~
`Vi l cu7~9GCE
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`19 98
`1- - - -- - -- s Eo : SR006523 1
`~ -
`T~! CURRE T OPIN ION
`l N CELL
`04 / 1 5 / 9 l:s
`
`Cell regulation
`Edited by Sara A Courtneidge and Alan Hall
`
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`
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`262
`
`Mechanisms and consequences of activation of protein
`kinase B/ Akt
`Ju I ian Downward
`
`Protein kinase B (PKB) /Akt is a growth-fa ctor-regulated
`serin e/threonine kinase wh ich contains a plecks trin homology
`domain. Binding of phosphoinositide 3-0H kinase produ cts
`to th e pleckstrin homology domain res ults in trans location
`of PKB/ Aki lo th e plas ma membrane where it is act ivated
`by phos phorylation by upstream kinases including th e
`phosphoinoside-dependent kinase 1 (PDK1 ). Activated
`PKB/ Akt provides a s urvival s ignal that protects cells from
`apoptosis induced by various stresses, and also mediates a
`number of metabo li c effects of insulin.
`
`Addresses
`Imperial Can cer Research Fund, 44 Lincoln's Inn Fields, London
`WC2 A 3PX, UK: e-mail : downward@icrf.i cnet. uk
`
`Current Opinion in Cell Biology 1998, 10:262 - 267
`
`http://biom ednet.com/elecref/09 5506740 I 0002 62
`
`© Current Biology Ltd ISS N 0955-0674
`
`Abbreviations
`BAD
`Bcl- XL/Bcl-2 associated dea th factor
`GSK3
`glycogen synthase kin ase 3
`IGF
`insulin -l1ko growth factor
`MAP
`mItog en activated protein
`MEK
`MAP kinase kin ase
`NGF
`nerve growth factor
`PDGF
`platelet-derived growth factor
`PDK1
`PIP3 -dependent kinase 1
`PFK-2
`6-ph osph ofru c to-2- kina se
`PH
`pleckstrin homology
`Pl 3-K
`phosphoinos1tide 3 -0 H kinase
`P1 (3,4)P2 ph osphat1dyl1nos1t ol-3,4-b1sphosphate
`Pl P3
`phosphat1dyl1n os1tol-3,4 ,5-trisphosphate
`PKB
`protein kinase B
`
`e ffec ts ol' in sulin and ol' seve ral growth fa ctors and s ci mu Ii,
`and wi ll address rece nt progress in understand ing h o \\
`l'KB//\ kt is reg ulate d by phos phoinos itid e ."l-O H kin a~c
`anu how it pla ys a key pa rt in protectin g cel ls
`l'rorn
`apopros i~.
`
`Regulation of protein kinase B
`In aduiti on to hav ing a se rin e/threonin e kin ase do ma in,
`PKB/1\k t co ntain s a pkckstrin homol ogy (Pl I) do mai n
`at its amin o- te rmin al e nd (a min o ac ids 1- 106), whi c h
`makes up the maj or part of th e amin o- re rminal reg uL1tor,
`domain (res idu es 1- 147). Th e kina se domain stretc h e~
`from amin o ac id 148 to 411 , with the ca rb oxy- term in al
`ta il
`regio n (amin o aciu s 41 2~80) acco untin g fo r rhc
`re maind e r of the prote in ( Fi g ure I). In add itio n to thc
`first charac terised me mb e r o f rh e famil y (P KB a, 1\ ktl ,
`R/\C -l'K a), two othe r ve ry close re lati ves have now been
`id e nci fl ed - R/\C- l'K~, also te rm e d PKB~ and /\kc2 HJ,
`an d R/\ C-PKy ISi. PKl3//\ kt is ac ti vated
`in
`res pon ~e
`to
`treat ment or cell s wit h a wide va ri ety of g ro\\'th
`srim ul i, including pl atelet-de ri ve d g rowth fa cto r (PDGF ).
`e pid e rm al grow th fa cto r ( EC F ),
`ins ulin, th rombin an d
`nerve growth fa cto r (NGl7 ). Several fi ndin gs indicate th at
`the lipi d kinase phosp hoinos ici de 3-0 1 I kin ase (Pl 3-K) i,
`involve d in reg ul ati on of PK B//\ kt: g rowth-f'a ctor-in duced
`acrivation of PKB//\ kt is inhibited by th e Pl 3-K inh ibi to r
`worcma nnin 16- 8 I, by
`the express io n of a dom inan t
`negat ive fo rm or l'l 3- K 171 anu, in the case ol' P DGI ·'.
`by 111 u rat ion ol' I' [) CJ F rece pto r cyros ines 740 ,111d 7 5 l .
