research article the p85 regulatory subunit of pi3k ... · research article the p85 regulatory...

Research ArticleThe p85 Regulatory Subunit of PI3K Mediates cAMP-PKA andInsulin Biological Effects on MCF-7 Cell Growth and Motility

E Di Zazzo1 A Feola23 C Zuchegna2 A Romano2 C F Donini4 S Bartollino1

C Costagliola1 R Frunzio2 P Laccetti2 M Di Domenico35 and A Porcellini2

1 Department of Medicine and Health Sciences University of Molise Campobasso Italy2 Department of Biology University Federico II of Naples Naples Italy3 Department of Biochemistry Biophysics and General Pathology Second University of Naples Italy4Department of Experimental Medicine Sapienza University of Rome Rome Italy5 Sbarro Institute for Cancer Research and Molecular Medicine Center for Biotechnology Temple UniversityPhiladelphia PA USA

Correspondence should be addressed to M Di Domenico didomenicomarinagmailcom

Received 18 April 2014 Revised 17 June 2014 Accepted 18 June 2014 Published 9 July 2014

Academic Editor Elisabetta Baldi

Copyright copy 2014 E Di Zazzo et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Recent studies have shown that hyperinsulinemia may increase the cancer risk Moreover many tumors demonstrate an increasedactivation of IR signaling pathways Phosphatidylinositol 3-kinase (PI3K) is necessary for insulin action In epithelial cells whichdo not express GLUT4 and gluconeogenic enzymes insulin-mediated PI3K activation regulates cell survival growth and motilityAlthough the involvement of the regulatory subunit of PI3K (p85120572PI3K) in insulin signal transduction has been extensively studiedthe function of its N-terminus remains elusive It has been identified as a serine (S83) in the p85120572PI3K that is phosphorylatedby protein kinase A (PKA) To determine the molecular mechanism linking PKA to insulin-mediated PI3K activation we usedp85120572PI3K mutated forms to prevent phosphorylation (p85A) or tomimic the phosphorylated residue (p85D)We demonstrated thatphosphorylation of p85120572PI3KS83modulates the formation of the p85120572PI3KIRS-1 complex and its subcellular localization influencingthe kinetics of the insulin signaling both onMAPK-ERK andAKTpathways Furthermore the p85120572PI3KS83 phosphorylation plays acentral role in the control of insulin-mediated cell proliferation cell migration and adhesionThis study highlights the p85120572PI3KS83role as a key regulator of cell proliferation and motility induced by insulin in MCF-7 cells breast cancer model

1 Introduction

Over the past decade several studies have shown that insulintherapy may increase the cancer risk [1 2] In additionseveral epidemiological studies have reported that obesepeople and those with type 2 diabetes have a higher riskof developing cancer [3 4] Insulin resistance that occursin obese people and those with type 2 diabetes induces acompensatory increase in insulin levels [5] Some cancercells have increased insulin receptor (IR) content and in thesetting of hyperinsulinemia certain tumorsmay demonstrateincreased activation of IR signaling pathways [6 7] Hyperin-sulinemia in a nonobese mousemodel of type 2 diabetes hasbeen reported to lead to increased mammary tumor growth

[7] Again blocking the insulin receptor (IR) tyrosine kinaseusing the inhibitor BMS-536924 reduced tumor growth inthese mice [7] Moreover reduction of circulating insulinlevels using a beta 3-adrenergic receptor agonist (CL-316243)or downregulation of the IR in cancer cells and xenograftsdecreased tumor growth [8] and reduced cell proliferationand metastasis [9]

The phosphoinositide 3-kinase (PI3K) signaling pathwaycritically regulates cell growth and survival [10 11] Class IAphosphoinositide 3-kinase (PI3K) is a heterodimeric enzymecomposed of a catalytic subunit (p110) and a regulatorysubunit (p85)The regulatory p85 subunit (p85120572PI3K) consistsof several domains including the SH3 domain two prolinerich fragments and two SH2 domains separated by the

Hindawi Publishing Corporatione Scientific World JournalVolume 2014 Article ID 565839 11 pageshttpdxdoiorg1011552014565839

2 The Scientific World Journal

inter-SH2 (iSH2) sequence [12] The p85 adaptor proteinemploys the iSH2 region to bind the p110 catalytic subunit andemploys SH3 and SH2 domains phosphotyrosine residuesand proline-rich motifs to interact with activated tyrosinekinases [13 14] adaptor proteins estrogen and androgenreceptors retinoic acid receptor and PKA [15ndash17] ThePI3K-Akt pathway is activated by binding of p85120572PI3K totyrosine-phosphorylated sites on growth factor receptors oron associated adaptor proteins such as IRS-1 (insulin receptorsubstrate 1) [18] Protein kinase B (PKB) also called Akt is awell-known target of PI3K and regulates multiple biologicalfunctions including gene expression cell cycle survivalinsulin-induced metabolic signals endocytosis and vasculartrafficking cell transformation and oncogenesis [19] TheERK and Akt pathways work coordinately or synergicallyto promote cell growth and progression through the cellcycle [20] The Akt pathway stimulates the uptake of glucose[21] whereas ERK activates immediate-early transcriptionfactors including c-Fos and Ets-1 Because the ERK and Aktpathways are pleiotropic and interconnected it is difficultto determine the distinct contribution of each to the overallproliferative response to growth factors Nonetheless bothphosphorylated Akt and ERK (pAkt and pERK resp) arefrequently used as readouts of proliferative or oncogenicsignalling [22 23]

PI3K plays a critical role in insulin signalling and thep85120572PI3K exerts both positive and negative effects on theinsulin transduction pathways [24 25] The activity of PI3Kis necessary to elicit many of the effects of insulin onglucose and lipidmetabolism indicating that it is an essentialdownstream effector of insulin signalling The principalmechanisms through which insulin activates PI3K appearto be via IRS-12 proteins [26] PI3K interacts with IRS-1and IRS-2 docking proteins and plays a key role in medi-ating insulin signal downstream from IRS-1 that results forexample in MCF-7 cell line in cell cycle progression [22]While necessary for the stability and membrane recruitmentof the p110 catalytic subunit of PI3K p85120572PI3K repressesthe basal activity of p110 in the absence of growth factorstimulation In some cell types p85 is in excess of p110 andfree monomeric p85120572PI3K acts as a negative regulator ofPI3K signalling [23 27] In its unbound form p85120572PI3Ksequesters the adaptor protein IRS-1 forming cytosolic focifree of p110 and therefore limits the extent of PI3K signalingdownstream of the insulin and IGF-1 receptors The SH3domain located in the N-terminal portion of p85120572PI3K isresponsible for the interaction with IRS-1 [28]

We have identified a serine (at codon 83) adjacent toN-terminus SH3 domain in the PI3K regulatory subunitp85120572PI3K that is critical for cell cycle progression cell survivaland migration p85120572PI3KSer83 is phosphorylated by proteinkinase A (PKA) in vivo and in vitro influencing the ability ofSH3 domain to interact with different partners [16 17 29]

The aim of this work was to analyze the role of the SH3domain of p85120572PI3K in insulin signalling used as modelthe breast cancer cell line MCF-7 For this purpose wetested whether phosphorylation of p85120572PI3KS83 modulatesthe ability of p85120572PI3K to form complex with IRS-1 or IRS-2

and therefore influences the insulin signaling in MCF-7 cellsFurthermore we analyzed the biological consequences of theexpression of mutant versions of p85120572PI3K in MCF-7 cellsfocusing on the role of p85120572PI3KS83 in cell motility growthand survival

2 Materials and Methods

21 Cell Cultures and Plasmid Transfections MCF-7 cellswere obtained from American Type Culture Collection(ATCC Rockville MD USA) and cultured as previouslydescribed [16] All experiments were performed in cells aftera serum starvation for 6 h and growing for the time indicatedin the legend of figures with 05 FCS and 2mML-glutamineDMEM (Lonza Verviers Belgium) For treatment MCF-7cells were grown in the same medium supplemented with10 nM insulin (Sigma-Aldrich Co St Louis MO USA) withor without 10 120583gmL PKA inhibitor P9115 (Sigma-AldrichCo St Louis MO USA) for 15 minutes

Plasmids carrying bovine p8WT tagged with FLAG(pSG5-FLAG-p85wt) or its S83 mutated forms p85A(pSG5-FLAG-p85A) and p85D (pSG5-FLAG-p85 D) wereobtained as previously described [16] Transfectionswere per-formed using Lipofectamine-GIBCO BRL Life Technologies(Rockville MD USA) following the manufacturerrsquos instruc-tions In all transfections pEGFPC3 plasmid was includedto determine and normalize transfection efficiency Experi-ments varying in the transfection efficiency above 20 werediscarded All data used derived from experiments in whichtransfection efficiency was greater than 55 Experimentswith difference in transfection efficiency between differentexperimental points above 20 were discarded

