This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and

Transcription

1 Ths artcle appeared n a journal publshed by Elsever. The attached copy s furnshed to the author for nternal noncommercal research and educaton use, ncludng for nstructon at the authors nsttuton and sharng wth colleagues. ther uses, ncludng reproducton and dstrbuton, or sellng or lcensng copes, or postng to personal, nsttutonal or thrd party webstes are prohbted. In most cases authors are permtted to post ther verson of the artcle (e.g. n Word or Tex form) to ther personal webste or nsttutonal repostory. Authors requrng further nformaton regardng Elsever s archvng and manuscrpt polces are encouraged to vst:

2 Analytcal Bochemstry 414 (2011) Contents lsts avalable at ScenceDrect Analytcal Bochemstry journal homepage: Study of the PDK1/AKT sgnalng pathway usng selectve PDK1 nhbtors, HCS, and enhanced bochemcal assays Alexandra Hofler a, Tm chols b, Stephan Grant a, Laura Lngardo a, Edward A. Esposto c, Scott Grdley c, Sean T. Murphy d, John C. Kath d, Carán. Cronn d, Mchelle Kraus d, Gordon Alton a, Zh Xe a, Scott Sutton d, Mke Gehrng e, Jacques Ermoleff a, a Department of ncology, Pfzer, La Jolla, San Dego, CA 92121, USA b Department of Drug Safety Research and Development, Pfzer, La Jolla, San Dego, CA 92121, USA c Blue Sky Botech, Worcester, MA 01605, USA d Department of Chemstry, Pfzer, La Jolla, San Dego, CA 92121, USA e Department of External Research Soluton CoE at Pfzer, La Jolla, San Dego, CA 92121, USA artcle nfo abstract Artcle hstory: Receved 21 ovember 2010 Receved n revsed form 21 February 2011 Accepted 8 March 2011 Avalable onlne 12 March 2011 Keywords: PDK1 AKT Knase nhbtor Sgnal transducton Templatedrected assembly (TDA) The PI3K/AKT sgnalng pathway has an mportant regulatory role n cancer cell growth and tumorgeness. Sgnal transducton through ths pathway requres the assembly and actvaton of PDK1 and AKT at the plasma membrane. n actvaton of the pathway, PDK1 and AKT1/2 translocate to the membrane and bnd to phosphatdylnostol(3,4,5)trsphosphate (PIP 3 ) through nteracton wth ther pleckstrnhomology domans. A bochemcal method was developed to measure the knase actvty of PDK1 and AKT1/2, utlzng nckelchelatng coated lpd vescles as a way to mmc the membrane envronment. The presence of these vescles n the reacton buffer enhanced the specfc actvty of the Hstagged PDK1 (fulllength, and the truncated knase doman) and the actvty of the fulllength Hstagged AKT1 and AKT2 when assayed n a cascadetype reacton. Ths enhanced bochemcal assay s also sutable for measurng the nhbton of PDK1 by several selectve compounds from the carbonyl4amnopyrrolopyrmdne (CAP) seres. ne of these nhbtors, PF , was further evaluated usng a hgh content cellbased assay n the presence of CH cells engneered wth GFPPDK1. Ó 2011 Elsever Inc. All rghts reserved. Correspondng author. Address: Scence Center Drve, San Dego, CA 92121, USA. Fax: Emal address: Jacques.ermoleff@pfzer.com (J. Ermoleff). 1 Abbrevatons used: PDK1, 3phosphonostdedependent proten knase 1; TRC2, mammalan target of rapamycn complex 2; PI3K, phosphonostde3 knase; RTK, receptor tyrosne knase; EGFR, epdermal growth factor receptor; IGFR, nsuln growth factor receptor; IR, nsuln receptor; PIP 2, phosphatdylnostol(3,4)bsphosphate; PIP 3, phosphatdylnostol(3,4,5)trsphosphate; PIF, PDK1 nteractng fragment; SGK, glucortcodstmulated proten knase; S6K, P70 rbosomal S6 knase; PTE, phosphatase and tensn homolog deleted on chromosome 10; ATP, adenosne 5 0 trphosphate; DTT, dthothretol; EDTA, ethylenedamnetetraacetc acd; TCEP, trs(2carboxyethyl)phosphne; TDA 2.0, TemplateDrected Assembly, second generaton proten assembly reagent. The phosphonostde3 knase (PI3K) 1 /AKT pathway s a crtcal cellular pathway nvolved n varous cell functons such as cell survval, cell dfferentaton, cell growth, and proten expresson. The actvaton of ths pathway starts at the cell membrane and s ntated on the bndng of growth factors to ther respectve tyrosne knase receptors (RTKs), such as the epdermal growth factor receptor (EGFR), the nsulnlke growth factor receptor1 (IGFR1), and the nsuln receptor (IR). n bndng, these RTKs actvate downstream PI3Ka, whch catalyzes the phosphorylaton of phosphatdylnostol(3,4)bsphosphate (PIP 2 ) to generate bologcally actve phosphatdylnostol(3,4,5)trsphosphate (PIP 3 ). The formaton of PIP 3 trggers membranebased colocalzaton of the 3 0 phosphonostde dependent knase1 (PDK1) and AKT, whch bnd to PIP 3 through ther pleckstrn homology (PH) domans. PDK1 s consttutvely actvated n the cell due to ts ablty to phosphorylate ts own Tloop; however, the mgraton of ths enzyme to the membrane helps to actvate AKT1 n conjuncton wth the mammalan target of rapamycn complex 2 (TRC2) through the phosphorylaton of three key resdues, Thr308, Ser473, and Thr450 [1,2]. Conversely, the entre PI3K/ AKT pathway s downregulated by the tumor suppressor PTE (phosphatase and tensn homolog deleted on chromosome 10), whch dephosphorylates PIP 3 and thus prevents the colocalzaton of AKT and PDK1. In addton, PDK1 has the ablty to be recruted n the nucleus. Ths mechansm s drven by the phosphorylaton of key resdues on the enzymes such as Ser396, Tyr9, and Tyr376, and by nuclear export sgnal (ES) n the PDK1 tself. Lastly, the SHP1 phosphatase has also been shown to assocate wth the tyrosne phosphorylated PDK1 to facltate ts entry to ths cellular compartment [3,4]. In the nucleus, PDK1 s suspected to phosphorylate specfc substrates and to provde protecton to the cells aganst proapoptotc stmul /$ see front matter Ó 2011 Elsever Inc. All rghts reserved. do: /j.ab