`whi ch bind to th e Pl 3-K regula to ry subunit l6,71. Pl 3- K
`;1crivicy is thu s require d for the reg ulati on of J> KB/t\k r
`by growt h fac tors, but it also appears to be s urti cicnc:
`ex press ion of' co nstituti ve ly ac ti va ted form s of Pl 3-1,
`n.:s ul ts in stimula tio n o f PKB//\ kt !9, 10, l l••, IZJ. As Pl 3- K
`is also stimul ated by direc t in teracti o n of th e small GTPase
`R,1s with the pl 10 ca tal yti c subun it I 13, 141, PKB/J\ kt is
`aho co ntroll e d by Ras [9, 10, 15 ]. Pl 3- K acti vity, and hence
`l'KB//\kt acti vity, has also been shown r...:centl y to be
`stimula[(;d by aulll:s io n of epithelia l ce ll s ro ex tracel lu la r
`mar ri x 116••, 17• I.
`
`Introduction
`T he se rine/rl ireo ni ne prore in kinase B (J>J,,:B )//\kt wa s
`idcn Lifi ed
`ind e prnd e nLl y
`in 199 1 by rhree difk rrnt
`g1rn 1ps. ' l\vo group s ide ntifie d Lil e kin:1~e ;1s ;1 res ult of'
`11 , homology co
`i>oLh pro Lei n kinase C (7.'\% similari ty
`to the kina se domain ol' l'KCE) ;111d pro re in kina se /\
`((>H% similariry to Lil e kina se dom:1in o r· J>J,,:J\ ), giving ri se
`to the names prore in kina se B (PKB ) 111 and l{/\C-PK
`(1e L1 Led lO ril e /\ ;1nd C kin ase,) 121. 1\ r rhe sa me rim e,
`In addi tion ro l'l 3-K-mcdiated reg ulation ol' PKl3//\kr.
`rhis kin:1,e w;1s id e ntif ie d as the pro du ct of' the oncoge ne
`another pathwa y for th e re gulati o n of PKB//\kc e xists rhat
`1·-1d ·1 of Lil e ;1c ute ly rraml'ormin g re tro virus /\K'f'8 found
`is not sensitive co Pl 3- K inhibi tors such as wo rtmannin .
`in ;1 ro de nL 'f'-cell ly111ph oma 131. The re rrov iral oncogene
`Ce ll ul ar stresses such as hea t s hoc k and hypcros mol:t rity,
`e ncode d a fu sion of th e ce llular 1\kr prote in ro rh e viral
`both of whi ch activate the p38/ l lOG I kinase cascade.
`srru ctural pro te in Ga g. The nom e nclarure
`l{ /\C -PK is
`arc ab le to stimul ate the activ ity of PKB//\kt [1 8 I. I !car
`now al'o id cd to preve nt confu sion wirh th e unrelated
`shock induces association of l'KCo with the PI I doma in
`l{lw -f;1111 il y (;TP:1,e Ra e, bur both J>KI\ and /\k t are
`of PKB//\kt; previ o usly it had been reported ch ar chc
`wi de ly use d. Thi s re vie w will s11 mm ;1rise th c ev ide nce
`kina se also associates wit h J>K CI;; I 191. The significan cc of
`Ll1;1 L I' K 1\//\ln is an i 111pon;1n t n1ed i:1 wr 01· th e phys iologica I
`PKB/1\ kt in stress signa llin g is c urrentl y unclear.
`This m aterial w ascopie<l
`atthe· NLM and m ay oe
`Suoj ect US Copyright Laws
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`NOVARTIS EXHIBIT 2041
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`

`Mechanisms and consequences of activation of protein kinasi; B/ Akt Downward
`
`263
`
`autophosphorylation rea cti o n . Th e sequences s urroundin g
`res idu es a rc very diffe rent,
`two phos phorylated
`the
`s ugges ting that two different kinases are probab ly invo lved
`in phosphorylacing th ese s ites. Ph osp horyla tion of t he two
`sites occ urs in dependently and ca uses strong activation
`of the kinase activity o f PKl3/Akt which can be reversed
`transloca(cid:173)
`by ph os ph atase treatment 17,20 1. l\tlembran e
`tion medi ted b y the PI I do main ca n be co nst itu tive ly
`mimi cked by addition of a m yri stoy iati o n sequence co
`thi s cau ses its ac ti vat io n due co co nstituti ve
`PKB/1\k t:
`p hosp horylatio n (24° 0 ,26 1.
