Experimental Cell Researdl 253, 210-229 (1999)
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`• _... .®
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`Article I D excr.1g99.4690, available online at http://wwyv.idealibrary.com on = == It Jt-L -
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`The Regulation and IA"ctivities of the ~v~ultifunctional
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`SeiinefThieonine Kinase AktfPKB
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`Eugene S. Kandel and Nissim Hay'
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`Department of Molecular Genetics. University of Illinoisat Chicago, Chicago. IIIinois60607
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`The serine/threonine kinaseAkt, or protein kinase 8
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`(PKB), has recently been a focusof intense research. It
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`appears that .A.ktfPt<.B iies in the crossroads of muiti(cid:173)
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`pie cellular signaling pathways and acts as a trans(cid:173)
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`ducer of many functions initiated by grov'fth factor
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`receptors that activate phosphatidy!inosito! 3-kinase
`(PI3-k:nase).AktlPKB is particularly important in me-
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`diating several metabolic actions of insulin Another
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`major activity of AktlPKB is to mediate cell survival.
`I n ad d it ion, the r ecen t d i scov er y of the tumor su r p r es(cid:173)
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`sor PTE N as an antagon I st of PI 3-k I n ase an d AktlPK B
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`kinase activity suggeststhat AktlPKB isa critical fac(cid:173)
`tor in the genesis of cancer Thus. elucidation of the
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`mechanisms of AktiPKB reguiation and its physioiog(cid:173)
`i ca i fu n ct ion s sh au i d be imp a rt ant fa r the un d er st and(cid:173)
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`ing of cellular metabolism, apoptoslS, and cancer
`© '999 AcauerYliC P, 03&&
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`INTRODUCTION AND HISTORICAL PERSPECTIVE
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`I II 1991 tVIiO independent lines of research converged
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`un the discovery of C;I eDNA encoding a novel sel inel
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`threonine kinase. One group cloned the ceiiuiar homo(cid:173)
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`logue of the v-akt oncogene from a transforming retro(cid:173)
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`virus (AKTS) in spontaneous thymoma of the AKR
`rnuuse and its prt .. vJud: \,vf!S cailed o-.A.kt [1,2]. ii'le Same
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`cD!'J,i>, vIas cloned by tV!O other groups searc.I-Jing for
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`novel members of the protein kinase C (PKC) and
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`protein kinaseA (PKA) superfamily as possible partic.(cid:173)
`ipants in Signa! tr"ansduGiior"! c.:f!seades [3, 4]. AC,,:,:r.Jru(cid:173)
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`ing!y, the nove! kinase vIas called R,Ll.C (re!ated to ,f.,
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`and C kinascs) or PKB (protein kinase B)-and in this
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`review we will refer to it as Akt/PKB. Eight years of
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`subsequent research has left !itt!edoubt that AktlPKB
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`plays a prominent role in both growth factor signaling
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`and oncogenesis. Currently, closeAkt homologues have
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`been identified in a variety of species, induding birds,
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`insects, nematodes, slime mold, and yeast and at least
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`iTo \vhom correspondence and reprint requests should be ad(cid:173)
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`dressed at t he Depart ment of M o! eru!ar Genetics (M ie 669), U niver(cid:173)
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`sity of Illinois in Chicago. 900 South Ashland Avenue, Chicago, lL
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`60607. Fax: (312) 355-2032. E-mail: nhay@.lic.edu.
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`some organisms have more than one gene for simiiar
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`yet distinct isoforms of thiS enzyme [5-11].
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`Interest in AktlPKB was piqued in 1995 when it was
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`shovvil to be a direct downstream eiiecio( of phospha(cid:173)
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`tidyiinositoi 3-kinase (Pi 3-kinase) [·12, i3j. When giy(cid:173)
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`cogen synthase kinase 3 (GSK3) was identified as an
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`Akt/PKB target [14], it established the current para(cid:173)
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`digm for insulin signaling in which Akt/PKB plays the
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`key roie. The finding that Pi 3-kinase activates Akil
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`PKB ied to studies showing that it is a major partici(cid:173)
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`pant in growth fador-mediated cell survival [15-18]. It
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`also became clear that AktlPKB is capable of linking
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`growth factor signaiing through Pi 3-kinase to basic
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`metabolic functions, such as protein and lipid synthe~
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`sis, carbohydrate metabolism, and transcription. As
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`the field evolved, new prospectives on the interplay
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`between ceil grQ\Nth, survivai, and metabolism, under
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`both normal and pathological conditions, have been
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`established. These concepts and several emerging
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`questions will be discussed in tillS review.