3 180 PDK1 and AKT sgnalng pathway / A. Hofler et al. / Anal. Bochem. 414 (2011) ot surprsngly, the consttutve actvaton of the PI3K/AKT pathway plays a major role n the development and the survval of varous types of cancers due ether to the loss of PTE actvty or to the ncrease of PI3K and/or AKT actvty [5 7]. For nstance, AKT1 gene amplfcaton and mutaton occur n gastrc and colorectal cancer, whle AKT2 gene amplfcaton has been observed n breast, ovaran, and pancreatc cancers [8]. In addton, mutatons n PI3Ka or PTE genes lead to aberrant prolferatve sgnals and cellular transformaton [9 11]. Currently, several AKT1, mtr, and PI3Ka nhbtors have been reported n the lterature, and a few are now ether n preclncal or n advanced clncal stages [12,13]. Whle no late stage and selectve nhbtor has been reported for PDK1, t nevertheless represents an attractve target for drug development. PDK1 belongs to the AGC knase famly and was frst dentfed by Phl Cohen s group n 1997 [14]. Ths enzyme has been characterzed as a master knase, due to ts propensty to actvate other mportant downstream AGC knases such as AKT, P70 rbosomal S6 knase (S6K), serum and glucortcodstmulated proten knase (SGK), atypcal and typcal PKC, and p90 rbosomal S6 knase (RSK). RA antsense targeted aganst PDK1 n PTE null cells sgnfcantly reduced ther prolferaton and survval [15], whle overexpresson of PDK1 n epthelal cells results n ther transformaton [16,17]. In addton, hypomorphc mutaton of PDK1 protected PTE / mce from developng a wde range of tumors [18]. Several nonselectve nhbtors for PDK1 have already been reported n the lterature [19] and have been shown to block survval of cancer cells. In the present study, we frst used a cell free model system composed of lpd vescles wth nckelchelatng head groups, TDA 2.0, that mmcs the cellular mcroenvronment. Controllng the exact composton of the vescles allowed us to study the mechansm of actvaton of AKT1 and AKT2 n the presence of PDK1 and mtr. Under these condtons, we have been able to study the role that a few key resdues play on the actvty and the stablty of the AKT enzymes and to observe the extent of PDK1 nhbton on AKT actvaton. Also, the potency of several novel nhbtors from the carbonyl4amnopyrrolopyrmdne (CAP) seres was evaluated aganst PDK1. Comparatve studes were conducted wth two dfferent assay formats and our data suggest that the presence of lpd partcles does not affect the potency of these compounds. verall, the addton of TDA 2.0 provdes an enhanced bochemcal assay method for measurng the actvty of membrane anchored proten knases and may be useful for knase drug dscovery and hghthroughput screenng platforms. Lastly, we used a GFPPDK1 engneered CH cell to hghlght the effect of PDK1 selectve nhbtors on the recrutment of PDK1 at the membrane, the phosphorylaton state of AKT1, and the translocaton of Fox03a from the cytoplasm to the nucleus. purchased from Lfe Technologes. TDA 2.0 proten assembly reagent was purchased from Blue Sky Botech, Inc. (Worcester, MA). Recombnant human Hstagged PDK1 catalytc doman (aa ) was made nhouse at Pfzer La Jolla. CHhIR cells (PDK CH) stably expressng human PDK1 (GenBank Accesson o. M_002613) coupled to the Ctermnus of enhanced Green Fluorescent Proten (EGFP) were purchased from Thermo Fsher Scentfc. Cells were mantaned n Ham s F12 meda wth 1% pencllnstreptomycn, 0.5 mg/ml Genetcn, and 10% heatnactvated FBS (Gbco). For the cell assay, the rabbt polyclonal antbodes that specfcally bnd to phosphoakt Thr, and Fox03a were all purchased from Cell Sgnalng Technology. Hoechst and goat antrabbt IgG conjugated to Alexafluor 532 were purchased from Invtrogen Lfe Technologes. For the Western blot assay, ant GST and antphosphoakt Thr308 and Ser473 antbodes were purchased from Cell Sgnalng (Beverly, MA). The antphospho AKT Thr450 antbody s from Abcam (Cambrdge, MA). The goat antrabbt IgGAP pab s from Vector Labs (Burlngame, CA), the goat antmouse IgGAP pab and the goat antrabbt IgGHRP antbody were from Jackson ImmunoResearch (West Grove, PA). The anths mouse mab was from Clontech (Mountan Vew, CA). All the knetc experments were conducted at room temperature (22 C) and the concentratons of reagents reported n the followng sectons are reported as fnal n the meda buffer. All the expermental data were generated n duplcate and were ftted usng a nonlnear regresson analyss software, GraphPad Prsm 5 [20]. Compound synthess and selectvty The synthess and selectvty of CAP compounds (Fg. 1) have been brefly descrbed by Murphy et al. [21] and wll be descrbed n further detals n a subsequent paper. Brefly, PF was submtted to a broad knase selectvty panel (see Supplementary Table 2) provded by Invtrogen and the Unversty of Dundee (UK) as a feeforservce and data were generated n the presence of 1 lm nhbtor aganst a panel of selected 60 knases. In addton, PF was also submtted to a smaller nhouse knase panel H 2 H H 2 H Materals and methods Reagents and general enzymatc assay condtons EDTA, Trs and Hepes buffer, dmethyl sulfoxde (DMS), ATP, DTT, magnesum chlorde, and Brj35 were all purchased from SgmaAldrch (St. Lous, M). Fluorescentlabeled AKT substrate (5FAMGRPRTSSFAEGCH2) and PDK1 substrate (5FAMARK RERTYSFGHHACH) were purchased from Calper LfeScences (Hopknton, MA). The PDK1 mna peptde (AcSoxPKTFCGTPEYL APEVRREPRILSEEEQEMFRDFDYIADH 2 ) was purchased from Invtrogen Lfe Technologes (Carlsbad, CA). The fulllength human recombnant nactve termnal Hstagged AKT1 (aa 1 480) was purchased from Cell Scences (Canton, MA). The fulllength human recombnant Hstagged PDK1, the fulllength human recombnant nactve Hstagged AKT2, and actve mtr (aa ) were H 2 PF H PF PF H 2 F H PF Fg.1. 2D structure of carbonyl4amnopyrrolopyrmdne (CAP) nhbtors. All the compounds used n ths study are enantomers except for PF whch was used as a racemc mxture.