`
`Recently, two gro ups have ind e pend e ntly re porte d the
`identification and c haracterisation of a kinase that ph os(cid:173)
`phory lates Thr308 of PKB//\kt res ultin g in its activation
`) . The activ ity of thi s kin ase, te rm ed POK l
`127 °0 ,28°0
`(P IP ,-dependent kinase I ) b y Aless i el a!.[27 ••], coward s
`i11 vitro b y P IP3 and
`PKB/Akt is strongly st imul ated
`P1(3,4) Pz (Figure 2) . Thi s po ints to a dua l role for PIP3 and
`P 1(3,4)Pz in reg u lating PKB/1\kt in the ce ll : fi rstl y, these
`Pl 3- K products bind to the PH d omai n o f PKB/Akt and
`ca use its trans location to the pla sma membran e, possibl y
`thereb y promotin g its interact io n with POK I. Seco ndl y,
`itse lf. T he
`th ey activate the kinase ac tivity o f POK I
`c ON/\ for POK I ha s now been c loned and encodes a
`ubiquitou s 556-res idu c kina se from the same fam il y as
`PKB/Akt, PKA and PK C, whi c h al so has a Pl I d o main
`near its carboxy terminus [29••) . Inte restingly, POK I is
`re lated to the Drosophila kina se DSTPK 6 1, whic h has
`been impli cated in the reg ul a ti o n of sex differentiation,
`ooge nesis and spe rm atoge nes is.
`
`/\ number o f iss ues remain to be reso lved about th e
`fun c ti o n of POK I. Do PIP1 and P l(3,4)P 2 bind to, and
`stimu late, POK I directly, or is the activatio n of POK I
`towards PK 13/Akt substrate driven ? In
`kina se act ivity
`och e r wo rd s, docs PIP.'\ o r P l(3,4)P 2 binding to PKB//\ kt
`ca use a co nformati o na l chan ge that res u lts in its actin g
`as a be tte r s ubstrate for POK I? S in ce del e ti o n o f th e
`PH domain o f POK I res ults in a 30-fo ld redu ctio n in
`its ab ility to act ivate PKB//\ kt i11 villo,
`it see m s like ly
`th at P IP.'\ a nd Pl (3,4 )P 2 d o bind to the PH d omain of
`POK I and stimulate its kina se activity independentl y
`of the s ubstrate, PKl3/Akt 129••1 . PDK I lacking a Pl I
`d o main still retains so me ca pac ity to be ac tivated coward s
`PKB/Akt b y P IP3, possib ly indi catin g an effect ste mmin g
`li pid ves ic les used
`t he
`to
`loca li sa ti on
`from PKB/A kt
`in ch is assay. H owever, deletion o f the Pl I d om ain o f
`PKl3/A kt res ulted in an en zyme that was activated i11 vi/lo
`th e
`b y POK ! in a P IP3-ind epe nd e nc manner. Poss ibl y,
`vesicle-based assay sys tem use d in these experiments does
`not adequately mimi c rhe co nditi o ns in the ce ll.
`
`Figure 1
`
`Human
`PKB a
`
`Human
`PKB~l
`
`Human
`
`rat
`
`PKBy
`
`1 - - AH ----,
`106 148
`
`PH
`
`PH
`
`PH
`
`~ .:s:::::::.
`454
`..---P-H-- ~,-,--k-in_a_s_e__._ __ J _ta-i~I j
`
`Current Opinion in Coll Biology
`
`Schematic representation of the four known PKB/Akt iso forms.
`PKB f31 and ~2 are alternatively spliced forms derived from the same
`gene. The sites of insulin-induced phosphorylation are shown. AH, Aki
`homology domain; PH, pleckstrin homology domain.
`
`The pleckstrin homology domain of PKB/Akt
`The integrity of the pl eckstrin ho m ology domain ha s
`to be esse nti al for activation of PKl3/A kt
`bee n found
`factors
`to vario us growth
`in response
`intac t ce lls
`in
`[6,201, a nd also in response to express ion of activated
`Pl 3- K [9, l 0, 11 .. , 12]. The m echan ism behind thi s ha s
`lipi ds produ ced by P l
`th e
`recentl y been e lucidated:
`3- K, phosphatidy li nos itol-3,4,5-tri sp hosp ha te (P IP3) and
`p hosp hacid yli nositol-3, 4-b isp hos phatc (P l[3,4]Pz), are able
`to bind to the PH domain of PKB//\kt with rel ativel y
`high affinity a nd s pecificity fl 1••,2 1••, 22•, 23" ]. Binding
`of PIP3, the immed iate produ c t o f P l 3-K in cells, to the
`PH d omain of PKB/Akt does not activate its kinase act ivity
`i11 vitro, although P I(3,4)P 2 is able to cause a m od es t
`in c rease in the ac tivity of PKB/Akt i11 vitro [ i J ••,22 °,23 •] .
`P robab ly the m os t impo rtant as pect o f the fun c tion of
`the P I I domain of PKl3/Akc is to med iate tran slocacion
`o f t he kinase from the cytoso l co the p las ma membran e
`fo ll o win g ac t ivat ion of P I 3-K in res ponse to treatment
`o f ce ll s wit h g rowth factor [24••] . It a ppears that thi s
`tra n s locat ion is requ ired in order to prese nt PKB/Akc co
`ups tream ac tivatin g kinases.
