Chinese Journal of Biochemistry and Molecular Biology. p70 S6 B (PKB) rapamycin

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1 ISSN CN ΠQ Chinese Journal of Biochemistry and Molecular Biology 19 (2) : p70 S6,,,,,,, 3,, (, ) (PMA), Western, mtor(mammalian target of rapamycin) rapamycin 23 (PI3K) LY PMA (CHL) p70 S6 (p70 S6K) B (PKB),PMA p70 S6K rapamycin PMA p70 S6K, PMA mtor2 p70 S6K, PKB, PMA PKB LY p70 S6K PKB, PMA p70 S6K PMA p70 S6K, p70 S6K PI3K, PMA p70 S6K PI3K p70 S6, B,mTOR, 23, Q591 2,Q253 P70 S6 Kinase Activated by Insulin and PMA Through Distinct Pathways in Protein Synthesis ZHAO Yong2mei, FU Wei, SONG Yu2tong, WANG Ya2jie, LIU Yi, CHEN Fei, ZONG Zhi2hong, YU Ai2ming, HAN Ya2ling, YU Bing2zhi 3 ( Department of Biochemistry, School of Basic Medical Science, China Medical University, Shenyang , China) Abstract Stimulation of serum2starved Chinese hamster lung (CHL) cells with either phorbol 122myristate 132acetate ( PMA) or insulin resulted in the activation of p70 S6 kinase All these effects were blocked by rapamycin, a specific inhibitor of mtor (mammalian target of rapamycin), confirming that mtor activation played an essential role in the activation of p70 S6 kinase stimulated by insulin and PMA Protein kinase B (PKB) was activated by insulin but not by PMA L Y294002, a specific inhibitor of phosphatidylinositol23 kinase ( PI3 K), could block the activation of p70 S6 kinase and PKB stimulated by insulin The activation of p70 S6 kinase stimulated by PMA was unaffected by LY These data suggested that p70 S6 kinase was activated by insulin and PMA through different pathways PMA could induce the activation of p70 S6 kinase independently without PI3 K and PKB, whereas insulin stimulated the activation of p70 S6 kinase through a mechanism that required PI3 K and might involve PKB : , : (No ) (No ) 3 Tel : , Fax : ,E2mail : com,, , ( ),, Received :July 30,2002 ;Accepted :October 18,2002 Supported by National Key Program of Basic Research 973( G ) and Key National Natural Science foundation of China ( ) 3 Corresponding author Tel : (024) , Fax : (024) , E2mail : com

2 Key words p70 S6 kinase,protein kinase B,mammalian target of rapamycin, phosphatidylinositol23 kinase, protein synthesis [1 ], 100 nmolπl rapamycin 45 min 50, molπl LY min, 100,mTOR (mammalian target of rapamycin) nmolπl 100 gπl PMA, 45 ( phosphoinositide kinase2 related kinases,pikks) / l [2 ] p70 S6 (p70 S6K) 4E 1 (4E2BP1) Na 3 VO 4,25 mmolπl NaF,25 mmolπl 2glycerophosphate,,mTOR 2 mmolπl EDTA, 2 mmolπl EGTA, 1 mmolπl DTT, 1 [3 ],mtor p70 S6K 23 (PI3K) B (PKB) PKB PI3K, PI3K Western ( 20 g) [4 ], PI3K mtor (phorbol 122myristate 132 [5 ] acetate,pma) PKC,, p70 S6K (1 600) PKB (1 400) PI3K mtor 2 h, HRP (1 LY PI3K, 5000) 1 h,ecl [ 6] rapamycin mtor, p70 S6K 200 l ( 200 rapamycin LY PMA g ) p70 S6K 4 l, 4 2 h, ( Chinese hamster A2 20 l,4 1 h lung cells, CHL), p70 S6K PKB, 3, S6 ( 50 PMA mmolπl MOPS,pH 712, 10 mmolπl MgCl 2, 10 mmolπl PI3K mtor, paranitrophosphate,1 mmolπl DTT) 1 S6 100 molπl ATP,20 Ci/ ml [ 2 32 P]ATP,5 molπl p70 S6K ( : RRRLSSLRA) p70 1 S6K 40 l, min, 25 l Whatman P CHL 10 % min p70 S6K DMEM, ml, 70 % DMEM,18 h 10 % DMEM, 100 nmolπl 211 p70 S6 K 100 gπl PMA, 45 min 30 min min 30 min A( 20 mmolπl Tris2HCl,pH 715,011 mmolπl mmolπl PMSF,2 gπml aprotinin,2 gπml leupeptin),,4, g 30 min, 10 %SDS2PAGE,, 5 %BSA TTBS 4, DMEM A2 (1 cm 2 cm),75 mmolπl 3, GIBCO ; PMA PKB ( 2B) 2 min, 10 ml Sigma ; rapamycin LY294002, Beckman cpm [ 7] Calbiochem ; p70 S6K PKB PKB HRP IgG HRP IgG ECL p70 S6K Santa Cruz ;[ 2 32 P]2ATP 40 l ( 50 mmolπl Tris2HCl,pH714,5 molπl A (protein kinase A inhibitor),100 molπl EGTA, 10 mmolπl Mg (Ac) 2, 1 molπl microcystin, molπl ATP,20 Ci/ ml [ 2 32 P]ATP,30 molπl H 2 B ) 2 CHL 18 h,