4 PDK1 and AKT sgnalng pathway / A. Hofler et al. / Anal. Bochem. 414 (2011) and showed K values >1 lm aganst mtr, AKT1, S6K, and PI3Ka [21]. Producton of polyhstagged PDK1 (aa ) knase doman A nucleotde sequence encodng amno acds of human PDK1 was cloned nto a custom baculovrus transfer vector that appended the cloned fragment wth an termnal polyhstdne purfcaton tag (MIYYHHHHHHDYGIPTTELYFQAL). Recombnant baculovrus was prepared usng the BactoBac method (Invtrogen) and used to nfect Sf9 nsect cells (at mo = 1). Infected cells were harvested after 48 h and stored at 80 C. The nsect cell pellet was lysed n 50 mm TrsHCl, ph 7.4, 200 mm acl, 0.25 mm TCEP, contanng one EDTAfree protease nhbtor tablet (Roche) per 75 ml buffer. The suspenson was centrfuged at 5000g for 1 h and the target bound to ProBond resn (5 ml; Invtrogen). The resn was washed overnght wth 50 mm TrsHCl, ph 7.4, 400 mm acl, 20 mm mdazolehcl, ph 7.4, 1 mm TCEP, and the bound PDK1 stepeluted by usng 50 mm TrsHCl, ph 7.4, 400 mm acl, 250 mm mdazolehcl, ph 7.4, 1 mm TCEP. PDK1 was concentrated to 2 ml by usng an Amcon Ultracel 10K (Mllpore) centrfugal concentrator and passed through a BoSep S3000 gel fltraton HPLC column (Phenomenex) equlbrated wth 25 mm TrsHCl, ph 7.4, 250 mm acl, 1 mm TCEP. The peak fractons were pooled and the PDK1 concentrated to 2.6 mg/ml. Proten concentraton was determned by usng the Coomasse Plus Proten Reagent (Perce) wth BSA (2 mg/ml ampoule; Perce) as standard. Complex formaton and organzatonal actvaton of PDK1 enzyme actvty by TDA 2.0 proten assembly reagent The actvty of PDK1 (fulllength and catalytc doman) was measured wth and wthout TDA 2.0 n 50 mm Trs buffer, 10 mm MgCl 2, 0.01% Tween 20, ph 7.4 wth 5% DMS and 1 mm ATP. PDK1 (50 nm) wth and wthout TDA 2.0 (5 pm, ntal concentraton) was serally dluted 2fold and added to Trs buffer. 5FAMlabeled PDK1 peptde (5 lm) was added n the reacton meda n a 96well Vbottom plate. The enzymatc reacton was ntated on addton of ATP (1 mm). An alquot of the assay mxture was then transferred to a low volume 384well black plate for determnaton of the relatve amounts of substrate peptde and product phosphopeptde usng a Calper EZreader from whch the rate of turnover was calculated (mcromolar per mnute). The substrate and product were separated on the bass of charge usng upstream and downstream voltages of 2250 and 500 V, respectvely, and a screenng pressure of 1.2 ps. AKT actvaton n the presence of mtr and PDK1 Actvatons of AKT1 and AKT2 were conducted n a smlar Trs buffer (see prevous secton) wth 2% DMS. The reacton was conducted wth 25 nm nactve AKT(1 and 2), 25 nm mtr, 2.5 nm PDK1, and 2.5 pm TDA 2.0. The reacton was ntated wth 5 lm AKT substrate and 1 mm ATP and the rate of substrate converson was measured on a LabChp EZReader. The nstrument was set up to collect alquots from the assay mxture at regular ntervals. The upstream, downstream voltages and the pressure were set to 2800 and 380 V, and 0.8 ps, respectvely. Determnaton of the apparent Mchaels Menten constants, K app m for PDK1 k app cat K app m and and kapp cat values for ATP were determned n the presence of 25 nm FLPDK1 and 2.5 pm TDA 2.0 n 50 mm Trs buffer, 10 mm MgCl 2, 0.01% Tween 20, ph 7.4, wth 5% DMS. The enzyme was ncubated for 10 mn n the assay buffer n the presence of 5 lm 5FAMPDK1 peptde n a 96well Vbottom plate. The reacton was then ntated by the addton of varous concentratons of ATP. Product phosphopeptde was determned as prevously descrbed (see prevous secton). K app m and kapp cat values for 5FAMlabeled peptde were determned usng the same expermental condtons n the presence of 1 mm ATP and varous concentratons of peptde. Enzyme nhbton Inhbton studes were conducted usng two assay formats, mna and Calper. For the mna assay, K app studes were conducted n the presence of 20 nm KDPDK1, 50 lm ATP, and 3 lm Sox peptde n a 50 mm Hepes, 5 mm MgCl 2, 0.01% Brj35, 1 mm DTT assay buffer at ph 7.4. The ncrease of fluorescence (k ex = 360 nm and k em = 485 nm) was recorded contnuously usng a Safre TECA plate reader. For the Calper assay, the K app constant for FLPDK1 alone was determned n the presence of 25 nm enzyme. For AKT1, the reacton was conducted wth 25 nm nactve AKT1, 25 nm mtr, 2.5 nm FLPDK1. Both sets of Calper nhbton studes were conducted wth 2.5 pm TDA 2.0, 1 mm ATP, and 5 lm peptde n 50 mm Trs buffer, 10 mm MgCl 2, 0.01% Tween 20, ph 7.4, wth 5% DMS. The enzyme, the peptde, and varous amounts of nhbtor were prencubated for 15 mn, pror to addton of ATP to the reacton meda. K app and K were calculated by fttng the expermental data to the followng equatons [22] 0 0 qffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffff V ¼ V o 1 ½EŠ o þ½iš o þ 11 Kapp ½EŠ o þ½iš o þ K app 2 4 ½EŠ o AA; ð1þ 2 ½EŠ o where K app ¼ K ð1 þ½a o Š=K m Þ: ð2þ [E] o and [I] o are the total actve enzyme and nhbtor concentratons, respectvely; K s the nhbton bndng constant; V and V o are the rates of peptde