`
`Upstream kinases for PKB/Akt
`In res pon se to growth facto r treatment o f ce ll s, PKB/Akt
`It is still unclear wh et he r o r no t POK I is enriched 1n
`beco mes phos phorylaced at two major sites, Thr308 in
`the p las ma m e mbrane : it wa s origina ll y iso lated from
`the kina se domain and Scr47 3 in t he ca rboxy- cerminal
`brain cytoso l, so membrane locali sati o n is ne ith e r consti -
`ta il 125 ••]. As mutants of PKB/Akt, in which the kinase
`tucive nor co mpl ete. \Vilh o ut so m e form o f prefe rential
`is in ac tivated, a lso become effic ientl y ph os phory lated at
`.local isatio n o f POK! to the membran e it is un c lear wh y
`th ese sites thi s is un like ly to represent an incramo le.c ular
`rn 1.s mate ri a·1 w as CO?l e-d
`at t he NLM and may l>e•
`Su l>j e-ct US Oo?y r ight Law s
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`

`264 Cell regulation
`
`Figure 2
`
`PIP3
`
`Pl(3,4)P 2
`
`PDK 1
`~
`
`p
`
`T308
`
`Kinase
`
`Active
`PKB/Akt
`
`Kinase
`
`PH
`
`Inactive
`PKB /Akt
`
`Glycolysis
`
`G lycogen
`syn th ase I
`
`translocation
`
`Protein
`synthesis
`
`Survival/
`proliferation
`
`\
`
`A model for th e ac tivation of PKB/Akt by Pl 3-K-dependent mechanism s. Following stimu lation of growth factor receptors, Pl 3-K is activat ed,
`resulting in the producti on of PIP3 and Pl(3,4 )P2. Th e binding of th e pleckstrin homology (PH) domain of PKB/Akt to th ese ph osphoinositides
`recru its PKB/Akt to th e plasma membrane. PKB /Akt is th en ph osphorylated on Thr308 by PDK1 and on Ser473 by PDK2. Th e activity, or
`possibly th e localisat ion, of PDK1 , and perh aps PDK 2, may also be reg ulated by Pl 3-K products. Activa ted PKB/Akt phosphorylates substrates,
`re sultin g in a vari ety of biological effects, in cluding suppression of apoptosis and control of metabolism. Pl 3-K, phosphatidyl inositol 3-OH
`kin ase; S6 K, p 70 S6 kin ase ; GS K3, glycogen syn thase kinase 3; POK, PIP3-dependent kinase; PFK-2, 6-phosphofructo-2-kinase.
`
`Curren t O pinion 111 C ell Biology
`
`me mbran e bindin g 01 Pl, B/;\kc, for e xamp le by artif-ici:il
`myri stoy lat ion, should be scimulawry. Other prote in s
`co uld be invol ved in direc tin g the locali sation of J>l)K I
`i11 vitro rcconscicu cion
`whi ch wo uld be mi ss in g from
`ex pe rim e nts. Th e iss ue of ll'h c th c r l'DK I is constitu ti vel y
`acti ve or scimu l:1tcd by gro\1·ch fa cto rs is on e of the
`bi gges t unreso lved qu es ti ons: when ovcrc xprcsse d
`in
`sc rum -starved 293 ce ll s, PDK I do cs not app ea r to be
`sri mu l:1rcd by treatm e nt of ce ll s with insuli n-lik e growth
`L1 cror- l ( IG F- 1) l29••] . le 1s, howe ver, poss ible th at chi s
`acti vit y is due to the lrn sal le vels of P IP 1 in the ce ll s, :1s
`th e pl10spl10ryl:1t ion o f e ve n mc mlir:1nc -t:1J"gcccd PKB/1\k t
`is redu ced by long- term exposure of ce ll s to l' I 3- K
`inhibitors 12-J•• I.
`
`rt!. , al so appears rn be und e r the contro l of Pf 3-K as p hos (cid:173)
`phorylation of Ser473 in res pon se w insulin is se nsitivc co
`wo rtmannin l25 ••] . A direct cargc t of p38/ I IOG I, mi rngcn
`activated protc in ki na sc activated protcin (J\,IA PK J\ P)
`kina sc 2, is ab le w phosph o rylate Se r4 73 i11 vitro [25•• ],
`and ma y the rc fore be ab le to contribu te to the acti vat io n
`of PKB/1\kt. I lowcve r, MAPKAP kin ase 2 is not ge nera ll y
`activated by stimuli that act ivate PK13/;\ kt, and vi ce vcrs:1,
`so it is ve ry un li ke ly that MAPK ;\P kinase 2 is rc sponsibk
`for thc phosphorylati on o f Sc r473 o f PKl3/A kt.