3 2 : p70 S6 231 mtor rapamycin PI3K 213 PMA PKB LY CHL,, p70 S6K PI3K LY CHL,, p70 S6K PMA,, 2189 rapamycin LY294002, p70 S6K LY294002, PKB, rapamycin LY294002, PI3K PKB p70 S6K, rapamycin LY p70 S6K,,LY PKB, 57 % 65 %( Fig 1) PKB PI3K,LY PKB, 3140,PMA PKB 69 %( Fig 3) Fig 1 Activation of p70 S6K stimulated by insulin was assessed by in vitro kinase assay control RAP : rapamycin ; LY: LY ; Ins : insulin These results are representative of three separate experiments Fig 3 Activation of PKB stimulated by insulin and PMA was assessed by in vitro kinase assay control LY: LY ; Ins : insulin These results are representative of three separate experiments 212 PMA p70 S6 K 3134 rapamycin, PMA, AP1 B p70 S6K, rapamycin PMA p70 S6K,LY CHL, p70 S6K PKB p70 S6K, PI3K, PMA p70 S6K, PMA p70 S6K ( Fig 2) 214 PMA p70 S6 K PKB PMA p70 S6K,, PMA,, PMA rapamycin PMA p70 S6K ( Fig 4), PKB, PMA PKB ( Fig 5) Fig 2 Activation of p70 S6K stimulated by PMA was assessed by in vitro kinase assay control RAP : rapamycin ; LY: LY These results are representative of three separate experiments Fig 4 Western blot analysis of p70 S6K using antibody against p70 S6K 1 :Control ; 2 : Control + rapamycin ; 3 : Control + LY ; 4 : Insulin ; 5 : Insulin + rapamycin ; 6 : Insulin + LY ; 7 : PMA ; 8 :PMA + rapamycin ; 9 :PMA + LY294002

4 Fig 5 Western blot analysis of PKB using antibody against PKB 1 :Control ; 2 :Control + LY ; 3 :Insulin ; 4 :Insulin + LY ; 5 :PMA ; 6 :PMA + LY PI3K PKB 3 p70 S6K, PMA p70 S6K p70 S6K 40 S S6, PI3K PKB S6 5 TOP ( 5 terminal [8 oligopyrimidine tract) mrnas ] 5 TOP, PKB Weng Qing2 mrnas mrnas 20 %, ping (10 nmolπl) CHO, [12, p70 S6K ] (rp) polya PMA,,, AP1 B [13,14 ], [9 ] PMA p70 S6K PKB,,p70 S6K, PMA p70 S6K rapamycin, PKB,, FK506 p70 S6K PKB (FKBP12),rapamycin2FKBP12 mtor ( mammalian target of PMA cpkcs (, 1, rapamycin) rapamycin mtor [15 2 ) npkcs(,,, ) ] PKC mtor C PI3K PI4K, mtor mtor (phosphoinositide kinase2related kinases,pikks) mtor /,, PKC mtor p70 S6K [10 ], Kumar PKC mtor24e2bp1 PMA p70 S6K, rapamycin,pkc mtor, 4E2 PMA p70 S6K, BP1, PMA mtor p70 PKC 4E2BP1 [16 ] Monfar S6K, PMA 22 p70 S6K mtor 4E2BP1 rapamycin2 wortmannin2 ( 4E 1) [17 ],, CHL PMA p70 S6K 4E2BP1 ( ) 4E2BP1 mtor npkc cpkc,, eif24e, eif2 PI3K PKB PKC p70 S6K 4E mrnas 5,, PI3K 3, PKB PI3K, PI3K ( References), mtor PKB, PKB mtor C 2446 (Thr 2446 ) 2448 (Ser 2448 ) [11 ] PI3K PKB p70 S6K, CHL,PKB, PMA PI3K LY PKB p70 S6K, LY PMA p70 S6K, PMA p70 S6K, PMA p70 S6K 1 Conlon I, Raff M Size control in animal development Cell, 1999, 96 :