phosphorylaton n the presence or n the absence of nhbtor, respectvely; [A] o s the total ATP concentraton. Western blots Enzyme concentratons for Western analyss were as follows 200 nm AKT1 or AKT2, 200 nm mtr, 20 nm FLPDK1, and 20 pm TDA 2.0. Samples from knase reactons were analyzed by SDS PAGE (4 20% acrylamde trsglycne, Lonza) usng standard methods. Antbodes used were anths (Clontech), PhosphoAKT (Thr308) (C31E5E) (Cell Sgnalng), PhosphoAKT (Ser473) (D9E) (Cell Sgnalng), antgst (91G1) (Cell Sgnalng), goat antrabbt IgGAP (Vector Labs), goat antmouse IgGAP (Jackson ImmunoResearch). Immunoreactve bands were vsualzed usng Western Blue stablzed substrate. Western blots were quanttated usng ImageJ software (v1.43, (see Supplemental Fg. S1). Hgh content cellular assay PDK1CH cells were plated out at 3000 cells/well n 384well plates (Aurora Boscences, San Dego, CA). After 24 h the cells were washed 3 tmes wth Ham s F12 contanng 1% pencllnstreptomycn, 5 mm Hepes 0.1% FBS, and 0.1% BSA, and cultured for 2 h. Compounds contanng 0.3% DMS fnal were added n a 4X volume n assay meda (.e., Hams F12 contanng 1% pencllnstreptomycn, 5 mm Hepes, and 0.1% BSA) and ncubated for 2 h. Assay meda wth or wthout 1 mg/ml (4X) recombnant human IGF1 were added to the cell culture usng a Janus lqud handler wth a 384well head from Perkn Elmer. The supernatants were mxed by ppettng and allowed to ncubate for 4 mn at ambent room

5 182 PDK1 and AKT sgnalng pathway / A. Hofler et al. / Anal. Bochem. 414 (2011) temperature. After ncubaton, the cells were fxed wth the addton of an equal volume of a neutral buffered 10% formaln soluton (SgmaAldrch). Followng fxaton, cells were permeablzed wth 0.5% Trton X100 n Dulbecco s PBS wthout calcum and magnesum for 30 mn and blocked wth 1.0% BSA n PBS for 2 h. Prmary anybodes were added n a stanng buffer (.e., PBS contanng 0.3% Trton X100 and 0.5% FBS) overnght at 4 C n a humdfed chamber. Plates were washed thoroughly usng a Botek ExL405 plate washer. Secondary antbodes n stanng buffer were added and ncubated 2 h at room temperature. Cells were washed n a soluton contanng 0.5 lg/ml Hoechst and 2 lg/ml HCS CellMask Deep Red. The plates were maged usng a Perkn Elmer pera equpped wth a UV lght source, 488, 532, and 633 nm lasers. Analyss of the mages was completed usng Acapella algorthms custom desgned for each assay. Results PDK1 and AKT1/AKT2 actvty n the presence of TDA 2.0 PDK1 actvty was measured usng a small 14mer 5FAMlabeled peptde (for peptde sequence see Materals and methods) n the presence and n the absence of TDA 2.0. As llustrated n Fg. 2a and b, the addton of lpdbased partcles n the assay buffer boosts the PDK1 enzyme actvty by 4 to 5fold for the catalytc doman and 20fold for the fulllength enzyme as compared to the enzyme alone. Also, data n Fg. 2c show that the actvaton occurs only n the presence of Hstagged PDK1. The actual effect of these artfcal vescles on the PDK1 actvty remans to be fully understood; however, TDA 2.0 contan 2 chelatng moetes creatng a template whch drects the assembly of purfed Hstagged protens whch are normally membrane assocated; ths approach has been utlzed by several research groups wth a broad range of proten classes [23]. Therefore, we speculate that t promotes assembly of relevant membraneassocated conformaton, stmulatng the transautophosphorylaton and subsequently the transactvaton of PDK1 va proten colocalzaton, thus replcatng the normal cellular effect of PIP 3 recrutment of PDK1 to the membrane va ts PH doman. Further knetc analyss was conducted wth FL PDK1 and TDA 2.0 to determne a K app m and k app cat values of 13.6 ± 2.7 lm and 0.72 ± mn 1 for ATP, respectvely, and 25.5 ± 5.7 lm and 1.8 ± 0.18 mn 1 for the 5FAMpeptde. Unfortunately, we were unable to measure and compare these same constants n the absence of TDA 2.0 due to the lack of sgnfcant PDK1 actvty toward the peptde substrate. The effect of TDA 2.0 was also evaluated on the actvaton of AKT1 and AKT2 by FLPDK1 and mtr. As llustrated n Fg. 3a and b, AKT s readly actvated when FLPDK1, mtr, and TDA 2.0 are smultaneously present n the reacton meda. Interestngly, our expermental data showed that a bref burst of AKT2 actvty was also recorded only n the presence of PDK1 and TDA 2.0 (Fg. 3b); however, the actvty of AKT2 plateaued very rapdly, wthn 20 mn, suggestng that enzyme stablty s negatvely affected when mtr s absent from the assay buffer. These results are n agreement wth prevous studes conducted by Facchnett et al. [24] that dentfy mtr as a key enzyme responsble for the foldng and the stablty of AKT. Western blot analyss Western blot analyss of phoshospecfc antbodes of samples from knase assays ndcates that addton of mtr and PDK1 wth AKT1 ncreases the level of phosphoser473 and phosphothr308 (Fg. 4). Addton of TDA 2.0 sgnfcantly ncreases phosphorylaton on these resdues as well. Surprsngly, Western blot analyss also showed that AKT1 and AKT2 seem to autophosphorylate