`
`Consequences of PKB/ Akt activation
`T he re
`is consid e rab le
`intcrt.:st
`in
`thc substrates ol
`PKB/A kt . Th e prt.:fcrrt.:d substra tt.: sequen ct.: fo r PKB/t\k 1
`to phosphorylate in a sy ntht.:tic pept idt.: is Rx Ryz(S/T )(hv)
`where x is an y amino acid , y and z are small
`rt.:sidu t.:)
`Ph ospl10ryh1tion of PKB/1\kt at Th r.108 do t.:s not result
`in ph osph ory lat ion ar St.:r473 , suggt.:sr in g that thi s site is
`ot ht.:r than glyc in t.:, and hy is a bu lky hydroph ob ic gro ur
`fi rst dirt.:ct i11 v iv o substrate to be icknri fi cc
`[30 [. Th t.:
`phusph orylatcd by a kin :tst.: orht.:r th:1n l' DK I or l'Kl3 /;\kr.
`was glycogrn sy nt hast.: kin asc 3 (GSK3) [3 1 ]. Stirnul:1tio1
`Thi s unidrntifi e d kina st.:, rdc rrcd lO as PD1' 2 hy Alessi rt
`Th is m at e ri a t w as co pi e-d
`atth 1> NLM a nd m ay be
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`Mechanisms and consequences of activation of protein kinase B/Akt Downward
`
`265
`
`in s ulin o r IGF- 1 leads co PKB//\ kt
`of 293 ce ll s w ith
`med iated phos phory lation of GS K3 at Ser9, re sultin g in
`its
`inac ti va ti on and co nsequ e nt activation o f g lycoge n
`sy nthes is [32 ]. GSK3 is in vo lved in regulati o n of seve ral
`incrace llu lar sig nallin g pathways inc lu ding co ntrol o f the
`tra nsc ription fa c tors /\Pl an d C REB (th e cyc li c AMP
`res po nse c le m e n t bindin g prote in) , the tumour suppresso r
`ge ne product AP C, t he wingless de ve lop m enta l pat hwa y
`in Ries and d o rsove ntral pattern in g in frogs 133 ]. GSK3
`i, reg u la te d by severa l diffe re nt mec ha ni sms. In additio n
`co GS K3, an other m etabo li c regula to ry e nzyme that is
`phos phorylatcd b y PKB//\kt is 6-p hosp ho fru cco-2-k ina se
`(P l.- K-2) 134•]. Another target for PKB/Akt is th e riboso mal
`pro te in S6 kin ase, p7QS6K , a lthough in this case there 1s no
`evi dence that it is a d irect s ub strate [7]. p7QS6K is res pon(cid:173)
`,ib le fo r alterin g the pattern o f protein sy nthesis following
`mirogcnic stimul ation of ce ll s . In addition , PKB/J\kt ha s
`to stim ulate g lu cose uptake an d GLUT4
`bt: t:n found
`tra n~ loca ti o n (GLUT4 is the in s u lin -respons ive glu cose
`transpo rte r isoform . It is translocatecl from intra ce llular
`com partments to th e p lasma mambranc in response to
`in~ ulin ) 135 1, and to indu ce adipocyte differt:ntiation 136]
`in 3T 3- LI ce lls. It is li),;:el y t hat PKB//\kt is respo nsiblt:
`fo r transducing a cons ide rab le num be r of the metabolic
`cff ec ts of ins u Ii n.
`
`Rece nt reports revea l t h at PK13//\kt is invo lved in regu (cid:173)
`la t in g ce ll surv iva l. It h as bee n dem o nstrated prev iously
`tha t inhibition of Pl 3-K b locks the ab ility of s urviva l
`types fro m programmed
`fac cors co protect var io us ce ll
`ce ll dea th [37,38]. Exp ression of activated forms of Pl
`.3- K or PKB/Akt protects Cos-7 ep ith e li al ce ll s from
`apopcos is in d uced b y ul travio let irradiati o n l.39•], protects
`neu rona l ce ll s from ce ll death in duced by withd rawa l of
`th e s urviva l fa cto rs IGF -1 [40•] o r NGF 14 1 •J, and protec ts
`from death
`in duced by remova l
`ha emacopo ictie ce ll s
`of inte rl euk in -3 [42 •]. /\ct ivatecl P l .3-K and PKB//\ kt
`tu protect 1\1 DCK epithe li al
`have a lso been s how n
`ce ll, from apoptosis indu ce d by detac hm en t of adh erent
`ce ll s from their extrace llular matrix ('anoikis') I 16••1 an d
`to protect Rat-! hbrob lasts from apopcos is in duced by
`w ichdrawa l of scrum facto rs while c-111yc is bein g artihcia ll y
`ex pre ss ed [43 •,44•1 . Dominant-negati ve forms of PKl3/A kt
`we re capa ble of indu c in g apoprosis in so me of rhese
`,yscems, and ot h er pathwa ys actin g downstream of P[
`3 -K, s uc h as p7QS6K and t he Rh o-fam il y GTPasc Rae,
`we re fou nd not to be in vo lved in sig nallin g ce ll surviva l.