22 Western Blot and Immunoprecipitation Lysates fromtransfected MCF-7 cells were separated by SDS-PAGE andimmunoblotted as previously described [17] Antibodiesagainst IRS-1 (sc-560) and 120573-actin were purchased fromSanta Cruz Biotechnology Inc (Santa Cruz CA USA)Antibody against FLAG tag (F7425) was from Sigma Aldrich(Co St Louis MO USA) Antibodies against AKT phos-phorylated AKT (Ser473) Erk12 phosphorylated Erk12(Thr202Tyr204 D13144E) and phospho-(SerThr) PKAsubstrate antibody (9621) were fromCell Signaling Technolo-gies (Beverly MA USA) Antibody against PI3K-p85120572 wasfromUpstate Biotechnology Inc (Lake Placid NYUSA) Forimmunoprecipitation cell lysates (1mg) were precleared with1 120583g of Normal IgG and then incubated with 4120583g of anti-bodies against p85120572PI3K (SC-1637 Santa Cruz BiotechnologyInc Santa Cruz CA USA) or against FLAG tag (A4596Sigma Aldrich Co St Louis MO USA) Immunocomplexeswere precipitated following the manufacturerrsquos instructionsand processed for Western blot analysis as described Imageanalysis for all gels was performedwith ImageJ software usingthe ldquoGel Plotrdquo plug-in

23 Confocal Laser Scanning Microscopy MCF-7 cellsexpressing p85120572PI3K WT or its mutants were seeded on12mmglass coverslips with serum starved for 6 h and treated

The Scientific World Journal 3

with 10 nM insulin for 10 and 20 minutes As internal controlMCF-7 cell lines were treated for 30 minutes with FBS Afterincubation cells were fixed for 20minwith paraformaldehyde(3 wv in PBS) permeabilized for 20minwith Triton X-100(02 vv in PBS) and incubated for 1 h with PBS containingFCS (1 vv) Coverslips were stained by incubation withanti-FLAG and anti-IRS-1 antibodies diluted 1 1000 in PBSfor 3 h followed by three washings with PBS Coverslips werethen incubated with Alexa-Fluor 488 anti-rabbit 1 1000 andAlexa-Fluor 633 anti-mouse 1 1000 in PBS for 1 h Cellsstained with Alexa-Fluor 488 anti-rabbit and Alexa-Fluor633 anti-mouse alone were used for the background control(Figure S1A in Supplementary Material available online athttpdxdoiorg1011552014565839) All coverslips werewashed three times in PBS incubated for 10min with PBScontaining Hoechst 33258 (Sigma) at a final concentrationof 1mgmL and finally washed three times with PBS Thecoverslips were mounted in Mowiol (Calbiochem CA) onglass slides All images were captured with Zeiss confocalmicroscope 510Themicrophotographs were analyzed for thecolocalization with ImageJ software using the colocalizationfinderThe JACoP plug-insandwas used for theVan Steenselrsquoscross-correlation function (CCF) (Figure S1B) and PearsonrsquosCoefficient (Figure 2) [30] The fluorophores were imagedseparately to ensure no excitationemission wavelengthoverlap For the transfection efficiency cells were stainedwith anti-FLAG primary antibody and Alexa-Fluor 488anti-mouse antibody as previously described and analysedby FACS

24 Clonogenic Assay Clonogenic assay was performed aselsewhere described [31] MCF-7 cells (3 times 102) expressingp85120572PI3K WT or its mutants were resuspended in DMEMsupplemented or not with 10 nM insulin and seeded into 6well plates and cultured for 15 days in 5 nM insulin untilcells have formed sufficiently large clones (at least 50 cells)Clones were counted after 301015840 fixing with a mixture of 6glutaraldehyde and 05 crystal violet with ImageJ softwareusing the ldquoanalyze particlesrdquo routine

25 Cell Growth Analysis Proliferation was assessed by cellcounting and by MTT assays using the 3-(45-dimeth-ylthiazol-2-yl)-25-biphenyltetrazolium bromide (MTT)(Sigma-Aldrich Co St Louis MO USA) as previouslydescribed [17]

26 Cell Cycle Analysis Cell cycle analysis was performedby fluorescence-activated cell sorting (FACS) as elsewheredescribed [17] Fluorescence was determined by using theFACScanto II Flow Cytometer (Becton Dickinson FranklinLakes NJ USA)

27 Wound Healing Assay Wound healing assay was per-formed as elsewhere described [31] Confluent MCF-7 cellswere scratched with a 200 120583L pipette tip and cellular debrishas been removed by washing gently Following woundingculture medium was replaced with fresh medium containing05 FCS and cells were exposed to insulin 10 nM for12 h Images were acquired on a phase contrast microscope

Axio Observer (Carl Zeiss Inc Oberkochen Germany)and wound diameter was measured at 0 and 12 h using thecomputing software Axio Vision (Carl Zeiss Inc)

28 Statistical Analyses All data are presented as the meansplusmn SD of at least three experiments in triplicates (119899 ge 9)Statistical significance between groups was determined usingStudentrsquos 119905-test (matched pairs test or unmatched test wereused as indicated in the figure legends) All statistical analyseshave been performed using JMP Software purchased byStatistical Discovery SAS Institute (119875 lt 005 statistical signif-icance 119875 lt 0001 high statistical significance)

3 Results31 Role of Phosphorylation of p851205721198751198683119870S83 in the Interactionwith IRS-1 We have previously identified a serine (at codon83) in the p85120572PI3K molecule that is phosphorylated invivo and in vitro by PKA [17] To demonstrate whetherS83 is phosphorylated in PKA-dependent manner followinginsulin treatment we have untreated and treatedMCF-7 cellswith insulin in the presence and in the absence of PKAinhibitor (PKAi) as described in material and methods Totalprotein extractwas immunoprecipitatedwith antibodies anti-p85120572 and probed with the phospho-(SerThr) PKA substrateantibody Figure 1(a) shows that the S83-p85120572 is slightlyphosphorylated in starved cells Insulin treatment (10 nM)for 10min greatly increases S83 phosphorylation in p85120572-PKA-dependent manner because it is prevented by 15minpretreatment with a selective inhibitor of PKA (PKAi frag-ment 14ndash22) To determine the relevance of this site in theformation of the sequestrating complex IRS-1p85120572PI3K [24]we have substituted Ser83 with alanine (p85A) to preventphosphorylation or with aspartic acid (p85D) to mimic thephosphorylated residue As this complex canmediate cAMP-PI3K effects on growth and survival we set out to determinethe formation of IRS-1p85120572PI3K complex in cells expressingwild type p85120572PI3K or p85A or p85D MCF-7 were trans-fected with p85120572PI3K wild type or p85A or p85D and 36 hafter transfection the cells were serum starved for 6 h before10min insulin (10 nM) Cell lysates were immunoprecipitatedwith anti-p85 antibody and then analyzed by Western blotwith anti-IRS-1 antibody In the presence of insulin p85120572PI3Kwild type efficiently was found associated in a protein com-plex with IRS-1 This association was significantly inhibitedin cells expressing p85A and stimulated in p85D expressingcells In the absence of insulin IRS-1p85120572PI3K complex wasbarely detectable in the p85A expressing cells In p85D-expressing cells however in absence of insulin a robustassociation of p85120572PI3K with IRS-1 suggests that Ser83 inp85120572PI3K cooperates with another insulin-mediated signal toregulate p85120572PI3K association with IRS-1 (Figure 1(b)) Theinteraction specificity was assessed using IgG immunopre-cipitates as negative control p85120572PI3K IRS-1 and 120573-tubulinexpression levels in input lysates were shown (Figure 1(c))Similar results were obtained in different immunoprecipitionexperiments performed with anti-IRS-1 and with anti-FLAGantibodies (Figure S2) Changes in the IRS-1p85 ratio in cells


Ip p85120572

++ +

+minus minus

minusminus ++ +

+minus minus

minusminusInsulinPKAi

InsulinPKAi

Ip control Ig

WB P-ST WB P-ST

p85120572 p85120572

InputWB p85120572 WB p85120572

(a)

Ip p85120572

+minus +minus +minus +minusInsulin

Ip control Ig

WB IRS1

p85120572

FLAG

Ctrl p85WTp85WT

p85Ap85A

p85Dp85D

(b)


WB IRS1

p85120572

FLAG

Ctrl p85WT p85A p85DInput

120573-Tubulin

(c)

0

50

100

150

200

250

Ctrl p85WT p85A p85D

+Insulin05 FCS

IRS1

p85

ratio

( o

f con

trol)

lowast

lowast

lowast

lowastlowast

∘ ∘

(d)