on Ser473 when TDA 2.0 s present n the reacton meda and that mtr can phosphorylate both resdues, Ser473 and Thr308. Lastly, resdue Thr450 on AKT1 and AKT2 appears to be already phosphorylated pror to addton of mtr and PDK1 to the meda. PDK1 and AKT1 nhbton A few nhbtors from the CAP seres (see lst Table 1) were evaluated aganst FLPDK1. The mechansm of nhbton of these nhbtors has been resolved by prevous crystallography studes [21] whch showed these compounds competng wth the ATP at the knase hnge regon. K values for these compounds are reported n Table 1. ne of these compounds, PF (Fg. 1), was further evaluated to prevent the actvaton of AKT1. Whle the ntal data set showed that the nhbtor can effectvely nhbt the PDK1 actvty n the nanomolar range at hgh concentratons of ATP (.e., K app and a K values of 31.5 ± 2.4 and 0.31 ± 0.04 nm were determned, respectvely), the compound s consderably less effectve n preventng the actvaton of AKT1 when used n a cascade assay (.e., K app = 1.53 ± 0.66 lm, a 70fold ncrease as compared to the K app for PDK1; see Fg. 5). PDK1 and Fox03a translocaton and phosphorylaton of AKT Thr308 n CH cells The PDK1 nhbtor PF was also evaluated n cells for ts ablty to modulate the nsulnlke growth factor1 (IGF1) dependent translocaton of PDK1 to the cell membrane and the phosphorylaton of Thr308AKT. For these experments, a hgh content cellbased assay was developed usng CH cells that were Rate (um / mn) a Rate (um / mn) b [KDPDK1] (nm) [FLPDK1] (nm) Fg.2. The presence of TDA 2.0 lposomes n the reacton meda boosts the actvty of PDK1 actvty. As llustrated n panel a, the actvty of KDPDK1 (catalytc doman) alone ncreases by 4 to 5fold n the presence of these lpdbased vescles (s) as compared to the reacton n the absence of t (d). A greater effect (20fold) was observed n the presence of FLPDK1 (fulllength) when mxed wth the same lpd (s) as compared to the reacton n the absence of t (d) (b). As a control study, panel c shows that the ncrease of PDK1 enzyme actvty can only be observed when the enzyme possesses a Hs tag. The last experment was conducted n the presence of 50 nm enzyme whle peptde, TDA 2.0, and ATP concentratons are smlar to the rest of ths study (see expermental secton).

6 PDK1 and AKT sgnalng pathway / A. Hofler et al. / Anal. Bochem. 414 (2011) a AKT1 AKT2 Rate (um / mn) AKT mtr PDK1 TDA 2.0 % Substrate Converson b tme (sec) Fg.3. The presence of TDA 2.0 lposomes n the reacton meda boosts the actvty of AKT actvty when combned wth PDK1 and mtr (a) used n a cascade assay. Interestngly, the combnaton of PDK1 and TDA 2.0 has a lmted mpact on the actvty of AKT2 (s, b), whle the addton of mtr to the mxture sgnfcantly boosts the stablty and the actvty of AKT2 (d). A smlar effect was observed wth AKT1 too (data not shown). Fg.4. Western blot analyss. The level of phosphorylaton of Ser473, Thr308, and Thr450 of AKT1 (a) and AKT2 (b) was evaluated n the presence of PDK1, mtr, and TDA 2.0. Results showed that commercal AKT enzymes are already phosphorylated on Thr450. By comparson, the level of phosphorylaton for resdues Ser473 and Thr308 vares n the presence of mtr, PDK1, and TDA 2.0. Surprsngly, these data suggest that AKT autophosphorylates on Ser473 (columns 2 and 6, panels a and b) and mtr phosphorylates both resdues, Ser473 and Thr308 (columns 7 and 8, panels a and b). Table 1 K values aganst PDK1 were determned wth a few CAP nhbtors (see Fg. 1) usng mna and Calper assay formats. PDK1 nhbtors K (nm, mna) K (nm, Calper) PF ± 1.4 (n = 2) 10.6 ± 0.5 (n = 2) PF ± 1.1 (n = 3) a 0.3 ± 0.04 (n = 2) PF ± 0.02 (n = 1) 0.6 ± 0.1 (n = 2) PF ± 0.55 (n = 4) a 1.8 ± 0.1 (n = 2) The K app was converted nto K value usng Eq. (2) for compettve nhbtor. a Data from Murphy et al. [19]. engneered to express GFPPDK1. n stmulaton wth IGF1, GFP PDK1 mgrated to the nner surface of the cell membrane (Fg. 6a, upper left panel). Pror treatment of the cells wth PF (50 lm) reduced the rato of membraneassocated versus cytosolc GFPPDK1 (Fg. 6b) after IGF1 stmulaton. A concentratondependent effect was observed for the effect of PF on the membrane/cytosol levels of GFPPDK1 after IGF1 stmulaton wth an IC 50 value of 2.23 ± 0.56 lm (Fg. 7a). Gven the hgh selectvty for PF for nhbton of PDK1 actvty, t s lkely that PF s able to modulate an autophosphorylaton step that s requred for ether translocatng PDK1 to the membrane and/or