`T he Raf-M EK-mi toge n acc ivatc cl protein (l\llAP ) kina se
`pathway was a lso s how n no t co p romote ce ll surviva l in
`e pichc lia l cel ls [1 6••J o r in hbroblasts 139•,44 •1; in face, t he
`.\ '1/\ P k in ase pathway indu ce d apoptos is in 111yr-cx pressing
`~e ru m -d e pri ved hbrob lasts I 44• j .
`
`at Ser l 36 of 13AD 146 .. ], crea tin g a bindin g site for
`14-.3-3 (a fam il y o f ubiquitou s hi ghl y exp resse d ad apto r
`prote ins): w hen BAD is b o und co 14-3-3 it is unabl e to
`h etcroclime ri se with and inhibit th e surviv al act iv ity of
`the prote in s Bc l-2 or 13e l-X1, [4 71. In b oth haematopo ietic
`precurso r ce ll s and fibrobl asts, ovt:rex prcss io n of BAD
`w ill
`induce cel l death, and this effect is reversed b y
`the ex press ion o f activated forms of PKB//\kt. Death
`induce d by non-p hosp horylarnb le 13/\0 with a serin e to
`al anin e mu tation at codo n 136 is not sup p ressed by
`activated PKB/Akt. Th e ev id e nce is good, therefore, that
`PKB/Akt ca n act to re ve rse the death-i ndu cin g accivity
`of 13/\D. I lowcvc r, it is fa r from clear that t hi s is th e
`on ly, or even the p rimary, way in wh ic h the surviva l s ignal
`from PKB//\kt is med iated. BAD is on ly ex presse d in a
`limi ted range of ti ss ue s a nd ce ll lin es, a nd almost all che
`work reported co elate on 13/\D ha s used overexpressed
`protein, ofte n in ce ll lin es rhar do nor normall y expre:;s
`it at a ll. /\t th e ve ry least, a re lative of 13/\0 mu se be
`postu lated to exp lain the ab ility of PKB/J\kt to protect
`cell s from apoptos is in the abse nce of BAD express ion,
`and it is quice possib le that PKI3//\kc al so acts on other
`unre lated components of apopcos is sig nallin g pat hways.
`It ma y be sign ihca nt that PKl3//\ k t has been reporcecl to
`tra nslocate to the nucl e us fo ll owing stimul at ion of ce ll s
`w ith growth facror, suggestin g that important substrates
`co uld ex ist in th is compa rtmcnr 124 .. ,48 1. It is not poss ible
`co say whethe r putative n uc lear targets for PKB//\kt are
`in vo lved in protection from apoptos is or other function s
`of PKB/Akt.
`
`The ability of PKB/J\kt to promote ce ll surv iva l in t he
`abse nce of normal protective c ue s may be c ritical
`to
`its funct ion as an oncogene in the AKT8 retrov irus 13 1.
`P KB- /\kt synergiscs stron g ly with the Raf/MAP kinase
`p at hwa y to cause cransformacion of N II I 3T3 ce ll s J 10 I and
`ma y also be ab le to dri ve ce ll -cyc le p rogression through
`indu ct ion of E2F transcriptional act ivi ty J,+9 1. In n,s-trans(cid:173)
`formcd ep ithelial cells, t he ab ili ty of the m s oncogene to
`promote ce ll surviva l in suspens ion is mediated through a
`Pl 3-K- PKB//\kt pat hwa y a nd not throug h t he Raf- M/\P
`k in ase pathwa y 11 6 .. ]. Ro ug hl y a quarter of a ll human
`ma lignancies arc ca rcin oma s that have undergone ms
`ac ti vation by mutati on. It is like ly t hat a m ajor co mponent
`of their abi li ty to grow in the absence of ad hes ion is due
`to t he s urviva l signa l tri ggered b y Ras actin g throu gh Pl
`3 -K to stimul ate PKB//\kt. /\nc ho rage- ind epc ncl enr growt h
`is one of t he key hal lm ark s of transformatio n. It mighc
`be expected, the refo re, that PKB//\kt und e rgoes activation
`in so m e human tumours;
`t hi s has ind ee d been fo und
`for PKI3~//\ k t2, which
`is amp lihed in 12% of ovarian
`ca rc in omas, 3% o f breasr carc ino m as and I 0% o f pancreatic
`carci no mas [50,5 1 I.