Figure 1 Phosphorylation of p85120572PI3KS83 modulates the IRS-1p85 interaction Total protein extract obtained from MCF-7 cells untreatedand treated with 10 nM insulin for 10min in the pretreated or not with 10 120583gmL PKAi for 15min was immunoprecipitated with anti-p85120572antibody and probed with anti-phospho (SerThr) PKA substrate antibody (P-ST) (a) MCF-7 cells transfected with p85WT or p85A or p85Dwere serum starved for 6 h and treated with 10 nM insulin for 10min Cell lysates were immunoprecipitated with anti-p85 antibody and thenanalysed by Western blot with anti-IRS-1 anti-p85 and anti-FLAG antibodies (b) p85120572PI3K IRS-1 and 120573-tubulin expression levels in inputlysates were shown (c) The upper bands in the insert of IRS-1 are not displaced by the control peptide and are considered nonspecific signals(IRS1-specific bands are indicated by the arrow) The histogram (d) represents the quantitation of IRS-1p85120572PI3K signals ratio obtained fromdifferent co-IP experiments Tominimize differences in transfection andor antibody efficiency the data shown in the panel (d) were obtainedby the comparison of different experiments performed with anti-p85120572 (three experiments) anti-IRS-1 (three experiments) and anti-FLAG((three experiments see Figure S2)The histograms show the fold variation relative to the untreated control cells of the ratio IRS-1p85 Valuesare expressed asmeanplusmnSD (119899 = 7) Differences between treatments were tested for statistical significance using Studentrsquos matched pairs 119905-test∘

119875 lt 001 versus 05 FCS treated cells lowast119875 lt 001 versus nontransfected cells lowastlowast119875 lt 001 versus p85120572PI3K wild-type or p85D expressingcells or control cells after insulin treatment

expressing the p85 mutants and determined by comparingall experiments are summarized in the histogram shown inFigure 1(d) Transfection efficiency hasmonitored bymeasur-ing the GFP expression (see Section 2) and only transfectionwith efficiency higher than 55 was taken (Figure S3A)These findings collectively suggest that phosphorylation by

cAMP-PKA is required for the modulation of interactionbetween endogenous IRS-1 and p85 in MCF-7 cells

32 p851205721198751198683119870S83 Phosphorylation Role in the SubcellularLocalization of p85120572 and IRS-1 Besides its conventional roleIRS-1 has been found in the nuclear compartment in several


Insulin(min)

Ctrl

FLAG

IRS1

Merge

0998400 10998400 20998400

(a)

0998400 10998400 20998400p85WT

(b)

0998400 10998400 20998400p85AInsulin

FLAG

IRS1

Merge

(min)

(c)

0998400 10998400 20998400p85D

(d)

Figure 2 Role of phosphorylation of p85120572PI3KS83 in the subcellular localization of p85120572 and IRS-1 MCF-7 cells transiently transfectedwith p85WT or p85A or p85D were seeded on 12mm glass coverslips with serum starved for 6 h and treated with 10 nM insulin for 10 and 20minutes As internal control cells were treated for 30minutes with FBS Immunofluorescence stainingwas performed as described inMaterialsandMethods After incubation cells were fixed permeabilized and stained with anti-FLAG and anti-IRS-1 antibodies Pearsonrsquos coefficient isshown in each panel overlapping images and Van Steenselrsquos cross-correlation functions (CCFs) were calculated for each experimental point(see Figure S1B) Transfection efficiency was determined by flow cytometry using anti-FLAG antibodies (see Section 2) and is shown in FigureS3B

cell types including breast cancer cells and breast tumors [3233] where it functions as transcriptional coregulator for RNApolymerases I and II [34] In vivo transgenicmousemodels ofbreast cancer showed that loss of IRS-1 enhances breast cancermetastasis supporting the hypothesis that IRS-1 may havea metastasis suppressor function [35] In addition nuclearIRS-1 may be a useful marker to predict tamoxifen responsein patients with early breast cancer because a reduction inthe nuclear localization of IRS-1 has a negative prognosisPrevious reports demonstrate that IRS-1 is chaperoned to thenucleus by other proteins [31 33]

In order to assess the relevance of p85120572PI3KS83 with thesubcellular localization of IRS-1MCF-7 were cells transientlytransfected with a construct containing p85120572PI3K wild typeor its S83 mutated forms seeded on 12-mm glass coverslipswith serum starved for 6 h and treated with 10 nM insulinfor 10 and 20 minutes (for transfection efficiency see FigureS3B) Results demonstrate that insulin induces a rapid andtransient formation of IRS-1p85120572PI3K colocalization foci atboth cytosolic and nuclear levels (Figure 2 and Figure S1B)In MCF-7 cells expressing p85120572PI3K wild type cytosolic-golgi as well as nuclear colocalization of IRS-1p85120572PI3K


can be observed IRS-1p85120572PI3K interaction occurs alreadyafter 10 minutes and disappears 20 minutes after expositionto insulin Interestingly in p85A expressing cells there isa marked reduction in the formation of cytosolic IRS-1aggregates and colocalization with p85120572PI3K is not observed(Figure 2) Conversely cell expressing p85D showed boththe IRS-1 cytosolic foci and IRS-1p85120572PI3K counteractionin the absence of insulin Treatment with 10 nM insulindid not increase the foci and the protein-protein complexformation significantly (Figure 2(d)) It is worth noting thatthe expression of both mutants (p85A or p85D) reduced thenuclear translocation of IRS-1 suggesting that the presence ofa serine at codon 83 is essential to the nuclear translocation ofIRS-1 and to the IRS-1p85120572PI3K interaction as that occurringin cells that express the mutant p85D

33 p851205721198751198683119870S83 Role in Signal Transduction of Insulin Wedetermined the phosphorylation of AKT and Erk 12 in celllines expressing p85WT or p85120572 mutant in the presence orabsence of insulin (10 nM) The P-AKTtotal AKT ratio aswell as the ratio P-Erktotal Erk a rather accurate index ofAKT and Erk12 activation was determined Cells expressingp85120572PI3KWT the p85Amutants or p85Dmutants were stim-ulated with insulin for various periods of time (see Figure 3)and tested forAKTandErk12 phosphorylation Results showthat insulin induced AKT and Erk12 phosphorylation andthat in cells expressing p85A AKT induction was delayedand Erk12 phosphorylation was anticipated (peak at 15mininstead of 10 minutes for P-AKT peak at 5min instead of10min for P-Erk) (Figure 3) The expression of p85D did notsignificantly affect the kinetics of AKT activation by insulinbut modified the absolute level of activation (Figures 3(a) and3(c) left panel) Induction of P-Erk12 by insulin was reducedand delayed in cells expressing p85D mutant (Figures 3(b)and 3(c) right panel) These data indicate that serine 83 inp85120572 PI3K is important for insulin induction of AKT andErk12 activation

34 Effects of p85120572 Phosphorylation on MCF-7 Cell Prolif-eration and Viability Growth is a complex and pleiotropicprocess which can be altered by different events Expressionof p85120572PI3K driven by a constitutive promoter might inducegrowth alterations per se It is well known that PI3K-AKTis one of the most important pathways promoting survivaland cell growth [10] To investigate the role of p85120572PI3Kmutants in the control of cell proliferation colorimetricMTTassay and cell cycle analysis by fluorocytometric absorbentcell sorter (FACS) have been performed on MCF-7 cellstransiently transfected with p85WT p85A or p85D or withthe empty vector untreated or treated with insulin (10 nM)Cells were counted at time 0 and at 24 48 72 and 96 hoursColorimetric MTT assay demonstrated that all cells overex-pressing p85120572PI3K showed an inhibition of cell proliferationbut the growth rate observed in cells overexpressing p85Awaslower with respect to that observed in cells overexpressingp85WT or p85D Insulin treatment had a reduced effect onMCF-7 transfected with p85WT Overexpression of p85WTnegatively regulates the insulin signaling (see Figure 3(c))

leading to a reduced growth response to the hormone Cellstransfected with p85A respond better to insulin treatmentbecause p85A unable to sequester IRS-1 and induce anearlier and powerful activation of Erk 12 (see Figures 3(b)and 3(c))

FACS analysis confirmed that cells expressing p85Ashowed a lower proliferation rate in basal conditions asdemonstrated by a robust reduction of the percentage of cellsaccumulated in S and G2 but are more sensitive to insulintreatment (Figure 4(b))

We also analyzed the ability to form colonies of the cellsexpressing the wild type and the mutant p85120572PI3K 200 cellsexpressing the various p85120572PI3K were seeded in sixwell platesin the presence of insulin (10 nM) Twenty-four hours laterinsulin was removed or lowered to 5 nM Fifteen days laterthe clones were counted and their cellularity was evaluated bycontrast microscopy Expression of p85A mutant induces theformation of a significant lower number of colonies than thecontrol and p85WT Cell expressing the p85Dmutant formeda large number of clones compared to the control and p85WTIn the presence of 5 nM insulin the control p85A and p85WTexpressing cells increased the number of clones while p85Dexpressing cells did not do so (Figure 4(c))

35 Role of Phosphorylation of p851205721198751198683119870S83 in the Control ofMotility Behavior In many cell types PI3K has multiple rolesin cellmigrationThe role of phosphorylation of p85120572PI3KS83in MCF-7 cellsrsquo motility was investigated by performingwound healing assays in presence or not of insulin (10 nM)According to our previously published data the resultsdemonstrated that expression of p85A had an effect per seonMCF-7 migration displaying a reduction of cell migration[31] Insulin treatment induced motility in cells overexpress-ing empty vector while it did not influence the motility ofcells overexpressing p85WT The better responsiveness toinsulin administration was detectable in cells overexpressingp85A (Figure 5) Our data demonstrated that p85120572PI3KS83was essential both for the control of motility behaviour andfor the induction of motility induced by insulin