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`THE AKT! PKB GE~~E FAMI LY
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`Three major isoforms of AktfPKB encoded by three
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`separate genes have been found in mammalian cells.
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`Akt1 or PK8c~ was the first isolated isoform; Akt2!
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`PKB{J and
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`'Nere subsequently cloned
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`through homology screen [19-22]. All th ree genes have
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`greater than 85% sequence identity and their protein
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`products share the same structural organization (Fig.
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`1). The first amino-terminal 100 amino adds possess a
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`pleckstrin homology (PH) domain that binds phospho(cid:173)
`lipids. A short glycine-rich region that bridges the PH
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`domain to the cata!yticdomain fo!!ov.JS the PH domain.
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`All AktlPKB isoforms are assumed to have identical or
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`similar substrate specificity but this has never been
`overtly tested. The last 70 amino acids of the carboxy(cid:173)
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`terminal tail contain a putative regulatory domain. In
`v-Akt, a truncated viral group-specific antigen, gag, is
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`fused in frame to the full-length Akt1 coding region
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`through a short 5' untranslated region of Akt1 [2]. All
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`three AktlPKB isoforms possess conserved threonine
`and serine residues (1308 and S473 in Akt1/PKBa)
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`that together with the PH domain are critical for Aktl
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`0014-4827/99 $30.00
`Copyright © 1999 by Academic Press
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`All rights of reproduction in any form reserved.
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`NOVARTIS EXHIBIT 2040
`Breckenridge v. Novartis, IPR 2017-01592
`Page 1 of 20
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`REGULATION AND ACTIVITIES OF AKT/PKB
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`211
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`Aktl!PKHu
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`,~~~, ~--~~-'---, ~
`J tregul.[
`I PH
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`l'Htalytic
`~ ',~---~,.
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`S.t71
`TJOZ
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`- - - - - , ~
`': ~el!ul.11
`(utah tit
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`rl\':>. I. Stj'uctural oiganization ofthettueemajor AktiPKB i50-
`forms is shown in comparison to virally encoded v-Akt and serum(cid:173)
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`and glu cocorti coid-i n dud ble ki nase SGK. All AktlPKB vari ants con(cid:173)
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`tain a plecsktrin homology domain (PH), a catalytic domain, and a
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`putative regulatory fragment at the C-terminus (regul). SGK has a
`similar structure and sequence, but lacks a p!eckstrin homology
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`domain v-Akt is an in-frame fusion of Akt-1 with a portion of
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`retroviral group-specific antigen (gag). Amino acid positions are
`shown for mouse proteins. Threonine and serine residues whose
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`pr'lOsphor"yiatiorl is requir"ed io induce adjvities of Hie enzyrnes are
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`indicated. See text for details.
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`PKB activation (see below). Equivalent threonine and
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`serine residues in a similar amino ac~d context are also
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`present in p70 56 kinase and in a!! PKC isoforms. !t is
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`noteworthy that the distanoe between the two phos(cid:173)
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`phorylated residues(~·160-170 aa) is also conserved in
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`these different protein kinases. Tvvo additional Akt
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`isoforms have been described and represent minor
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`splioe variants of human Akt2 and rat Akt3 [19, 23].
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`These isoforms exhibit a carboxy-therminal insertion
`of 40 aa and a partial deletion of 25 aa in the car boxy(cid:173)
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`terminal regulatory domain (including 8473), respeCJ(cid:173)
`tively. The biological significanoe of these isoforms re(cid:173)
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`mains undear.
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`A close relative of AktlPKB is serum- and gluoooor(cid:173)
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`ticoi d-i nduci ble ki nase (SGK) t hat was shown to h ave a
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`substrate specificity similar to that of AktlPKB [24,
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`25]. SGK has extensive secuenoe homology to AkllPKB
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`in the catalytic domain and possesses residues equiv(cid:173)
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`alent toT308andS4730fAkt1, but lacksa PH domain.