`
`T h e m ec h a ni s m by which PKB /J\kt protects ce ll s from
`Conclusions
`p rog ramme d ce ll deat h h as been the subject of mu ch
`PKB//\ kt has emc riscd as a c ri t ica l d ownst rea m ta rget for
`inves cigati o n recen tl y. PKB/Akt can phosphory late the
`Pl .3- K, being regul ated b y a co mbinati o n o f direct binding
`pro-a poptotic Bcl -2 fam il y member BAD [45 .. ,46 .. ], b oth
`i11 v itro a nd in intac t ce ll s. The phosphory lat ion occ urs
`o f P l
`.3-K-p rod uccd
`li pids
`its p lcckstrin ho mo logy
`to
`Th i.s m ate ri a I w as co~i e-d
`at the NLM and m ay t>e
`Su t>j e.ct US Co~yright Laws
`
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`266 Cell regulation
`
`d o main and phos phory lati o n b y upstn.:;1m kina ses s uc h as
`POK I rhat a re al so conrro llcd b y th esc lipid s. i\e civ acc d
`PKB/i\kr m c diate s a numbc r or rh c me tabo li c c ffec rs o r
`in sulin and al so provides a survival sig nal co cell s ch ar
`p ro tec ts chem fro m a po p cos is; ac ri vari o n o f PKB/i\kr b y
`ge ne a mplificat io n o r b y Ras mu ta ti o n m ay co nrribure co
`th e ge nc rari o n o r m ali g nanc ies char ca n fl o uri s h wirh o ur
`ad e qu ate ex traccllu lar s urviva l sig nal s. C ellular s ubstrates
`for PKB/i\kc include rh e metabo li c regul a tors G SK3 and
`PFK -2, a nd th e Bc l-2 -rcl a te d apo ptosis rcg ul aro ry prote in,
`13/\D. O th e r physio logi cal ly imp ortant targcts fo r PKB/i\kt
`a re likel y to e xist.
`
`References and recommended reading
`Papers of particular interest, published within th e annual period of review,
`have been highlighted as:
`
`•
`of special interest
`•• of outstanding interes t
`
`1.
`
`2.
`
`3.
`
`4.
`
`5.
`
`6.
`
`7.
`
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`
`Jones PF, Jakubowicz T, Pitassi FJ, Maure r F, Hemmings BA :
`Molecular cloning and identification of a serine/threonine
`protein kinase of the second -messenger subfamily. Proc Natl
`Acad Sci US A 199 1, 88 : 4 17 1-41 75.
`Bellacosa A, Testa JR, Staal SP, Tsichlis PN : A retroviral
`oncogene, akt, encoding a serine-threonine kinase contai ning
`an SH2-like region. Science 199 1, 254 :274-277.
`Jones PF, Jakubowicz T, Hemmings BA : Molecular cloning of a
`second form of rac protein kinase. Ce// Regul 199 1, 2:1001 ·
`1009.
`
`Konishi H, Kuroda S, Tanaka M, Matsuzaki H, Ono Y, Kameyama K,
`Haga T, Kikkawa U: Molecular cloning and characterization of a
`new member of the RAC protein ki nase fam ily: association of
`the pleckstrin homology domain of three types of RAC protein
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`subun its of G proteins. Biochem Biophys Res Commun 1995,
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`Franke TF, Yang S-1, C han TO, Datta K, Kazlauskas A,
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`encoded by the Akt proto-oncogene is a target of the PDGF(cid:173)
`activated phosphatidylinositol 3-kinase. Ce// 1995, 81 :727-736.
`Burgering BMT, Cofler PJ : Protein kinase B (c-akt) in
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`3-OH kinase as a direct target of Ra s. Nature 1994, 370 :5 27-
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`factors , v-src and v-ha-ras, in Sf9 and mammalian-cells. J Biol
`Chem 1996, 271 :30835-30839 .
`Khwaja A, Rodriguez-Viciana P, W ennstrom S, W arn e PH,
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`activate a phosphoinositide 3-OH kinase and protein kinase
`B/Akt cellular survival pathway. EMBO J 1997, 16 :2783-2793.
`Adhesion of epith elial cells to matrix is shown to activate PKB/Akt and pro(cid:173)
`tect th em from apoptosis. In additi on, PKB/Akt is shown to be th e pathway
`used by oncogenic Ras to protect epithelial cells from apoptosis induced by
`membrane detachment.
`17.
`King WG, Mattaliano MD, Chan TO, Tsic hlis PN , Brugge JS:
`Phosphatidylinositol 3-kinase is required for integrin(cid:173)
`stimulated AKT and Raf-1 /mitogen -activated protein kinase
`pathway activation. Mo/ Cell Bio/ 1997, 17:4406-4418.
`Adh esion of epithelial cells to matrix activa tes PKB/Akt.
`18.
`Konishi H, Matsuzaki H, Tan aka M, Ono Y, Tokunaga C, Kuroda S,
`Kikkawa U: Activation of RAC-protei n kinase by heat shock
`and hyperosmolarity stress through a pathway independent
`of phosphatidylinositol 3-kinase. Proc Natl Acad Sci USA 1996,
`93 :7639-764 3.
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`19.
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`20.
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`Konishi H, Shinomura T, Kuroda S, Ono Y, Kikkawa U: Molecular
`cloning of rat RAC protein kinase alpha and beta and their
`association with protein kinase C zeta. Biochem Biophys Res
`Commun 199 4, 205 :817-825.