4 Conclusions

Most cancers express both the insulin receptor and the IGF1Rgenes At the cellular level signaling downstream of insulinreceptors and hybrid receptors is similar but not identicalIn each case the kinase activity of the receptor leads tophosphorylation ofmembers of the insulin receptor substrate(IRS) family of proteins and this leads to activation of PI3KAKT and various downstream networks [36] Howeverdifferent cell types use this control system to regulate differentprocesses For example two major consequences of pathwayactivation in liver are the inhibition of gluconeogenesis andthe activation of glycogenosynthesis By contrast epithelialcells do not express gluconeogenic enzymes and the activa-tion of this pathway leads to the stimulation of proliferationand the inhibition of apoptosis The data presented hereinindicated the relevance of p85120572PI3KS83 in the response to


+Insulin(min)

Ctrl0 5 10 15 0 5 10 15 0 5 10 15 0 5 10 15

p85WT p85A p85D

P-AKT

AKT

(a)

P-ERK12

ERK12

120573-Actin

+Insulin(min)

Ctrl0 5 10 15 0 5 10 15 0 5 10 15 0 5 10 15

p85WT p85A p85D

(b)

00

02

04

06

08

10

12

14

16

0 5 10 15 0 5 1015 0 5 1015 0 5 10 15

Ratio

+Insulin(min)

+Insulin(min)Ctrl p85WT p85A p85D

P-AKTAKT

lowast

lowast

lowastlowast

P-ERKERKlowast

lowast

lowastlowast

00

05

15

10

20

25

30

0 5 10 15 0 5 1015 0 5 1015 0 5 1015

Ratio

Ctrl p85WT p 85A p85D

(c)

Figure 3 Phosphorylation of p85120572PI3KS83modulates postreceptorial transductionMCF-7 cells were transiently transfectedwith a constructcontaining p85WT or its S83 mutated forms and treated with 10 nM insulin for 5 10 or 15min AKT (a) and Erk12 (b) phosphorylation weredetermined through Western blot experiments with antibodies against AKT phosphorylated AKT (Ser473) Erk12 and phosphorylatedErk12 (Thr202Tyr204 D13144E)The histograms in the lower panel (c) show the densitometric analysis of the P-AKTAKT and P-ErkErkratio relative to 120573-actin derived from three independent experiments performed in triplicate (119899 = 9) Differences between treatments weretested for statistical significance using Studentrsquos matched pairs 119905-test lowast119875 lt 001 compared to the untransfected cells (same treatment sametime) Transfection efficiency was monitored by measuring the GFP expression (see Section 2) and only transfection with efficiency higher55 was taken (Figure S3A)

the activation of insulin receptor pathways Activation of PI3-kinase in the human breast cancer cell lineMCF-7 by insulin-like growth factor-I results in cell cycle progression and tyro-sine phosphorylation of IRS-1 [37] Experimental and clinicaldata implicate the IGF-IR and PI3K in breast cancer etiology[38] Recently frequent occurrence of p85120572PI3K mutations inglioblastoma and in colorectal pancreatic and breast tumoursamples has been reported [39 40] However the ability androle of these mutations in promoting oncogenesis are notstill understood In primary breast tumors the IGF-IR isoverexpressed and hyperphosphorylated in correlation withunresponsiveness to radiotherapy and tumor relapse [41]

A stable physical interaction between IRS-1 and p85120572PI3Khas been demonstrated in different cell lines This study

demonstrates that IRS-1 interaction with p85120572PI3K dependson the presence of p85120572PI3KS83 and that the phosphorylationat this residue plays an important role in the fine regulationof the bindingThe experiment performedwith p85120572mutantssuggests that p85120572PI3K phosphorylated at S83 by PKA forms acomplexwith the IRS-1 Indeed the substitution of Ser83withalanine (p85A) which prevents the phosphorylation by PKAabolishes the ability of p85120572PI3K to form a complex with IRS-1 both in basal conditions and after stimulation with insulinInsulin treatment induces the IRS-1 phosphorylation result-ing in the binding between IRS-1 and p85120572PI3K Accordingto a previous report [29] the presence of an excess of p85reduces insulin signaling by removing IRS-1 from p85-p110-IRS-1 trimers In MCF-7 the expression of a recombinant


0

2

4

6

8

10

12

0 24 48 72 96

Ctrl

+Insulin

lowastlowast

lowastlowast

lowast

lowast

lowast

lowast lowastlowast

05 FCS+Insulin05 FCS

p85WT

Cel

l gro

wth

(fol

d of

bas

al)

Time (hrs)

0

2

4

6

8

10

12

0 24 48 72 96

Ctrl

+Insulin05 FCS

+Insulin05 FCS

p85D

Cel

l gro

wth

(fol

d of

bas

al)

Time (hrs)

0

2

4

6

8

10

12

0 24 48 72 96

Ctrl

+Insulin05 FCS

+Insulin05 FCS

p85AC

ell g

row

th (f

old

of b

asal

)

Time (hrs)

(a)

6069

27 35

0

20

40

60

80

100

120

+Insulin

G2MS

G1

0

20

40

60

80

100

120

G2MS

G1

0

20

40

60

80

100

120

G2MS

G1

0

20

40

60

80

100

120

G2MS

G1


29 34 29 33

6268 6268

24 37

5874

lowast lowast

Cell

s (

)

05 FCS +Insulin05 FCS +Insulin05 FCS +Insulin05 FCS

(b)

0

20

40

60

80

100

120

140

Col

ony

num

ber

+Insulin

lowast

lowast

lowast

Ctrl

p85WT

p85A

p85D

+Insulin05 FCS

05 FCS


(c)

Figure 4 Effects of p85120572PI3KS83 phosphorylation on MCF-7 cells proliferation and viability (a) MCF-7 cells transiently transfected withp85WT p85A or p85D or with the empty vector were treated with 10 nM insulin or with 05 FCS For the colorimetric MTT assay (A)cells were counted after 0 24 48 72 and 96 hours ( lowastlowast119875 lt 001 compared to the control lowast119875 lt 001 compared to p85WT or p85D) (b) Cellcycle analysis was performed by fluorocytometric absorbent cell sorter (FACS) to determine the percentage of cells in G2M S and G1-phase( lowast119875 lt 001 compared to empty vector or p85WT or p85D) The data are the mean of three independent experiments performed in triplicate(119899 = 9) (c) For the clonogenic assay transfected cells were seeded into 6 multiwell plates in the presence of 10 nM insulin Twenty-fourhours later insulin was removed or lowered to 5 nM Fifteen days later the clones were counted and their cellularity was evaluated by contrastmicroscopy Clones were counted after 301015840 fixing with a mixture of 6 glutaraldehyde and 05 crystal violet The histogram represents theaverage number of colonies of four separate experiments performed in duplicate (119899 = 8) ( lowast119875 lt 001 versus empty vector)



05 FCS

+Insulin12h

(a)

0

10

20

30

40

50

60

70

80

90


+Insulin

lowast

lowast

lowast

05 FCS

Wou

nd h

ealin

g (

)

(b)

Figure 5 Role of phosphorylation of p85120572PI3KS83 in the control of migratory behaviourWound healing assay onMCF-7 cells overexpressingp85WT or mutants untreated or treated with insulin 10 nM for 12 h The white lines indicate the limits of the wound the black lines indicatethe limits after 12 h in the presence of 05 FCS or in the presence of 10 nM insulinThe red lines in the lower part of the upper panel representthe predicted limits of the wound without insulin Histogram represents quantification of the insulin treatment effect on cell motility ( ofwound healing) calculated as ratio between the space delimited by thewhite lines and those delimited by the black ones ( lowast119875 lt 001 comparingversus empty vector) Images are representative of three separate experiments performed in triplicate (119899 = 9) These results were confirmedby a transwell migration assay (data not shown)

p85120572PI3K reduces the insulin signal by attenuating Erk12activation and delaying the activation of AKT Converselythe p85 mutants modify the insulin response in differentextentWe hypothesize that p85A is unable to bind IRS-1 andtherefore make the cells more sensitive to RAS-ERK-MAPKpathway activation by insulin In p85D expressing cells theconstitutive IRS-1p85D interaction partially increases theinsulin sensitivity due to the ability of the p85D mutant tobind and activate the p110PI3K catalytic subunit [15] but shiftsthe insulin response to PI3K-AKT pathway

IRS-1 and p85120572 form a complex influencing the insulinresponse and their subcellular localization [42] The datapresented here demonstrate that in MCF-7 cells expressingp85120572PI3K insulin induces the colocalization of IRS-1 andp85120572PI3K and the formation of IRS-1 cytosolic foci in a timedependent manner The serine at codon 83 has a relevanteffect on the formation of the IRS-1p85120572PI3K complex asdemonstrated by the reduced ability of the p85A and the highefficiency of the p85D mutant to induce an insulin-mediatedIRS-1p85120572PI3K colocalization of IRS-1 and the formation ofcytosolic foci