5 2 : p70 S Keith C T, Schreiber S L PIK2Related Kinases : DNA repair, recombination, and cell cycle checkpoints Science, 1995, 270 : Schmelzle T, Hall M N TOR, a central controller of cell growth Cell, 2000, 103 : Proud C G, Denton R M Molecular mechanisms for the control of translation by insulin Biochem J, 1997, 328 : Flynn A, Proud CG Insulin and phorbol ester stimulate initiation factor eif24e phosphorylation by distinct pathways in Chinese hamster ovary cells overexpressing the insulin receptor Eur J Biochem, 1996, 236 : Kim J E, Chen Jie Cytoplasmic2nuclear shuttling of FKBP122 rapamycin2associated protein is involved in rapamycin2sensitive signaling and translation initiation Proc Natl Acad Sci USA, 2000, 97 : Cross D A E, Alessi D R, Cohen P, Hemmings B A Inhibition of glycogen synthesis kinase23 by insulin mediated by protein kinase B Nature, 1995,378 (6559) : Jefferies H B J, Fumagalli S, Dennis P B, Reinhard C Rapamycin suppresses 5 TOP mrna translation through inhibition of p70 s6k EMBO J, 1997, 16 : Nasmyth K Retinoblastoma protein Another role rolls in Nature, 1996, 382(6586) : Shuji Isotani, Kenta Hara, Kazuyoshi Yonezawa Immunopurified mammalian target of rapamycin phosphorylates and activates p70 S6 kinase in vitro J Biol Chem, 1999, 274 : Sekulic A, Hudson C C, Homme J L, Yin P, Otterness D M, Karnitz L M, Abraham R T A direct linkage between the phosphoinositide 32 kinase2akt signaling pathway and the mammalian target of rapamycin in mitogen2stimulated and transformed cells Cancer Res, 2000, 60(13) : Weng Qing2ping, Kozlowski M, Belham C, Zhang Ai2hua Regulation of the p70 S6 kinase by phosphorylation in vivo J Biol Chem, 1998, 273 (26) : Borner C, Hollt V, Kraus J Involvement of activator protein21 in transcriptional regulation of the human mu2opioid receptor gene J Mol Pharmacol, 2002,61 (4) : Tsakiridis T, Bergman A Actin filaments facilitate insulin activation of the Src and collagen homologousπmitogen2activated protein kinase pathway leading to DNA synthesis and c2fos expression J Biol Chem, 1998, 273(43) : Rasmussen H, Isales C M, Calle R, Throckmorton D Diacylglycerol production, Ca 2 + influx, and protein kinase C activation in sustained cellular responses Endocr Rev, 1995, 16 (5) : Kumar V, Pandey P, Sabatini D, Kumar M, Majumder P K, Kharbanda S Functional interaction between RAFT1ΠFRAPΠmTOR and protein kinase C in the regulation of cap2dependent initiation of translation EMBO J, 2000, 19 : Monfar M, Lemon K P, Grammer T C, Cheatham L, Blenis J Activation of pp70π85 S6 kinases in interleukin222responsive lymphoid cells is mediated by phosphatidylinositol 32kinase and inhibited by cyclic AMP Mol Cell Biol, 1995, 15 :