`Andj elkovic M, Jakubowi cz T, Cron P, Ming XF, Han JW,
`Hemmings BA: Activation and phosphorylation of a pleckstrin
`homology domain containing protein kinase (RAC-PK/ PKB)
`promoted by serum and protein phosphatase inhibitors. Proc
`Na tl Acad Sci US A 1996, 93 :5699-5 70 4.
`James SR, Downes CP, Gigg R, G rove SJA, Holmes AB,
`Alessi DR : Specific binding of Akt-1 protein kinase to
`phosphatidylinositol 3,4,5-trisphosphate without su bsequent
`activation. Biochem J 1996, 315 :709-713.
`Th e PH domain of PKB/Akt is shown to bind directly to PIP3 and Pl (3, 4)P2.
`In thi s case, lipid binding does not stimulate th e kinase activity of PKB/Akt.
`(Compare Fran ke el al., 1997 (11 ••]. Klippel el al., 1997 (2 2•]. Frech el al.,
`199 7 (23•) .)
`
`21.
`••
`
`22.
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`8.
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`9.
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`1 O.
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`11 .
`••
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`12.
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`Kohn AD, Kovacina KS, Ro th RA : Insulin stimulates the kinase
`activity of RAC -PK, a pleckstrin homology domain containing
`ser/thr kinase. EM BO J 1995, 14:4288-4295.
`Klippel A, Reinhard C , Kavanaugh WM, Apell G, Escobedo MA,
`Williams LT: Membrane localization of phosphatidylinositol
`3-kinase is sufficient to activate multiple signal transducing
`kinase pathways. Mo/ Cell Biol 1996, 16 :4 11 7-41 27.
`Marte BM, Rodri guez-Viciana P, Wennstrsm S, Warne PH,
`Downward J: Pl3K and PKB/ Akt act as an effector pathway for
`R-Ras. Curr Biol 1997, 7:63-70.
`Franke TF, Kaplan DR, Cantley LC, Taker A: Direct regulation of
`the akt protooncogene product by phosphatidylinositol-3,4-
`bisphosphate. Scien ce 1997, 275 :665-668.
`Th e PH domain of PKB/Akt is shown to bind directly to PIP3 and Pl(3,4) P2.
`P1 (3, 4)P2 is shown to activate the kinase ac tivity of PKB/Akt, at least weakly,
`in vitro.
`Didichenko SA, Tilton B, Hemmings BA, Ballmerh ofer K, Thelen M :
`Constitutive activation of protein -kinase -b and phosphorylation
`of p47(phox) by membrane-targeted phosphoinositide 3-
`kinase. Curr Biol 1996, 6: 1 27 1-1 278.
`Rodriguez-Viciana P, W arn e PH, Dhand R, Van haesebroeck B,
`Gout I Fry MJ W aterfield MD Downward J: Phosphatjdylinosi.tol-
`.
`'
`'
`'
`This material w as copied
`atthe• NLM and may oe(cid:173)
`Subj ect USCopyr ight Laws
`
`Klippel A, Kavanaugh WM , Pot D, Williams LT: A specific product
`of phosphatidylinositol 3-kinase directly activates the protein (cid:173)
`kinase akt through its pleckstrin homology domai n. Mo/ Cell
`Biol 1 99 7, 17: 338-344.
`The PH domain of PKB/Akt binds directly to PIP3 and Pl (3, 4)P2, with
`P1(3,4)P2 stimulatin g the kinase activity of PKB/Akt moderately.
`23.
`Frech M, An djelkovic M, lngley E, Reddy KK, Falck JR,
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`and phosphoinositides to the pleckstrin homology domain
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`The PH domain of PKB/Akt binds directly to PIP3 and P1 (3.4)P2, with
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`An djelkovic M, Alessi DR, Meier R, Fern andez A, Lamb
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`B. J Biol Chem 199 7, 272 :3 1 51 5-3 15 24.
`A detailed and conclusive study showing that PKB/Akt translocates to th e
`plasma membrane, and even tu ally to th e nucleus, following growth _ factor
`stimulation of cells. Membrane translocation is shown to be suff1c1ent to
`cause phosphorylation of regulatory sites on PKB/ Aki by upstream kinases.
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`Al essi DR, Andj elkovic M, Caudwell B, Cron P, Morri ce N,
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`The identification of the sites phosphorylated on PKB/Akt in response to
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`27.
`
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`Mechanisms and consequences of activation of protein kinase B/Akt Downward
`
`267
`
`kinase (PDK1) that phosphorylates and activates protein
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`The identification and chracteri sation of POK 1 as the upstream kinase th at
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`Stakoe D, Stephens LR, Copeland T, Gaffney PR, Reese CB,
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`DSTPK61 kinase. Curr Biol 1997, 7:776-789.
`The re port of the cloning of POK 1 . The kinase has a PH domain at its
`carboxyl 1erminu s and is from the same family as PKB/Akt itself.
`30
`Al essi DR, Caudwell FB, Andjelkovic M, Hemmings BA, Cohen
`P: Molecu lar basis for the substrate specificity of protein