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`SGK is more similar to the Akt homologues, Ypk1 and
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`Ypk2lYkr2, in budding yeast [26], suggesti ng that SGK
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`might be doser to the ancestral prototype of the Aktl
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`PKB famiiy,
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`All three AktlPKB isoforms are ubiquitously ex(cid:173)
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`pressed in mammals, although the levels of expression
`vary among tissues [-19-23,27]. Aki I/PKBa IS the pre(cid:173)
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`dominant isoform in most tissues. The highest expres(cid:173)
`sion of Akt2/PKB{3 was observed in the insulin-respon(cid:173)
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`sive tissues: skeletal muscle, heart, liver, and kidney
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`[20], suggesting thai this isoform is important for in(cid:173)
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`suiin signaiing. This is further substantiated by the
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`observation that Akt2IPKB{3 expression In developing
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`embryos is also highest in the insulin-responsive tis(cid:173)
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`sues, including iiver, brown fat, and skeletal rnusde
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`[28j. A pecuiiar paiiern of AktiiPKB" expression was
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`detected in brain, vvhere it is markedly increased in
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`regenerating neurons [29]. Akt1/PKBu is also the pre(cid:173)
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`UUrTllflcUli Isoform III muuse emDIYu fibrubia~is (VV.
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`Cnen and N.H., unpublished). Unlike two other iso(cid:173)
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`forms, Akt3fPBK)i shovvs a more restricted pattern of
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`expression. Higher levels of Akt3!PKBl' were detected
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`Ifl testis cHIll or cUll i::IrH.i iU\f'/ ieveis III the i::Illuit pclllCl'ei::lS,
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`heart, and kidney [2'1-23]. The expression pattern of
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`the three isoforms may not alvv-ays reflect their activi(cid:173)
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`t ies. Different levels of ki nase activit ies of t he different
`isofo((ns have been obse(vetj if! c:er'iaif! tissues and
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`during differentiation, whic.~ is not necessarily corre(cid:173)
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`lated v.;ith their level of expression [30, 31].
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`THE iViECHAi'JiSiViS OF AKT/PKB ACTiVATiON
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`Activation by PI 3-Klnase
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`The viral gag domain in v-A.kt possesses a myristoy!(cid:173)
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`atian signa! that mediates its targeting to the plasma
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`membrane and renders the enzyme constitutively ac(cid:173)
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`tive. This suggested that membrane association might
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`be important in the activation process of c-Akt and
`subsequent studies bore out this hypothesis. ! ndeed,
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`while CJ-Akt is localized primarily to the cytoplasm, a
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`large proportion of v-Akt is localized to the plasma
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`membrane [32]. Only upon stimulation does a fraction
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`of c-,.6.,kt migratetothe membrane and attach there via
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`its PH domain [33]. The presenoe of a PH domain
`together wit h the observation t hat the kinase activities
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`of both Akt1!PK8a and Akt2!PK8f3 can be rapidly ac(cid:173)
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`tivated by PDGF in rodent fibroblasts led to studies
`showing that AktlPKB is a direct target of PI 3-kinase
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`[14,13]. PI 3-kinase is activated by growth factor re(cid:173)
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`oeptors through binding of its regulatory subunit to
`phosphotyrosine residues in the reoeptor. Upon activa(cid:173)
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`tion the catalytic subunit of PI 3-kinase phosphory(cid:173)
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`lates phosphoinositides (PI) at the 3-position of the
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`inositol
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`PI (3,4,S)P3 (see review by B. van H aesebroeke and M.