7 184 PDK1 and AKT sgnalng pathway / A. Hofler et al. / Anal. Bochem. 414 (2011) V/Vo [Inhbtor] (um) Fg.5. Inhbton of PDK1 and AKT1 by PF PF s a selectve and potent nhbtor of PDK1 (see Supplementary data) wth K app n the nm range (s). Surprsngly, the nhbtor has a lmted effect downstream, on the AKT actvty, and exhbts and a K app n the lm range (d) aganst ths enzyme. mantanng PDK1 at the membrane. Ths s consstent wth prevously publshed reports that PDK1 s autoactvated by dmerzaton and transphosphorylaton at the plasma membrane [25]. In the same cells, IGF1 nduced the phosphorylaton of Thr308AKT whch was blocked when the cells were cotreated wth PF (Fg. 6c and d). The modulaton of IGF1stmulated phosphothr308akt levels by PF followed a concentratondependent response (Fg. 7b) wth an IC 50 value of 1.65 ± 0.3 lm that was consstent wth the nhbton of IGF1nduced translocaton of GFPPDK1 to the membrane. To further nvestgate the mpact of our nhbtor on the AKT pathway, the translocaton of Fox03 from the nucleus to the cytoplasm was also evaluated. As llustrated n Fg. 6e and f (lower panel) and Fg. 7b, the compounds prevent the mgraton of Fox03 to the cytoplasm wth IC 50 values of 6.71 ± 1.3 lm. Interestngly, the supermposton of the dose response curves for pthr308 and Fox03 translocaton clearly shows that the modulaton of these bomarkers s well correlated, wth smlar md ponts n the mcromolar range. Dscusson n actvaton by RTKs, the recrutment of PDK1 to the membrane trggers a cascade of events that ncludes the autoactvaton of PDK1. In turn, PDK1 phosphorylates and actvates several downstream knases such as AKT, SGK3, and S6K. As descrbed by Wck et al. [25], PDK1 s autoactvated through a seres of wellcoordnated events that requres the dmerzaton of the enzyme through the PH doman and transautophosphorylaton n the actvaton loop (.e., Aloop). Several studes have revealed that dockng stes such as the PIF doman located on the PDK1termnal doman can also play a crtcal role n the regulaton of the enzyme actvty [26,27]. In partcular, the nteractons between ether large peptdes or small lgands wth these dockng stes nduce changes n the proten conformaton and lead to an ncrease of enzyme actvty [28 30]. Interestngly, we have also been able to enhance the enzyme actvty by addng TDA 2.0, n the reacton meda. These vescles were added n order to mmc the cellular envronment and to reproduce the cascade of events that leads to the PDK1 actvaton. As reported n ths study, a 4 to 5fold and 20fold ncreases of enzyme actvty were observed n the presence of a small artfcal peptde (14mer) wth ether the catalytc doman or the fulllength PDK1, respectvely. Although the mechansm of actvaton of ths enzyme remans unclear, t s lkely that PDK1 bnds to TDA 2.0 through the Hstag and establshes dmers, or hgher order olgomerc structures. The dmerzaton of ths enzyme would be followed by transautophosphorylaton and autoactvaton. The effect of TDA 2.0 was also studed usng a more ntrcate bochemcal assay that was desgned specfcally to study the actvaton of nactve AKT by PDK1 and mtr knases. As reported n Fg. 3a and b, the presence of these artfcal vescles sgnfcantly boosted the actvaton of AKT1 and AKT2 actvty. Both AKT enzymes showed a burst of actvty that quckly plateaued f coupled wth PDK1 alone. However, AKT dsplayed a greater and more Fg.6. Hgh content analyss of CH cells transfected wth GFPPDK1. The effect of the PF was evaluated n a cellular envronment by measurng the translocaton of GFPPDK1 to the membrane (upper panels, a and b), the level of phosphorylaton of Thr308 (mddle panels, c and d), and translocaton of Fox03a to the nucleus (lower panel, e and f). The modulaton of these bomarkers was evaluated n the absence (left panels) and n the presence of 50 lm PF (left panels).

8 PDK1 and AKT sgnalng pathway / A. Hofler et al. / Anal. Bochem. 414 (2011) Translocaton to the plasma membrane (PDK1) 5 a [PF ] (um) Integrated Spot Intentsty (AKT phospho Thr308) b [PF ] (um) % uclear Translocaton (Fox03a) Fg.7. The effect of PF was evaluated aganst the translocaton of PDK1 to the membrane (d, a) and Thr308 AKT (d, b) and Fox03a translocaton to the nucleus (s, b). For each of these bomarkers an IC 50 values were measured and are reported under Results. lnear rate level of actvty when both enzymes, PDK1 and mtr, were added to the assay (Fg. 3b). Conversely, these two enzymes have very lmted mpact on the AKT actvaton n the absence of these lpds vescles. To further understand ths mechansm of actvaton, a Western blot analyss was conducted n order to dentfy the phosphorylaton state of the key amno acd resdues that have been reported to regulate the enzyme actvty. The results generated are n agreement wth prevous studes [31], whch show that PDK1 phosphorylates resdue Thr308 n the Aloop of AKT. The phosphorylaton of ths amno acd resdue alone s suffcent to actvate AKT to a lmted extent; however, the full actvaton of ths enzyme requres the phosphorylaton of addtonal resdues such as Ser473 n the Ctermnal hydrophobc motf and Thr450 n the turn motf by mtr and other knases. As prevously reported by Facchnett et al. [24], the phosphorylaton of resdues Thr450 and Ser473 plays an mportant role n the stablty of the enzyme whch seems to be consstent wth our knetc and data. Also and smlar to Facchnett s group, the present study shows that AKT autophosphorylates ts own Ser473 resdue. Surprsngly, the last pece of data provded by the Western blot analyss suggests that mtr has the ablty to phosphorylate