Recent studies highlighted the importance ofp85120572PI3KS83 in the control of cell growth in different cellularmodels such as FRTL-5 [17] NIH3T3 [16] LNCaP cells [43]and vascular SMCs [44] but it is worth noting that the samephosphorylation leads to opposing phenotypes dependingon the cell type In fact the phosphorylation of p85120572PI3KS83inhibits cell proliferation in fibroblasts and VSMC while itis essential for the correct cell cycle progression in thyroidcells and does not affect cell proliferation of endothelialcells [16 17 44] Our results show that phosphorylation

of p85120572PI3KS83 is essential for MCF-7 cell survival andproliferation as demonstrated by the low growth rateobserved in cells overexpressing the p85A mutant S83 seemsto play a role in mediating the growth effects of insulin Inour model we demonstrate that the expression of p85120572PI3Kmutants is responsible for cell cycle progression inducedby insulin treatment due to a significant reduction in theIRS-1p85120572PI3K complex formation

PI3K isoforms have multiple roles in cell migration inmany cell types and systems but the relative contributionof PI3Ks to different steps of migration depends on the cellstate and the combinations of stimuli to which it is exposed[10] Direct PI3K activation is sufficient to induce cell motilityand invasion [45] To date the role of p85120572PI3KS83 in theinsulin-regulatedmigratory behavior of cells has not yet beenreported This study demonstrates that the overexpression ofp85A induces a highly significant suppression of MCF-7 cellsmotility indicating that the phosphorylation of p85120572PI3KS83is a critical regulator of this process Moreover our datademonstrate that insulin treatment causes a significantincrease of motility only in cells expressing p85A underlyingthat S83 is essential also for insulin-mediated migration

We suggest that serine 83 phosphorylation mediated bycAMP-PKA induces a conformational change in the PI3Kcomplex resulting in a facilitated binding to insulin receptorand to IRS-1 with modulations of PI3K activity p85120572 cAMP-PKAmediated phosphorylation is necessary to modulate theinsulin response in the MCF-7 cells S83 has a pivotal rolein subcellular localization of p85120572 and IRS1 in the timingof AKT and ERK activation cell survival proliferation andmotility We believe that this site is a nodal point whereinformation from several receptors is channeled to PI3K


Conflict of Interests

The authors declare no competing financial interests

Authorsrsquo Contribution

Di Zazzo E and Feola A have equally contributed to thiswork

Acknowledgments

This work was partly supported by University Federico IIof Naples and by POR Campania FSE 2007ndash2013 ProjectCREMe that supported C Zuchegna and A Romanorsquos post-doctoral fellowship CIISOmdashConsorzio InteruniversitarioInternazionale per lo Sviluppo dellrsquoOncologia EpigenomicsFlagship Project-EPIGEN CNR

References

[1] L G Hemkens U Grouven R Bender et al ldquoRisk of malig-nancies in patients with diabetes treated with human insulin orinsulin analogues a cohort studyrdquo Diabetologia vol 52 no 9pp 1732ndash1744 2009

[2] C J Currie C D Poole and E A M Gale ldquoThe influence ofglucose-lowering therapies on cancer risk in type 2 diabetesrdquoDiabetologia vol 52 no 9 pp 1766ndash1777 2009

[3] S S Coughlin E E Calle L R Teras J Petrelli andM JThunldquoDiabetes mellitus as a predictor of cancer mortality in a largecohort of US adultsrdquoTheAmerican Journal of Epidemiology vol159 no 12 pp 1160ndash1167 2004

[4] E E Calle C Rodriguez K Walker-Thurmond and M JThun ldquoOverweight obesity and mortality from cancer in aprospectively studied cohort of US adultsrdquo The New EnglandJournal of Medicine vol 348 no 17 pp 1625ndash1638 2003

[5] G M Reaven ldquoPathophysiology of insulin resistance in humandiseaserdquo Physiological Reviews vol 75 no 3 pp 473ndash486 1995

[6] V Papa V Pezzino A Costantino et al ldquoElevated insulinreceptor content in human breast cancerrdquo Journal of ClinicalInvestigation vol 86 no 5 pp 1503ndash1510 1990

[7] R Novosyadlyy D E Lann A Vijayakumar et al ldquoInsulin-mediated acceleration of breast cancer development and pro-gression in a nonobese model of type 2 diabetesrdquo CancerResearch vol 70 no 2 pp 741ndash751 2010

[8] Y Fierz R Novosyadlyy A Vijayakumar S Yakar and DLeRoith ldquoInsulin-sensitizing therapy attenuates type 2 diabetes-mediatedmammary tumor progressionrdquoDiabetes vol 59 no 3pp 686ndash693 2010

[9] H Zhang D H Fagan X Zeng K T Freeman D Sachdev andD Yee ldquoInhibition of cancer cell proliferation andmetastasis byinsulin receptor downregulationrdquo Oncogene vol 29 no 17 pp2517ndash2527 2010

[10] L CCantley ldquoThephosphoinositide 3-kinase pathwayrdquo Sciencevol 296 no 5573 pp 1655ndash1657 2002

[11] R J Cain and A J Ridley ldquoPhosphoinositide 3-kinases in cellmigrationrdquo Biology of the Cell vol 101 no 1 pp 13ndash29 2009

[12] M A Krasilnikov ldquoPhosphatidylinositol-3 kinase dependentpathways the role in control of cell growth survival andmalignant transformationrdquo Biochemistry vol 65 no 1 pp 59ndash67 2000

[13] N J Hellyer K Cheng and J G Koland ldquoErbB3 (HER3)interaction with the p85 regulatory subunit of phosphoinositide3-kinaserdquo Biochemical Journal vol 333 no 3 pp 757ndash763 1998

[14] C J McGlade C Ellis M Reedijk et al ldquoSH2 domains of thep85120572 subunit of phosphatidylinositol 3-kinase regulate bindingto growth factor receptorsrdquo Molecular amp Cellular Biology vol12 no 3 pp 991ndash997 1992

[15] I Ciullo G Diez-Roux M Di Domenico A Migliaccio andE V Avvedimento ldquocAMP signaling selectively influences Raseffectors pathwaysrdquo Oncogene vol 20 no 10 pp 1186ndash11922001

[16] C Cosentino M Di Domenico A Porcellini et al ldquop85 regu-latory subunit of PI3K mediates cAMP-PKA and estrogensbiological effects on growth and survivalrdquoOncogene vol 26 no14 pp 2095ndash2103 2007

[17] G De Gregorio A Coppa C Cosentino et al ldquoThe p85 regu-latory subunit of PI3K mediates TSH-cAMP-PKA growth andsurvival signalsrdquoOncogene vol 26 no 14 pp 2039ndash2047 2007

[18] A P French S Mills R Swarup M J Bennett and T PPridmore ldquoColocalization of fluorescent markers in confocalmicroscope images of plant cellsrdquo Nature Protocols vol 3 no4 pp 619ndash628 2008

[19] T F Franke C P Hornik L Segev G A Shostak and C Sugi-moto ldquoPI3KAkt and apoptosis sizemattersrdquoOncogene vol 22no 56 pp 8983ndash8998 2003

[20] P J Coffer J Jin and J R Woodgett ldquoProtein kinase B (c-Akt)a multifunctional mediator of phosphatidylinositol 3-kinaseactivationrdquo Biochemical Journal vol 335 no 1 pp 1ndash13 1998

[21] B D Manning ldquoBalancing Akt with S6K implications for bothmetabolic diseases and tumorigenesisrdquo Journal of Cell Biologyvol 167 no 3 pp 399ndash403 2004

[22] A D Kohn S A Summers M J Birnbaum and R A RothldquoExpression of a constitutively active Akt SerThr kinase in 3T3-L1 adipocytes stimulates glucose uptake and glucose transporter4 translocationrdquoThe Journal of Biological Chemistry vol 271 no49 pp 31372ndash31378 1996

[23] J G Jackson M F White and D Yee ldquoInsulin receptorsubstrate-1 is the predominant signaling molecule activatedby insulin-like growth factor-I insulin and interleukin-4 inestrogen receptor-positive human breast cancer cellsrdquo TheJournal of Biological Chemistry vol 273 no 16 pp 9994ndash100031998

[24] J Luo and L C Cantley ldquoThe negative regulation of phosphoin-ositide 3-kinase signaling by p85 and itrsquos implication in cancerrdquoCell Cycle vol 4 no 10 pp 1309ndash1312 2005

[25] J Luo S J Field J Y Lee J A Engelman and L C Cantley ldquoThep85 regulatory subunit of phosphoinositide 3-kinase down-regulates IRS-1 signaling via the formation of a sequestrationcomplexrdquo Journal of Cell Biology vol 170 no 3 pp 455ndash4642005

[26] C M Taniguchi J Winnay T Kondo et al ldquoThe phosphoinos-itide 3-kinase regulatory subunit p85120572 can exert tumor sup-pressor properties through negative regulation of growth factorsignalingrdquo Cancer Research vol 70 no 13 pp 5305ndash5315 2010