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`Waterfield in this issue). PI 3-kinases are classified
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`NOVARTIS EXHIBIT 2040
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`Page 2 of 20
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`212
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`KANDEL AND HAY
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`intothreemajorgroups.lnthisreviewwewill usethe
`term PI 3-kinase to refer to the heterodimeric enzyme
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`composed of p85 regulatory subunit and pi iO catalytic
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`subunit. Two major observations strongly suggested
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`that the activation of AktlPKB is dependent on PI
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`3-kinase. First, activation of AkilPKB was shown to
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`depend on tyrosines Y740 and Y75i in the PDGF re(cid:173)
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`oeptor that had been identified as the binding sites for
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`the p85 regulatory subunit of PI 3-kinase [13]. Second,
`the Pi 3-kinase inhibitors worimannin and L Y94002
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`couid diminish the activation of AktJPKB by growth
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`factors [12-14, 34J. These Initial observations were
`follovliOO by experiments shCNving that over-expression
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`of a constitutiveiy activated PliO catalytic subunit of
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`Pi 3-kinase can activate AkiiPKB [35]. in addition, it
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`vv'assho'vvn that point mutationsin thePH domain that
`reduce phospholipid binding abrogate the ability of
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`AktfPKB lU ue activClted by gruwth facturs, anu a mu(cid:173)
`tation that increases phospholipid binding superacti(cid:173)
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`vates the enzyme [13, 36]. Further 5t udies sh oVved til at
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`both PI (3,4)P2 and PI (3,4,5)P3 bind with high affi nity
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`tu the PH dumClln uf AktlPKB [37-39], Huwevel, the
`reiative contribution of each 3-phosphory!ated phos(cid:173)
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`phoinositide species to AktJPKB activation in vivo re(cid:173)
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`mai ns u nelear, Exposu re of cells to syntheti e phospho(cid:173)
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`lipids si-]cn."!ljfj lhai Pi (3,4)P2 is a belle! aCliv8io( in
`some experiments [38, 39], \!Vhereas other experiments
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`sho'v'vcd higher binding affinity and better activation by
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`PI (3,4,5)P3 [37, 40, 41] The latter experiments "vere
`corroborated by observations that the SH2-containing
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`5-phosphatase
`(8H! P),
`v.Jhich
`converts
`inositol
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`PI (3,4,5)P3 to PI (3,4)P2, is a potent inhibitor of AktJ
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`P~<B activity in vivo ([42, 431, and see belovli),
`Upon binding of 3-phosphory!ated phosphoinosi(cid:173)
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`tides, the PH domain of ,11,ktlPKB facilitates dimeriza-
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`tion of the enzyme [44, 38], Experimental evidence
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`suggests that Akt/PKB exists in vivo as a dimer or a
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`trimer and this mu!timerization is required for the
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`regulation of ,.o.,ktlPKB activity. The interaction be(cid:173)
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`tween monomers within such a complex may well ex(cid:173)
`plain the behavior of at least some dominant-negative
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`forms of the enzyme (reviewoo in [45]),
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`Because AktlPKB activity is dependent on P! 3-ki(cid:173)
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`nase, any mechanism that activates PI 3-kinase can
`theoretically lead to stimulation of AktlPKB activity.
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`! ndeed, activation of AktfPK8 through P! 3-kinase is
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`not restricted to growth factors. For example, AktlPKB
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`is activated by integrins through activation of focal
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`adhesion kinase, which in turn binds and activates PI
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`3-kinase and subsequently AktlPKB [46-48]. Other
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`oeli surfaoe reoeptors that activate AktlPKB via PI
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`3-kinase indude CD28 and CD5 in T oelis, B oell re(cid:173)
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`oeptor (BCR) In B oells, G-protein-coupled reoeptors,
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`and the wopioid reoeptor [42, 49-54]. Angiotensin II
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`and hydrogen peroxide were also reported to activate
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`Akt/PKB through PI 3-kinase [55-57]. Among viral
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`proteins that activate Akt/PKB via PI 3-kinase are
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`polyomavirus middle-T antigen and H IV Tat protein
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`[58, 59]. in addition, AkiiPKB was shown to be acti(cid:173)
`vated by the oncogenic Ras through PI 3-kinase (re(cid:173)
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`viewed in [60]). The GTP-bound Ras binds and acti(cid:173)
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`vates the caiai ytic su bu nit p-l -lOaf Pi 3-ki nase and Ras
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`muiani ihat is noi ableio bind piiO could not activaie
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`AktlPKB [61]. It is not clear, however, whether the
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`activation of AktlPKB by activated Ras is as strong as
`the activation by growth fadors and activated p-l-10,
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`and whether it is universai or dependent on the ceii
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`type.
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`Fi ii all y, as discussed below 1 th ere is some evidence of
`Pi 3-kinase-independent rnecr'lanisrns of AktfPK8 adi(cid:173)
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`vat i on.