both resdues Ser473 and Thr308 on AKT (Fg. 4a and b, columns 7 and 8). The data generated wth these lposomes ndcate that we have been able to reproduce, to a lmted extent and n a chemcally defned n vtro assay, the cascade of events that lead to the n vvo actvaton of AKT. In agreement wth recent studes [32], these data also suggest that the presence of PIP 3 and the PH doman are not needed for actvaton of PDK1 or AKT. Therefore, we propose that AKT actvaton s ntated on bndng to TDA 2.0 whch provdes a crtcal membrane context that leads to the exposure of the Aloop and the hydrophobc motf of the Ctermnus, conformatonally alterng AKT to become an optmal substrate for PDK1 and mtr. However, snce HsPDK1 can be substtuted by FLAG(DPH)PDK1 (see Supplemental Fg. S2), and snce GSTtagged mtr also more effcently phosphorylates AKT, the membrane envronment afforded by assocaton wth TDA 2.0, and the conformatonal changes mparted by that assocaton, are lkely to be the crtcal molecular events responsble for actvaton and pharmacology observed here. Separately, mtr phosphorylates Ser473 resultng n full actvaton and ncrease stablty of AKT [33]. The effect of lposomes on the PDK1 actvty was also evaluated n the presence of PDK1 nhbtors from the carbonyl4amnopyrrolopyrmdne seres. A comparatve study was conducted n two dfferent assay formats, mna knetc assay and Calper moblty shft assay. The K values obtaned usng the mna assay were determned wthout TDA 2.0 as opposed to the values determned usng the Calper assay. As reported n Table 1, the values are the same between both assays whch demonstrate that whle nanopartcles ncrease the actvty of the knases, the bndng and nhbton of that actvty by smallmolecule nhbtors remaned unperturbed. ne selectve PDK1 nhbtor from the carbonyl4amnopyrrolopyrmdne seres, PF , was also evaluated to prevent the actvaton of AKT usng a cascade bochemcal assay. Ths compound nhbts PDK1 wth K values n the nanomolar range n the presence and n the absence of lpd vescles. Ths nhbtor was used as a tool to evaluate the nhbton of PDK1 on downstream bomarkers such as the actvaton of AKT. Surprsngly, our bochemcal data show that ths nhbtor does not seem to affect the actvaton of AKT to the same extent; ths compound s actually 70fold less potent n preventng the actvaton of AKT1 n a bochemcal cascade assay. The loss of potency from PDK1 to AKT s unclear; however, the Western blot data suggest an alternatve mode of actvaton for the AKT enzymes whch could be drven by the combnaton of ether PDK1 mtr or by a mechansm of AKT autophosphorylaton and mtr whch was also shown to phosphorylate both resdues, Thr308 and Ser473. Under these crcumstances, selectve nhbton of PDK1 could only have a lmted effect on the rest of the AKT pathway. PF was also ncubated wth CH cells to study the modulaton of several bomarkers such as the translocaton of PDK1 to the membrane, the translocaton of Fox03a to the nucleus, and the phosphorylaton of Thr308 AKT. The study was conducted usng a hgh content cellbased assay. The actvaton of CH cells by IGF shows the mgraton of GFPPDK1 at the nner surface of the cellular membrane. However, the mgraton of PDK1 to the membrane was prevented when the cells were ncubated n the presence of nhbtor. A smlar effect was observed wth Fox03a, whch remaned n the nucleus, suggestng that these compounds can negatvely affect the endogenous cellular AKT actvty and prevent the phoshorylaton of Fox03a. Lastly and smlarly to the work of Sched et al. [3], GFPPDK1 seems to accumulate n the nucleus; however, the presence of PF n the meda has lmted or no effect on the localzaton of PDK1 n the nucleus as llustrated n Fg. 6a and b. In concluson, ths study nvestgated the mechansm of actvaton of PDK1 and AKT n the presence of TDA 2.0. We showed that these artfcal lposomes enhance the PDK1 actvty and could be used n a pseudo n vtro cellular assay to study the actvaton and/or nhbton of the knases from the PI3K/AKT pathway. A new class of potent nhbtors of PDK1 was also nvestgated usng two bochemcal assay formats and our expermental data showed that addton of the nckelchelatng lposomes s sutable for assayng knase sgnalng pathway n the presence of nhbtors. Lastly, the effect of ths compound was also nvestgated n a cellular envronment on the modulaton of several bomarkers such as the translocaton of PDK1 to the membrane, the translocaton of Fox03a n the nucleus, and the phosphorylaton of Thr308 AKT. Appendx A. Supplementary data Supplementary data assocated wth ths artcle can be found, n the onlne verson, at do: /j.ab

