‘with phosphatase abolished the ability of PKB to phosphorylate substrate. Furthermore, after phosphatase treatment all PKB ‘was present as the faster migrating form (Fig. 30) ‘As with PKB, activation of p70“* by PDGF is inhibited by wortmannin and the Ap85 mutant'® (Fig. 2c). In addition, the various receptor mutants (AKi: Y740F/Y7S1F and Y7S1F) demonstrate reduced activation of PI(3)K, PKB and p70". In FIG. 2 1(3)K activity is essential for PKB activa tion. 2, Inibition of PDGF induced HA-PAB activ- @ PGR" __ Ki sw ation “by wortmannin, HAPKB actiy was + = 200 100 $0 wortmannin (OM) ec Trea tar poor ae ‘analysed from extracts of transfected Rat-1 cells ss 5 POG Dretreated with the indicated concentration of —_so- PKB eee ee” ‘wortmannin and subsequently stimulated with POGF. b, HA-PKB and HA-MAP kinase activity was measured in transfected NMUMG or PAE cells PKB MAPK = ve = s6 740/751 YAOF Y7SiF Poser" aki _SW fost post vost 20 PIOK er expressing diferent PDGF-receptor (POGFR) ‘mutants. Cells were stimulated before extraction with the indicated growth factors, In the case of the PAE cells expressing the various POGF recep- tor mutants, PU)K actwity present in antipnos- phoiyrosine immunopreciptates was measured In parallel and is expressed as fold induction {thie panel. , Inibition of POGF induced HA. PKB and HAp70™, but not HAMAP kinase, ‘activation by ApBS. The POGF:induced activities Of HAPKB, HILMAPK and HA-p70™™ were measured in the presence or absence of 8 yg ‘A085 plasmid METHODS. Wortmannin pretreatment was for £10 min witn the amounts indicated. POGF teat ‘ment, cell ysis, mmunopreciitation and kinase assay were as Fig. 1. Cultunng and character 'stcs of PDGF signaling of the various NMUMG and PAE, cell lines have been described previously***. Determination of $6kinase activ ty has been described previously". Phosphor ted $6 protein was separated by electrophoresis on 125% SOS-PAGE and detected by ‘autoradiography. NATURE - VOL 376 » 17 AUGUST 1995 = POGF LETTERS TO NATURE » “8 «+ s tp as pose ea eax. 8 J [a ares © eee Sati roe se sox [a= FIG. 3 in vio phosphoryaton of HA-PKS. a, POGF-induced phosphor ation of PKB and the effect of wortmannin were analysed by immuno: precipitation of in vivo *P-labelled HA-PKB from transfected Rat-1 cells ‘reated with the Ingicated stimul. After separation af proteins by poly ‘acrylamide gel electrophoresis and blotting of proteins 10 poywinylia- ‘enefuaride (PVOF) membrane, labelled HAPKB was visualized by ‘autoradiography (top). The position of HA-PKB was determined by immunoblotting (bottom). b, The phospho-amino acid (PAA) content of PKB was determined in parallel experiments in which Plabelled HA. PKB was excised fram the PVDF membrane fllowed by acid hydrolysis ‘and twodimensional chromatography. The position of the phospho: serine, phosphothreonine and phosphotyrosine standards are indica- ted. The autoradiogragh shown is the PAA analysis of the upper phosphonyated band from POGF stimulated cells. PAA analysis results (of all other bands were essentially identical to the one shown here. ‘The effect of treatment wit caf intestinal phosphatase (CIP) on HA-PKE _acthity and get-shift was measured by incubating mmunopeeciptates of ‘active HA.PKB with either CIP oF heatinactvated CIP (BCI), followed by an in vitro kinase assay or analysis on SDS-PAGE. METHODS. Transfected Rat-i cells were labelled overnight wth 1. mCi P.orthophosphate per ml medium. Following stimulation with POGF (20ng mi’) for 7 min either with or without wortmannin (50 nM) pre ‘reatment (10 min), cells were lysed and HA-PKE was precipitated as in Fig 4. After separation of proteins by SOS-PAGE on 8% polyacryiam. ide ges, proteins were transferred to PVDF membranes. The position Of labelled proteins was frst determined by autoradiography and in parallel by incubation of the transferred protein with polyclonal ant KB serum and detection with HRP-conjugated secondary antibody and fenhanced chemiluminescence (Amersham). PAA analysis was. per formed according to standard procedures". In c, active HA-PKE was, immunapreciptates from PDGF treated HA-PKB transfected Rat cals Precintates were incubated with ether 5 units of CIP for 5 min at 37 -C fr the same amount of heatinactvated CIP. After CIP incubation, ‘samples were washed extensively and assayed for kinase activity @s in Fig 1. Paralet samples were analysed by gel electrophoresis. FIG. 4 PKB activates p70" at a site upstream of rapamycin inhibition. 2, Gag: PRB activity was analysed from extracts of untreated Rat-1 cells land compared with unstimulated and POGF-stimulated HA-PKB activity Exoression of Gag-PKB and PKB was similar as determined by western blotting of total extracts with ant:PKB serum, Comparison of total pro- tein extracts and Immunogreciptated protein showed that HAPKB ang Gag-PKB were preciitated with equal eciency (not shown). HA-p70™ land HAMAP kinase activity were analysed from extracts of untreated Ratt cells or cells treated with PDGF. This was compared with activity present in extracts of Rat-1 cells that before analysis were cotransfected Wwith either pSG5-GagPKB or pSG5-Gag as a contol. b, The effect of the immunosuppressant rapamycin on HA‘p7O™" activity and HA‘PKB was compared by measuring the actviy of untreatee, POGF treated or Gag-PKB cotransfected cells either with or without pretreatment with rapamycin METHODS. The plasmid pSG5-PKEGag was constructed by ligating an '800-bp MoMuLV cDNA fragment encoding the p30 capsid protein’ in frame with the initiation codon of PKB utilizing an engineered Neo! site. This was subsequently cloned into pSGS and, upon transfection of mammalian cells, generates a fusion protein of M.~B5K as detected by western bloting of total protein extracts with both ant-PKB and ant ag antibodies (not shown). Actwity measurements using MBP as. substrate were performed on immunoprecipitated protein as in Fig. 1. ‘The Gag-PKE fusion protein was immunopreciptated using a MoMuLV- Gag speciic antiserum. ina, HA‘p70"™ or HA-MAP kinase were catans: fected with 3 ug of ether pSGB, pSG5.GagPKB or pSG5.Gag plasmic. In b, Ratd cells wansfected with HA-PKB or HA-p70™ were pretreated with 25 ng mI-® rapamycin for 10 min followed by POGF (20 ng mi") tweatment for 10 min. n the case of Gag-PKB cotransfection, rapamycin (25 ng mi") treatment was also for 10 min. NATURE + VOL 376 » 17 AUGUST 1995 a oe ene eee eee |=se me a se | | 270 ‘anti p70/85 5 ware b cgamgen ee — 56 PK 55x st orks” = fas se 01, LETTERS TO NATURE contrast the Y740F mutant, which also displays reduced PI(3)K and PKB activation, activates p70" normally, indicating that, in this cell-type, Y740F activates a PI(3)K-independent pathway to activate p70 (ref, 12), To investigate whether PKB mediates PIG3)K-

    You might also like