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`Activation by Phosphorylation: The PDKs
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`,l\ctivation of ,l\ktiPi{B in vivo by exposure to grO'Nth
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`factors or synthetic phospholipids is preceded by an
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`increase in seri n e and t h jeoni ne phosphorylat i on oft he
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`killClse itseif. SUllie residues such i::lS ser IIle 124 ClfJ(j
`threonine 450 in the mouse Akti/Pf{BH' are consiilu-
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`ttvely phosphorylated in a grO'vvth factor-independent
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`manner and 'vvere predicted to render the protein re(cid:173)
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`sponsive lo subsequent 8c:l.ivalion ever'lls [36]. Twu
`other residues that are rapidly phosphorylated upon
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`exposure to gro'vvth factors and arc the most ctiticai for
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`full activation of Akt/P~<B are threonine 308 (T308)
`and serine 473 (8473). T308 resides within the activa(cid:173)
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`tion loop of the kinase domain and 8473 lies in the
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`carboxy-terminal tai I. \Nhen these tv.o amino acids are
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`mutated to nonphosphorylatable residues activation of
`the kinase is abolished, vl,lhereas mutations to acidic
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`residues render the kinase more active even in the
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`8bsence of growth factors [62. 36]. The phosphorylntion
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`of T308 and S473 induoed by I GF-1 or insulin is sen(cid:173)
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`sitive to wortmannin, suggesting that this process is
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`dependent on P! 3-kinase [62]. TI.'vo possible exp!ana(cid:173)
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`tions for this phenomenon are that the binding of phos(cid:173)
`pholipids to the PH domain of Akt/PKB is a prerequi(cid:173)
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`site for its availability to other kinases or that the
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`kinases that phosphorylate T308 and 8473 are also
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`dependent on PI 3-kinase. It turns out that both expla(cid:173)
`nations are correct. The enzyme that phosphorylates
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`T308 was purified and c~onoo [63, 64, 40, 41]. The
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`ability of the enzyme to phosphorylate T308 is depen(cid:173)
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`dent on the presenoe of synthetic PI (3,4,5)P3 in vitro
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`and therefore it was termed 3-phosphoinositide-depen(cid:173)
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`dent kinase (PDK1) [64, 41]. PDK1 possesses a PH
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`domain in its carboxy-terminus and binds with high
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`affinity to PI (3,4,5)P3 and more weakly to PI (3,4)P2.
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`Deletion ofthePH domain ofPDK1 and mutatlonsthat
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`decrease binding to PI(3,4,5)P3 strongly decrease its
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`ability to activate Akt1/PKB" [65]. However, the ki(cid:173)
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`nase activity of PDK1 is tolerant to low conoentrations
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`
`NOVARTIS EXHIBIT 2040
`Breckenridge v. Novartis, IPR 2017-01592
`Page 3 of 20
`
`

`

`REGULATION AND ACTIVITIES OF AKT/PKB
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`213
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`of wortmannin. This is likely to be explained by a
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`relatively high affinity of the PH domain of PDK1 to
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`PI(3,4,5)P3 [4i, 65]. Alihough ii was reporied ihai
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`PDK1 can be translocated to the plasma membrane
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`upon growth factor stimulation [66], other studies us(cid:173)
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`Ing Immunoeiedron mlcrosoopy, confocai microscopy,
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`and a green fluoresoent protein-PDKi chimera ciearly
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`show that it is mostly cytosolic and remains so upon
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`stimulation [65]. Nevertheless a small portion ofPDK1
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`was always found in the piasma membrane even in
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`unsti muiated cei is and this may be aiso due to t he high
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`affinity of its PH domain to PI (3,4,5)P3. Alternatively
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`another unknovvn factor is requirecl for the binding of
`PDKI to the piasrna rnernbrane.
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`Ii is possible ihai ihe membrane-bound PDKi may
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`be required tor the phosphorylation ot Akt/PKB and
`other membrane-localized substrates, whereas the cy(cid:173)
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`iusuiir..: fUl'm IS requlreu ful' the phospflUlyiclliufl uf cy(cid:173)
`tosoiic proteins such as prO S6 kinase. Like l\ktlPKB,
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`PDK1 is evolutionarily conserved [64,67] and genetic
`studies in Caenorhabditis elegans confirm that PDK1
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`lies upstreaHl uf Akt bui duwf1~il'ealTl uf Pi 3-kim:lse.
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`PDKi gain-or-function mutant bypasses the require(cid:173)
`ment of PI 3-kinase for Akt activation [67].