9 186 PDK1 and AKT sgnalng pathway / A. Hofler et al. / Anal. Bochem. 414 (2011) References [1] R.W. Matheny, M. Adamo, Current perspectves on AKT AKTvaton and AKTons, Exp. Bol. Med. 234 (2009) [2] Y. Lao, M.C. Hung, Physologcal regulaton of AKT actvty and stablty, Am. J. Transl. Res. 2 (2010) [3] M.P. Sched, M. Parsons, J.R. Woodgett, Phosphonostdedependent phosphorylaton of PDK1 regulates nuclear translocaton, Mol. Cell. Bol. 25 (2005) [4] C.F. Sephton, D. Zhang, T.M. Lehmann, P.R. Pennngton, M.P. Sched, D.D. Mousseau, The nuclear localzaton of 3 0 phosphonostdedependent knase 1 s dependent on ts assocaton wth the proten tyrosne phosphatase SHP1, Cell Sgnal. (2009) [5] D.A. Altomare, J.R. Testa, Perturbatons of the AKT sgnalng pathway n human cancer, ncogene 24 (2005) [6] S.B. Vestey, C. Sen, C.J. Calder, C.M. Perks, M. Pgnatell, Z.E. Wnters, Actvated AKT expresson n breast cancer: correlaton wth P53, Hdm2 and patent outcome, Eur. J. Cancer 41 (2005) [7] R.L. Dllon, D.E. Whte, W.J. Muller, The phosphatdyl nostol 3knase sgnalng network: mplcatons for human breast cancer, ncogene 26 (2007) [8] A. Carracedo, P. Pandolf, The PTEPI3K pathway: of feedbacks and crosstalks, ncogene 27 (2008) [9] A. Denley, S. Kang, U. Karst, P.K. Vogt, ncogenc sgnalng of class I PI3K soforms, ncogene 27 (2008) [10] Y. Samuels et al., Hgh frequency of mutatons of the PIK3CA gene n human cancers, Scence 304 (2004) 554. [11] A. Carnero, C. BlancoAparco,. Renner, W. Lnk, J.F. Leaf, The PTE/PI3K/AKT sgnalng pathway n cancer, therapeutc amplfcatons, Curr. Cancer Drug Targets 8 (2008) [12] S.V. Bhagwat, A.P. Crew, ovel nhbtors of mtrc1 and mtrc2, Curr. pn. Invest. Drugs 11 (2010) [13] P. Workman, P.A. Clarke, F.I. Raynaud, R.L. van Montfort, Druggng the PI3 knome: from chemcal tools to drugs n the clnc, Cancer Res. 15 (2010) [14] P. Cohen, D.R. Aless, D.A. Cross, PDK1, one of the mssng lnks n nsuln sgnal transducton?, FEBS Lett 410 (1997) [15] P. Flynn, M. Wongdagger, M. Zavar,.M. Dean, D. Stokoe, Inhbton of PDK1 actvty causes a reducton n cell prolferaton and survval, Curr. Bol. 10 (2000) [16] Z. Xe, X. Zeng, T. Waldman, R.I. Glazer, Transformaton of mammary epthelal cells by 3phosphonostdedependent proten knase1 actvates betacatenn and cmyc and downregulates caveoln1, Cancer Res. 63 (2003) [17] X. Zeng, H. Xu, R.I. Glazer, Transformaton of mammary epthelal cells by 3 phosphonostdedependent proten knase1 (PDK1) s assocated wth the nducton of proten knase Calpha, Cancer Res. 62 (2003) [18] J.R. Bayascas,.R. Lesle, R. Parsons, S. Flemng, D.R. Aless, Hypomorphc mutaton of PDK1 suppresses tumorgeness n PTE(/ ) mce, Curr. Bol. 25 (2005) [19] C. Pefer, D.R. Aless, Smallmolecule nhbtors of PDK1, ChemMedChem 3 (2008) [20] GraphPad Prsm, verson 5.01, GraphPad Software Inc., San Dego. [21] S. Murphy, S. Baley, S. Bergqvst, B.J. Burke, S. Greasley,. Kablaou, J.C. Kath, S. Kazmrsk, S. Kupchnsky, M.A. Marx, D. Rchter, K. Tran, W. Verner, G. Alton, S. Bax, J. Ermoleff, L. Lngardo, J. Xe, M.J. Yn, ovel, Potent and Selectve Small Molecule Inhbtors of PDK1, Abstracts of the AACR Annual Meetng, Washngton, DC, USA, 2010, Abstract 753. [22] J.F. Morrson, Knetcs of the reversble nhbton of enzyme catalysed reactons by tghtbndng nhbtors, Bochm. Bophys. Acta 185 (1969) [23] S. Grdley, A.L. Shrout, E.A. Esposto, Challenges and approaches for assay development of membrane and membraneassocated protens n drug dscovery, Prog. Mol. Bol. Transl. Sc. 91 (2010) [24] V. Facchnett et al., The mammalan target of rapamycn complex 2 controls foldng and stablty of Akt and proten knase C, EMB J. 27 (2008) [25] M.J. Wck, J.R. Fresnda, H. Chen, M.J. Quon, L.Q. Dong, F. Lu, Mouse 3 phosphonostdedependent proten knase1 undergoes dmerzaton and transphosphorylaton n the actvaton loop, J. Bol. Chem. 44 (2003) [26] A. Mora, D. Komander, D.M.F. van Aalten, D.R. Aless, PDK1, the master regulator of AGC knase sgnal transducton, Semn. Cell Dev. Bol. 14 (2004) [27] R. Bond, Phosphonostdedependent proten knase 1, a sensor of proten conformaton, revew, Trends Bochem. Sc. 3 (2004) [28] M. Engel et al., Allosterc actvaton of the proten knase PDK1 wth low molecular weght compounds, EMB J. 25 (2006) [29] V. Hnde, A. Stroba, H. Zhang, L.A. LopezGarca, L. Idrssova, S. Zeuzem, D. Hrschberg, F. Schaeffer, T.J.D. Jorgensen, M. Engel, P.M. Alzar, R.M. Bond, Structure and allosterc effects of lowmolecularweght actvators on the proten knase PDK1, at. Chem. Bol. 5 (2009) [30] R.M. Bond, P.C. Cheung, A. Casamayor, M. Deak, R.A. Curre, D.R. Aless, Identfcaton of a pocket n the PDK1 knase doman that nteracts wth PIF and the Ctermnal resdues of PKA, EMB J. 19 (2000) [31] D.R. Aless, S.R. James, C.P. Downes, A.B. Holmes, P.R. Gaffney, C.B. Reese, P. Cohen, Characterzaton of a 3phosphonostdedependent proten knase whch phosphorylates and actvates proten knase Ba, Curr. Bol. 7 (1997) [32] Z. Dng, J. Lang, Y. Lu, V. Aryaratna, Z. Lu, M.A. Daves, J.K. Westwck, G.B. Mlls, Physcal assocaton of PDK1 wth AKT1 s suffcent for pathway actvaton ndependent of membrane localzaton and phosphatdylnostol 3 knase, PLoS E 5 (2010) [33] Y. Kawakam, H. shmoto, J. Ktaura, M. MaedaYamamoto, R.M. Kato, D.R. Lttman, M. Letges, D.J. Rawlngs, T. Kawakam, Proten knase C betaii regulates Akt phosphorylaton on Ser473 n a cell type and stmulusspecfc fashon, J. Bol. Chem. 279 (2004)