[27] P R Shepherd D J Withers and K Siddle ldquoPhosphoinositide3-kinase the key switch mechanism in insulin signallingrdquoBiochemical Journal vol 333 part 3 pp 471ndash490 1998

[28] K Ueki P Algenstaedt F Mauvais-Jarvis and C R KahnldquoPositive and negative regulation of phosphoinositide 3-kinase-dependent signaling pathways by three different gene productsof the p85120572 regulatory subunitrdquoMolecular and Cellular Biologyvol 20 no 21 pp 8035ndash8046 2000


[29] Y Ikegami K Inukai T Awata T Asano and S Katayama ldquoSH3domain of the phosphatidylinositol 3-kinase regulatory subunitis responsible for the formation of a sequestration complexwith insulin receptor substrate-1rdquo Biochemical and BiophysicalResearch Communications vol 365 no 3 pp 433ndash438 2008

[30] C L Neal J Xu P Li et al ldquoOverexpression of 14-3-3120577 in cancercells activates PI3K via binding the p85 regulatory subunitrdquoOncogene vol 31 no 7 pp 897ndash906 2012

[31] C F Donini E Di Zazzo C Zuchegna et al ldquoThe p85120572 regu-latory subunit of PI3K mediates cAMP-PKA and retinoicacid biological effects on MCF7 cell growth and migrationrdquoInternational Journal of Oncology vol 40 no 5 pp 1627ndash16352012

[32] OGMorali VDelmas RMoore C Jeanney J PThiery andLLarue ldquoIGF-II induces rapid120573-catenin relocation to the nucleusduring epithelium to mesenchyme transitionrdquo Oncogene vol20 no 36 pp 4942ndash4950 2001

[33] T Kabuta F Hakuno T Asano and S Takahashi ldquoInsulinreceptor substrate-3 functions as transcriptional activator in thenucleusrdquo Journal of Biological Chemistry vol 277 no 9 pp6846ndash6851 2002

[34] A P Soler K AThompson R M Smith and L Jarett ldquoImmu-nological demonstration of the accumulation of insulin but notinsulin receptors in nuclei of insulin-treated cellsrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 86 no 17 pp 6640ndash6644 1989

[35] Z Ma S L Gibson M A Byrne J Zhang M F White andL M Shaw ldquoSuppression of insulin receptor substrate 1 (IRS-1)promotes mammary tumor metastasisrdquoMolecular and CellularBiology vol 26 no 24 pp 9338ndash9351 2006

[36] D Leroith ldquoInsulin-like growth factor I receptor signalingmdashoverlapping or redundant pathwaysrdquo Endocrinology vol 141no 4 pp 1287ndash1288 2000

[37] G Razzini C P Berrie S Vignati et al ldquoNovel functional PI3-kinase antagonists inhibit cell growth and tumorigenicity inhuman cancer cell linesrdquo The FASEB Journal vol 14 no 9 pp1179ndash1187 2000

[38] EM Fox TWMiller J M Balko et al ldquoA kinome-wide screenidentifies the insulinIGF-I receptor pathway as a mechanismof escape from hormone dependence in breast cancerrdquo CancerResearch vol 71 no 21 pp 6773ndash6784 2011

[39] SNQuayle J Y Lee LW T Cheung et al ldquoSomaticMutationsof PIK3R1 Promote Gliomagenesisrdquo PLoS ONE vol 7 no 11Article ID e49466 2012

[40] L Li S J Plummer C LThompson T C Tucker andG Caseyldquo Association between phosphatidylinositol 3-kinase regulatorysubunit p85alpha Met326Ile genetic polymorphism and coloncancer riskrdquoClinical Cancer Research vol 14 no 3 pp 633ndash6372008

[41] G Hildebrandt J Zierski G Csecsei H W Mueller andH Stracke ldquoResults of continuous long term intravenousapplication of octreotide via an implantable pump system inacromegaly resistent to operative and X-ray therapyrdquo ActaNeurochirurgica vol 117 no 3-4 pp 160ndash165 1992

[42] K L Kelly and N B Ruderman ldquoInsulin-stimulated phos-phatidylinositol 3-kinase Association with a 185-kDa tyrosine-phosphorylated protein (IRS-1) and localization in a low densitymembrane vesiclerdquo Journal of Biological Chemistry vol 268 no6 pp 4391ndash4398 1993

[43] A Feola A Cimini F Migliucci et al ldquoThe inhibition ofp85120572PI3KSer83 phosphorylation prevents cell proliferation and

invasion in prostate cancer cellsrdquo Journal of Cellular Biochem-istry vol 114 no 9 pp 2114ndash2119 2013

[44] D Torella C Gasparri G M Ellison et al ldquoDifferentialregulation of vascular smooth muscle and endothelial cellproliferation in vitro and in vivo by cAMPPKA-activatedp85120572 PI3Krdquo The American Journal of PhysiologymdashHeart andCirculatory Physiology vol 297 no 6 pp H2015ndashH2025 2009

[45] P J Keely J K Westwick I P Whitehead C J Der and L VParise ldquoCdc42 and Rac1 induce integrin-mediated cell motilityand invasiveness through PI(3)Krdquo Nature vol 390 no 6660pp 632ndash636 1997

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


inter-SH2 (iSH2) sequence [12] The p85 adaptor proteinemploys the iSH2 region to bind the p110 catalytic subunit andemploys SH3 and SH2 domains phosphotyrosine residuesand proline-rich motifs to interact with activated tyrosinekinases [13 14] adaptor proteins estrogen and androgenreceptors retinoic acid receptor and PKA [15ndash17] ThePI3K-Akt pathway is activated by binding of p85120572PI3K totyrosine-phosphorylated sites on growth factor receptors oron associated adaptor proteins such as IRS-1 (insulin receptorsubstrate 1) [18] Protein kinase B (PKB) also called Akt is awell-known target of PI3K and regulates multiple biologicalfunctions including gene expression cell cycle survivalinsulin-induced metabolic signals endocytosis and vasculartrafficking cell transformation and oncogenesis [19] TheERK and Akt pathways work coordinately or synergicallyto promote cell growth and progression through the cellcycle [20] The Akt pathway stimulates the uptake of glucose[21] whereas ERK activates immediate-early transcriptionfactors including c-Fos and Ets-1 Because the ERK and Aktpathways are pleiotropic and interconnected it is difficultto determine the distinct contribution of each to the overallproliferative response to growth factors Nonetheless bothphosphorylated Akt and ERK (pAkt and pERK resp) arefrequently used as readouts of proliferative or oncogenicsignalling [22 23]

PI3K plays a critical role in insulin signalling and thep85120572PI3K exerts both positive and negative effects on theinsulin transduction pathways [24 25] The activity of PI3Kis necessary to elicit many of the effects of insulin onglucose and lipidmetabolism indicating that it is an essentialdownstream effector of insulin signalling The principalmechanisms through which insulin activates PI3K appearto be via IRS-12 proteins [26] PI3K interacts with IRS-1and IRS-2 docking proteins and plays a key role in medi-ating insulin signal downstream from IRS-1 that results forexample in MCF-7 cell line in cell cycle progression [22]While necessary for the stability and membrane recruitmentof the p110 catalytic subunit of PI3K p85120572PI3K repressesthe basal activity of p110 in the absence of growth factorstimulation In some cell types p85 is in excess of p110 andfree monomeric p85120572PI3K acts as a negative regulator ofPI3K signalling [23 27] In its unbound form p85120572PI3Ksequesters the adaptor protein IRS-1 forming cytosolic focifree of p110 and therefore limits the extent of PI3K signalingdownstream of the insulin and IGF-1 receptors The SH3domain located in the N-terminal portion of p85120572PI3K isresponsible for the interaction with IRS-1 [28]

We have identified a serine (at codon 83) adjacent toN-terminus SH3 domain in the PI3K regulatory subunitp85120572PI3K that is critical for cell cycle progression cell survivaland migration p85120572PI3KSer83 is phosphorylated by proteinkinase A (PKA) in vivo and in vitro influencing the ability ofSH3 domain to interact with different partners [16 17 29]

The aim of this work was to analyze the role of the SH3domain of p85120572PI3K in insulin signalling used as modelthe breast cancer cell line MCF-7 For this purpose wetested whether phosphorylation of p85120572PI3KS83 modulatesthe ability of p85120572PI3K to form complex with IRS-1 or IRS-2

and therefore influences the insulin signaling in MCF-7 cellsFurthermore we analyzed the biological consequences of theexpression of mutant versions of p85120572PI3K in MCF-7 cellsfocusing on the role of p85120572PI3KS83 in cell motility growthand survival

2 Materials and Methods

21 Cell Cultures and Plasmid Transfections MCF-7 cellswere obtained from American Type Culture Collection(ATCC Rockville MD USA) and cultured as previouslydescribed [16] All experiments were performed in cells aftera serum starvation for 6 h and growing for the time indicatedin the legend of figures with 05 FCS and 2mML-glutamineDMEM (Lonza Verviers Belgium) For treatment MCF-7cells were grown in the same medium supplemented with10 nM insulin (Sigma-Aldrich Co St Louis MO USA) withor without 10 120583gmL PKA inhibitor P9115 (Sigma-AldrichCo St Louis MO USA) for 15 minutes