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`I n addition to T308, the phosphorylation of S473 is
`aiso ftt~uifed rOf !na),-:.!(nai activation or AidJPKB [62,
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`36]. The findings that PDK1 cannot phosphorylate
`8473 in vitro or in cotransfcction experiments sug(cid:173)
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`gested that a distinct kinase activity termed PDK2 is
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`responsible for this function [63,40,41] but the iden(cid:173)
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`tity of this kinase has remained elusive. !t has been
`reported that integrin-linkcd kinase (I LK-1) is capable
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`of phosphorylating 8473 in vitro and in cotransfection
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`experiments [68]. However, others failed to reproduce
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`these results [69]. Recent!y it \AJas shO'Nn that PDK1
`interacts specifically in vitro and in vivo 'Nith the Glr(cid:173)
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`boxy-terminus region of protein kinase C-related ki(cid:173)
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`nase (PRK2) that was termed POK 1-interacting frag(cid:173)
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`ment (P! F) [69]. The interaction of PDK1 \!t/ith P! F
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`converts it to an enzyme that can phosphorylate both
`T308 and 8473 residues in AktlPKB [69]. The possibil(cid:173)
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`ity exists therefore that PDK1 can phosphorylate both
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`residues in vivo depending on postrans!ationa! confor-
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`mational change and/or interaction with another oellu(cid:173)
`lar protein. A related observation may be the finding
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`that one point mutation in POK1 of C. e!egans is suf(cid:173)
`ficient to bypass the requirement of PI 3-kinase for
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`AktlPKB activation [67]. It remains to be determined,
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`however, whether this constitutively active form of
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`PDK1 is capable of phosphorylating both T308 and
`8473. Interestingly, the minimal functional fragment
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`of PI F contains the putative PDK2 recognition site
`with senne substituted With the negatively charged
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`aspartate [69]. Thus, PI F may be considered a mimicof
`PDK2 substrate. While modulation of PDK1 specificity
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`by endogenous PRK2 remainsto bedemonstrated, it is
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`tempting to speoulate that onoe 8473 or an equivalent
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`residue in other enzymes is phosphorylated it will
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`serve as a caialysi ihai pri mes PDK2 act ivii yin PD K i.
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`Another possibility that cannot be completely exciuded
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`at present is that T308 phosphorylation permits auto(cid:173)
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`phosphoryiation of S473 by AktlPKB itseif. This POSSI(cid:173)
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`biliiy cannoi be ruled oui by ihe observaiion thai a
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`kinase-deficient mutant of AktlPKB can still be phos(cid:173)
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`phorylated on 8473 [62], because multimerization with
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`the wiid-type Akt/PKB in vivo may enabie the phos(cid:173)
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`phoryiation of 8473 of the mutant protein.
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`Negative Regulation of Aktl PKB Aciivity
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`Similar io oiher protein kinases, AkiiPKB is subject
`to negative regulation. The PH domain of AktJPKB tor
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`example may act both as a negative and as a positive
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`reyuii:lim uf ihe enzyme. Deietiun uf ihe PH UUrTlCllfl
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`rendering the enzyme incapable of interaction 'Nith
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`3-phosphoinositldes leads to a slightly higher basal
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`kinase activity than that of itvild type, but this activity
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`lGIn siiii be [lUl'rllCliiy eievaied in CI Pi 3-kinCise-uepen(cid:173)
`dent manner [34,64]. This suggests that in itsinaciive
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`form (not bound to 3-phosphoinositides) the PH do(cid:173)
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`main may confei a conformation that is not accessible
`lo PDKs. Anolhef possibility is thal lhe PH uOf"IHtin
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`interacts "·"lith a cellular protein that negatively regu(cid:173)
`lates the kinase and binding to 3-phosphoinositidcs
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`relieves this interaction.
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`is also
`The subcellular
`localization of AktlPKB
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`tightly regulated and may provide a mechanism for
`regulating cytoplasmic AktfPK8 activity. After 2 min
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`of stimulation ilvith IGF-1, Akt1lPK8(}' is transloc.ated
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`to the plasma membrane in a PH domain-dependent
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`manner but following this ,.t..ktlPKB is translocated to
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`the nucleus by ~ln unknown mec."anism [33]. ~4uclear
`translocation is probably independent of the PH do(cid:173)
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`main or kinase activity because both a mutant that
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`lacks the PH domain and a kinase-deficient mutant of
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`Akt/PKB are also found in the nucieus [33]. The phys(cid:173)
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`iological significance of the nudear localization is not
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`c~ear. !t might be required for phosphorylation of nu(cid:173)
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`dear proteins. A!ternative!y, the sequestration in the
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`n ud eus could be a way to I i mit the exposure of cytosol i c
`substrates to the kinase and might serve as a mecha(cid:173)
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`nism that indirectly negatively controls the kinase. !t
`has to be noted that Akt/PKB targeted to the plasma
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`membrane via a myristoylation signal exhibits consti(cid:173)
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`tutively active phenotype in regard to all known func(cid:173)
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`tions of activated wild-type enzyme, but fails to trans(cid:173)
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`locate to the nudeus [33].