Plasmids carrying bovine p8WT tagged with FLAG(pSG5-FLAG-p85wt) or its S83 mutated forms p85A(pSG5-FLAG-p85A) and p85D (pSG5-FLAG-p85 D) wereobtained as previously described [16] Transfectionswere per-formed using Lipofectamine-GIBCO BRL Life Technologies(Rockville MD USA) following the manufacturerrsquos instruc-tions In all transfections pEGFPC3 plasmid was includedto determine and normalize transfection efficiency Experi-ments varying in the transfection efficiency above 20 werediscarded All data used derived from experiments in whichtransfection efficiency was greater than 55 Experimentswith difference in transfection efficiency between differentexperimental points above 20 were discarded

22 Western Blot and Immunoprecipitation Lysates fromtransfected MCF-7 cells were separated by SDS-PAGE andimmunoblotted as previously described [17] Antibodiesagainst IRS-1 (sc-560) and 120573-actin were purchased fromSanta Cruz Biotechnology Inc (Santa Cruz CA USA)Antibody against FLAG tag (F7425) was from Sigma Aldrich(Co St Louis MO USA) Antibodies against AKT phos-phorylated AKT (Ser473) Erk12 phosphorylated Erk12(Thr202Tyr204 D13144E) and phospho-(SerThr) PKAsubstrate antibody (9621) were fromCell Signaling Technolo-gies (Beverly MA USA) Antibody against PI3K-p85120572 wasfromUpstate Biotechnology Inc (Lake Placid NYUSA) Forimmunoprecipitation cell lysates (1mg) were precleared with1 120583g of Normal IgG and then incubated with 4120583g of anti-bodies against p85120572PI3K (SC-1637 Santa Cruz BiotechnologyInc Santa Cruz CA USA) or against FLAG tag (A4596Sigma Aldrich Co St Louis MO USA) Immunocomplexeswere precipitated following the manufacturerrsquos instructionsand processed for Western blot analysis as described Imageanalysis for all gels was performedwith ImageJ software usingthe ldquoGel Plotrdquo plug-in

23 Confocal Laser Scanning Microscopy MCF-7 cellsexpressing p85120572PI3K WT or its mutants were seeded on12mmglass coverslips with serum starved for 6 h and treated











Ip p85120572

++ +

+minus minus

minusminus ++ +

+minus minus


InsulinPKAi

Ip control Ig

WB P-ST WB P-ST

p85120572 p85120572

InputWB p85120572 WB p85120572

(a)

Ip p85120572


Ip control Ig

WB IRS1

p85120572

FLAG

Ctrl p85WTp85WT

p85Ap85A

p85Dp85D

(b)


WB IRS1

p85120572

FLAG


120573-Tubulin

(c)

0

50

100

150

200

250


+Insulin05 FCS

IRS1

p85

ratio

( o

f con

trol)

lowast

lowast

lowast

lowastlowast

∘ ∘

(d)







Insulin(min)

Ctrl

FLAG

IRS1

Merge

0998400 10998400 20998400

(a)

0998400 10998400 20998400p85WT

(b)

0998400 10998400 20998400p85AInsulin

FLAG

IRS1

Merge

(min)

(c)

0998400 10998400 20998400p85D

(d)












4 Conclusions



+Insulin(min)

Ctrl0 5 10 15 0 5 10 15 0 5 10 15 0 5 10 15

p85WT p85A p85D

P-AKT

AKT

(a)

P-ERK12

ERK12

120573-Actin

+Insulin(min)

Ctrl0 5 10 15 0 5 10 15 0 5 10 15 0 5 10 15

p85WT p85A p85D

(b)

00

02

04

06

08

10

12

14

16

0 5 10 15 0 5 1015 0 5 1015 0 5 10 15

Ratio

+Insulin(min)


P-AKTAKT

lowast

lowast

lowastlowast

P-ERKERKlowast

lowast

lowastlowast

00

05

15

10

20

25

30

0 5 10 15 0 5 1015 0 5 1015 0 5 1015

Ratio


(c)






0

2

4

6

8

10

12

0 24 48 72 96

Ctrl

+Insulin

lowastlowast

lowastlowast

lowast

lowast

lowast

lowast lowastlowast


p85WT

Cel

l gro

wth

(fol

d of

bas

al)

Time (hrs)

0

2

4

6

8

10

12

0 24 48 72 96

Ctrl

+Insulin05 FCS

+Insulin05 FCS

p85D

Cel

l gro

wth

(fol

d of

bas

al)

Time (hrs)

0

2

4

6

8

10

12

0 24 48 72 96

Ctrl

+Insulin05 FCS

+Insulin05 FCS

p85AC

ell g

row

th (f

old

of b

asal

)

Time (hrs)

(a)

6069

27 35

0

20

40

60

80

100

120

+Insulin

G2MS

G1

0

20

40

60

80

100

120

G2MS

G1

0

20

40

60

80

100

120

G2MS

G1

0

20

40

60

80

100

120

G2MS

G1


29 34 29 33

6268 6268

24 37

5874

lowast lowast

Cell

s (

)


(b)

0

20

40

60

80

100

120

140

Col

ony

num

ber

+Insulin

lowast

lowast

lowast

Ctrl

p85WT

p85A

p85D

+Insulin05 FCS

05 FCS


(c)




05 FCS

+Insulin12h

(a)

0

10

20

30

40

50

60

70

80

90


+Insulin

lowast

lowast

lowast

05 FCS

Wou

nd h

ealin

g (

)

(b)













Acknowledgments


References























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology











Ip p85120572

++ +

+minus minus

minusminus ++ +

+minus minus


InsulinPKAi

Ip control Ig

WB P-ST WB P-ST

p85120572 p85120572

InputWB p85120572 WB p85120572

(a)

Ip p85120572


Ip control Ig

WB IRS1

p85120572

FLAG

Ctrl p85WTp85WT

p85Ap85A

p85Dp85D

(b)


WB IRS1

p85120572

FLAG


120573-Tubulin

(c)

0

50

100

150

200

250


+Insulin05 FCS

IRS1

p85

ratio

( o

f con

trol)

lowast

lowast

lowast

lowastlowast

∘ ∘

(d)







Insulin(min)

Ctrl

FLAG

IRS1

Merge

0998400 10998400 20998400

(a)

0998400 10998400 20998400p85WT

(b)

0998400 10998400 20998400p85AInsulin

FLAG

IRS1

Merge

(min)

(c)

0998400 10998400 20998400p85D

(d)












4 Conclusions



+Insulin(min)

Ctrl0 5 10 15 0 5 10 15 0 5 10 15 0 5 10 15

p85WT p85A p85D

P-AKT

AKT

(a)

P-ERK12

ERK12

120573-Actin

+Insulin(min)

Ctrl0 5 10 15 0 5 10 15 0 5 10 15 0 5 10 15

p85WT p85A p85D

(b)

00

02

04

06

08

10

12

14

16

0 5 10 15 0 5 1015 0 5 1015 0 5 10 15

Ratio

+Insulin(min)


P-AKTAKT

lowast

lowast

lowastlowast

P-ERKERKlowast

lowast

lowastlowast

00

05

15

10

20

25

30

0 5 10 15 0 5 1015 0 5 1015 0 5 1015

Ratio


(c)






0

2

4

6

8

10

12

0 24 48 72 96

Ctrl

+Insulin

lowastlowast

lowastlowast

lowast

lowast

lowast

lowast lowastlowast


p85WT

Cel

l gro

wth

(fol

d of

bas

al)

Time (hrs)

0

2

4

6

8

10

12

0 24 48 72 96

Ctrl

+Insulin05 FCS

+Insulin05 FCS

p85D

Cel

l gro

wth

(fol

d of

bas

al)

Time (hrs)

0

2

4

6

8

10

12

0 24 48 72 96

Ctrl

+Insulin05 FCS

+Insulin05 FCS

p85AC

ell g

row

th (f

old

of b

asal

)

Time (hrs)

(a)

6069

27 35

0

20

40

60

80

100

120

+Insulin

G2MS

G1

0

20

40

60

80

100

120

G2MS

G1

0

20

40

60

80

100

120

G2MS

G1

0

20

40

60

80

100

120

G2MS

G1


29 34 29 33

6268 6268

24 37

5874

lowast lowast

Cell

s (

)


(b)

0

20

40

60

80

100

120

140

Col

ony

num

ber

+Insulin

lowast

lowast

lowast

Ctrl

p85WT

p85A

p85D

+Insulin05 FCS

05 FCS


(c)




05 FCS

+Insulin12h

(a)

0

10

20

30

40

50

60

70

80

90


+Insulin

lowast

lowast

lowast

05 FCS

Wou

nd h

ealin

g (

)

(b)













Acknowledgments


References























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Insulin(min)

Ctrl

FLAG

IRS1

Merge

0998400 10998400 20998400

(a)

0998400 10998400 20998400p85WT

(b)

0998400 10998400 20998400p85AInsulin

FLAG

IRS1

Merge

(min)

(c)

0998400 10998400 20998400p85D

(d)












4 Conclusions



+Insulin(min)

Ctrl0 5 10 15 0 5 10 15 0 5 10 15 0 5 10 15

p85WT p85A p85D

P-AKT

AKT

(a)

P-ERK12

ERK12

120573-Actin

+Insulin(min)

Ctrl0 5 10 15 0 5 10 15 0 5 10 15 0 5 10 15

p85WT p85A p85D

(b)

00

02

04

06

08

10

12

14

16

0 5 10 15 0 5 1015 0 5 1015 0 5 10 15

Ratio

+Insulin(min)


P-AKTAKT

lowast

lowast

lowastlowast

P-ERKERKlowast

lowast

lowastlowast

00

05

15

10

20

25

30

0 5 10 15 0 5 1015 0 5 1015 0 5 1015

Ratio


(c)






0

2

4

6

8

10

12

0 24 48 72 96

Ctrl

+Insulin

lowastlowast

lowastlowast

lowast

lowast

lowast

lowast lowastlowast


p85WT

Cel

l gro

wth

(fol

d of

bas

al)

Time (hrs)

0

2

4

6

8

10

12

0 24 48 72 96

Ctrl

+Insulin05 FCS

+Insulin05 FCS

p85D

Cel

l gro

wth

(fol

d of

bas

al)

Time (hrs)

0

2

4

6

8

10

12

0 24 48 72 96

Ctrl

+Insulin05 FCS

+Insulin05 FCS

p85AC

ell g

row

th (f

old

of b

asal

)

Time (hrs)

(a)

6069

27 35

0

20

40

60

80

100

120

+Insulin

G2MS

G1

0

20

40

60

80

100

120

G2MS

G1

0

20

40

60

80

100

120

G2MS

G1

0

20

40

60

80

100

120

G2MS

G1


29 34 29 33

6268 6268

24 37

5874

lowast lowast

Cell

s (

)


(b)

0

20

40

60

80

100

120

140

Col

ony

num

ber

+Insulin

lowast

lowast

lowast

Ctrl

p85WT

p85A

p85D

+Insulin05 FCS

05 FCS


(c)




05 FCS

+Insulin12h

(a)

0

10

20

30

40

50

60

70

80

90


+Insulin

lowast

lowast

lowast

05 FCS

Wou

nd h

ealin

g (

)

(b)













Acknowledgments


References























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology









4 Conclusions



+Insulin(min)

Ctrl0 5 10 15 0 5 10 15 0 5 10 15 0 5 10 15

p85WT p85A p85D

P-AKT

AKT

(a)

P-ERK12

ERK12

120573-Actin

+Insulin(min)

Ctrl0 5 10 15 0 5 10 15 0 5 10 15 0 5 10 15

p85WT p85A p85D

(b)

00

02

04

06

08

10

12

14

16

0 5 10 15 0 5 1015 0 5 1015 0 5 10 15

Ratio

+Insulin(min)


P-AKTAKT

lowast

lowast

lowastlowast

P-ERKERKlowast

lowast

lowastlowast

00

05

15

10

20

25

30

0 5 10 15 0 5 1015 0 5 1015 0 5 1015

Ratio


(c)






0

2

4

6

8

10

12

0 24 48 72 96

Ctrl

+Insulin

lowastlowast

lowastlowast

lowast

lowast

lowast

lowast lowastlowast


p85WT

Cel

l gro

wth

(fol

d of

bas

al)

Time (hrs)

0

2

4

6

8

10

12

0 24 48 72 96

Ctrl

+Insulin05 FCS

+Insulin05 FCS

p85D

Cel

l gro

wth

(fol

d of

bas

al)

Time (hrs)

0

2

4

6

8

10

12

0 24 48 72 96

Ctrl

+Insulin05 FCS

+Insulin05 FCS

p85AC

ell g

row

th (f

old

of b

asal

)

Time (hrs)

(a)

6069

27 35

0

20

40

60

80

100

120

+Insulin

G2MS

G1

0

20

40

60

80

100

120

G2MS

G1

0

20

40

60

80

100

120

G2MS

G1

0

20

40

60

80

100

120

G2MS

G1


29 34 29 33

6268 6268

24 37

5874

lowast lowast

Cell

s (

)


(b)

0

20

40

60

80

100

120

140

Col

ony

num

ber

+Insulin

lowast

lowast

lowast

Ctrl

p85WT

p85A

p85D

+Insulin05 FCS

05 FCS


(c)




05 FCS

+Insulin12h

(a)

0

10

20

30

40

50

60

70

80

90


+Insulin

lowast

lowast

lowast

05 FCS

Wou

nd h

ealin

g (

)

(b)













Acknowledgments


References























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


+Insulin(min)

Ctrl0 5 10 15 0 5 10 15 0 5 10 15 0 5 10 15

p85WT p85A p85D

P-AKT

AKT

(a)

P-ERK12

ERK12

120573-Actin

+Insulin(min)

Ctrl0 5 10 15 0 5 10 15 0 5 10 15 0 5 10 15

p85WT p85A p85D

(b)

00

02

04

06

08

10

12

14

16

0 5 10 15 0 5 1015 0 5 1015 0 5 10 15

Ratio

+Insulin(min)


P-AKTAKT

lowast

lowast

lowastlowast

P-ERKERKlowast

lowast

lowastlowast

00

05

15

10

20

25

30

0 5 10 15 0 5 1015 0 5 1015 0 5 1015

Ratio


(c)






0

2

4

6

8

10

12

0 24 48 72 96

Ctrl

+Insulin

lowastlowast

lowastlowast

lowast

lowast

lowast

lowast lowastlowast


p85WT

Cel

l gro

wth

(fol

d of

bas

al)

Time (hrs)

0

2

4

6

8

10

12

0 24 48 72 96

Ctrl

+Insulin05 FCS

+Insulin05 FCS

p85D

Cel

l gro

wth

(fol

d of

bas

al)

Time (hrs)

0

2

4

6

8

10

12

0 24 48 72 96

Ctrl

+Insulin05 FCS

+Insulin05 FCS

p85AC

ell g

row

th (f

old

of b

asal

)

Time (hrs)

(a)

6069

27 35

0

20

40

60

80

100

120

+Insulin

G2MS

G1

0

20

40

60

80

100

120

G2MS

G1

0

20

40

60

80

100

120

G2MS

G1

0

20

40

60

80

100

120

G2MS

G1


29 34 29 33

6268 6268

24 37

5874

lowast lowast

Cell

s (

)


(b)

0

20

40

60

80

100

120

140

Col

ony

num

ber

+Insulin

lowast

lowast

lowast

Ctrl

p85WT

p85A

p85D

+Insulin05 FCS

05 FCS


(c)




05 FCS

+Insulin12h

(a)

0

10

20

30

40

50

60

70

80

90


+Insulin

lowast

lowast

lowast

05 FCS

Wou

nd h

ealin

g (

)

(b)













Acknowledgments


References























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

2

4

6

8

10

12

0 24 48 72 96

Ctrl

+Insulin

lowastlowast

lowastlowast

lowast

lowast

lowast

lowast lowastlowast


p85WT

Cel

l gro

wth

(fol

d of

bas

al)

Time (hrs)

0

2

4

6

8

10

12

0 24 48 72 96

Ctrl

+Insulin05 FCS

+Insulin05 FCS

p85D

Cel

l gro

wth

(fol

d of

bas

al)

Time (hrs)

0

2

4

6

8

10

12

0 24 48 72 96

Ctrl

+Insulin05 FCS

+Insulin05 FCS

p85AC

ell g

row

th (f

old

of b

asal

)

Time (hrs)

(a)

6069

27 35

0

20

40

60

80

100

120

+Insulin

G2MS

G1

0

20

40

60

80

100

120

G2MS

G1

0

20

40

60

80

100

120

G2MS

G1

0

20

40

60

80

100

120

G2MS

G1


29 34 29 33

6268 6268

24 37

5874

lowast lowast

Cell

s (

)


(b)

0

20

40

60

80

100

120

140

Col

ony

num

ber

+Insulin

lowast

lowast

lowast

Ctrl

p85WT

p85A

p85D

+Insulin05 FCS

05 FCS


(c)




05 FCS

+Insulin12h

(a)

0

10

20

30

40

50

60

70

80

90


+Insulin

lowast

lowast

lowast

05 FCS

Wou

nd h

ealin

g (

)

(b)













Acknowledgments


References























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



05 FCS

+Insulin12h

(a)

0

10

20

30

40

50

60

70

80

90


+Insulin

lowast

lowast

lowast

05 FCS

Wou

nd h

ealin

g (

)

(b)













Acknowledgments


References























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






Acknowledgments


References























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article the p85 regulatory subunit of pi3k ... · research article the p85 regulatory...

Documents