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`As described above phosphorylation of AktlPKB is
`reqUired for its aciivation. It appears that this phOS(cid:173)
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`phoryl ation is tightly controlled. The facts that the key
`phosphoserine and phosphothreonine residues in Akt/
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`PKB havea relatively short half-life and that phospha-
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`NOVARTIS EXHIBIT 2040
`Breckenridge v. Novartis, IPR 2017-01592
`Page 4 of 20
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`214
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`KANDEL AND HAY
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`tase i nh i bitors such as vanadate and okadai c acid were
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`shown to augment AktlPKB kinase activity both indi(cid:173)
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`cate that the enzyme is negatively regulated by de(cid:173)
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`phosphorylation [70,71]. Phosphatase 2A (PP2A) may
`be the key enzyme associated with dephosphorylation
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`of AktiPKB in vitro and in vivo [70-72]. Hyperosmoiic
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`shock rapidly inactivates AktlPKB and this is preceded
`by dephosphorylation of T308 and S473. The dephos(cid:173)
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`phorylation and the decrease in AktlPKB activity can
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`be prevented by calyculin A, a relatively specificinhib(cid:173)
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`itor of PP2A [72].
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`It was also shown that the major Srcfamily tyrosine
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`kinase in hematopoeitic celis, Lyn, antagonizes the
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`activation and phosphorylation of AktlPKB by BCR in
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`B cells r731. It is possible that Lyn exerts its effect by
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`activation of serine/threonine phosphatases or by acti(cid:173)
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`vation of a 3-phosphoinositide-preferring phosphatase
`that antagonizes PI 3-kinase activity.
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`A 3-phosphoinositide-specific phosphatase activity
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`was found to reside in thetumor suppressor PTEN (for
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`phosphatase and tensin homologue deleted from chro(cid:173)
`mosome 10), which is mutated or deleted in a wide
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`range of human cancers (reviewed in [74]). PTEN
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`shares homology with dual-specificity phosphatases
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`that can dephosphorylate serine, threonine, and ty(cid:173)
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`rosine residues. However, attempts to oonfirm PTEN
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`as a protein phosphatase revealed only relatively weak
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`activity r75, 76], suggesting that PTEN is a specialized
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`phosphatase for certain proteins and/or it possesses a
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`different activity. I ndeed it was found that PTEN is a
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`potent lipid phosphatase [77, 78]. Overexpression of
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`PTEN significantly reduced PI (3,4,5)p3 production in(cid:173)
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`duced by insulin, and PTEN-null cells have higher
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`levels of PI (3,4,5)P3 [79, 77, 80]. A recombi nant PTEN
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`dephosphorylates 3-phosphoinositides specifically at
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`position 3 of the InOSitol ring and has highest speaflG(cid:173)
`ity for PI (3,4,5)P3 [77]. Since PTEN antagonizes the PI
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`3-kinase activity its role in tumor suppression may
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`involve AktlPKB (see disaussion below). Experiments
`in tumor cell lines with inactive PTEN and in PTEN(cid:173)
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`null fibroblasts showed that these cells exhibit high
`basal activity of Akt/PKB [78-82]. These results. to(cid:173)
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`gether with genetic studies in C. elegans demonstrat(cid:173)
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`ing that PTEN lies in the same pathway with PI 3-ki(cid:173)
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`nase/Akt and inhibits Akt [83], established PTEN as a
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`bona fide negative regulator of AktlPKB.
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`Another lipid phosphatase that can negatively regu(cid:173)
`lateAkt/PKB activity isSHIP-an inositol 5' phospha(cid:173)
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`tase that hydrolyzes PI (3,4,5)P3 to PI (3,4)P2. Overex(cid:173)
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`pression of SHIP was shown to inhibit AktlPKB
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`activity and SHIP-null cells exhibit prolonged activa(cid:173)
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