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<?xml version="1.0" ?> <tei> <teiHeader> <fileDesc xml:id="0"/> </teiHeader> <text xml:lang="en"> <p>Temporal regulation of c-Myc protein accumulation is essential for<lb/> normal cell proliferation. Complete loss of c-Myc or N-Myc function<lb/> results in embryonic lethality <ref type="biblio">1,2</ref> , and studies using conditional c-Myc<lb/> knockout alleles show that cells lacking c-Myc cease to proliferate and<lb/> exit the cell cycle <ref type="biblio">3,4</ref> . In contrast, overexpression of c-Myc in cultured<lb/> cells or transgenic animals blocks differentiation, induces neoplastic<lb/> transformation and can initiate apoptosis <ref type="biblio">5,6</ref> . Moreover, a wide variety<lb/> of naturally occurring tumours exhibit both chromosomal transloca-<lb/>tions and amplification of the c-myc locus that result in constitutive<lb/> overexpression of c-Myc protein <ref type="biblio">7–9</ref> .<lb/></p> <p>Myc protein is stabilized after activation of Ras, allowing it to accu-<lb/>mulate to high levels <ref type="biblio">10</ref> . Ras promotes stability of c-Myc through at<lb/> least two effector pathways: the Raf–MEK–ERK kinase cascade, and the<lb/> phosphatidylinositol-3-OH kinase (PI(3)K)–Akt pathway that inhibits<lb/> glycogen synthase kinase-3β (GSK-3β) <ref type="biblio">11</ref> . The ERK and GSK-3β<lb/> kinases phosphorylate two sites near the amino terminus of c-Myc that<lb/> are highly conserved in all mammalian c-Myc isoforms <ref type="biblio">11–14</ref> . These<lb/> phosphorylation sites, Thr 58 and Ser 62, exert opposing control on c-<lb/>Myc degradation through the ubiquitin-proteasome pathway <ref type="biblio">11</ref> . Thus,<lb/> after a growth stimulatory signal, c-myc gene transcription is increased<lb/> and newly synthesized c-Myc protein is phosphorylated on Ser 62,<lb/> resulting in its stabilization. Phosphorylation at Ser 62 is also required<lb/> for the subsequent phosphorylation of c-Myc at Thr 58 by GSK-3β,<lb/> which is associated with c-Myc degradation <ref type="biblio">11–14</ref> . During early G1<lb/> phase, however, GSK-3β activity is regulated by Ras-mediated activa-<lb/>tion of the PI(3)K/Akt pathway (which phosphorylates and inhibits<lb/> GSK-3β), facilitating stabilization of c-Myc <ref type="biblio">15</ref> . Later in G1 phase, when<lb/> Akt activity declines, GSK-3β becomes active and phosphorylates c-<lb/>Myc on Thr 58, which is important for c-Myc turnover.<lb/></p> <p>Thr 58 phosphorylation contributes to degradation of c-Myc<lb/> through the ubiquitin pathway, as mutation of Thr 58 to alanine<lb/> results in a stable and more oncogenic c-Myc protein <ref type="biblio">11–13,16,17</ref> .<lb/> Moreover, the c-Myc T58A mutant is no longer a substrate for ubiquiti-<lb/>nation in vivo <ref type="biblio" >11</ref> . Nevertheless, how Thr 58 phosphorylation facilitates<lb/> c-Myc degradation remains unclear. We now show that Ser 62 dephos-<lb/>phorylation is important for ubiquitin-mediated degradation of<lb/> c-Myc and that Thr 58 phosphorylation promotes dephosphorylation<lb/> of Ser 62. In addition, we show that PP2A dephosphorylates Ser 62 on<lb/> c-Myc and that the Pin1 prolyl isomerase, which has previously been<lb/> shown to enhance PP2A function <ref type="biblio">18,19</ref> , also regulates c-Myc turnover in<lb/> a process that is dependent on phosphorylation of Thr 58. We propose<lb/> that a sequence of Ras-initiated signalling events is required to regulate<lb/> c-Myc protein stability and demonstrate that a stabilized c-Myc protein<lb/> can replace SV40 small T antigen in the transformation of human cells.<lb/></p> <head>RESULTS<lb/></head> <head>Ubiquitinated c-Myc is phosphorylated on Thr 58, but not on<lb/> Ser 62<lb/></head> <p>Newly synthesized c-Myc is phosphorylated on Ser 62, allowing the<lb/> subsequent phosphorylation of Thr 58 that is necessary to promote<lb/> ubiquitin-mediated degradation of c-Myc <ref type="biblio">11–14</ref> . However, treatment of<lb/> cells with a proteasome inhibitor results in the accumulation of species<lb/> of c-Myc with a larger relative molecular mass higher (M r ) that is rec-<lb/>ognized by an antibody specific for Thr 58-phosphorylated c-Myc,<lb/> but not by an antibody specific for Ser 62-phosphorylated c-Myc <ref type="biblio">11</ref> .<lb/> This observation suggested that ubiquitinated forms of c-Myc might<lb/> be phosphorylated selectively on Thr 58. To address this possibility,<lb/> we examined the phosphorylation status of ubiquitinated c-Myc.<lb/></p> <p>A recombinant adenovirus containing the c-myc gene under the<lb/> control of the cytomegalovirus (CMV) enhancer (Ad-Myc) was used<lb/> to produce c-Myc protein in otherwise quiescent cells, an approach<lb/> we have employed previously to control the conditions in which<lb/> c-Myc accumulates <ref type="biblio">10,11</ref> . We have shown that c-Myc is phosphorylated<lb/> at Ser 62 as well as Thr 58 under these conditions, and that serum<lb/> stimulation or mutation of Thr 58 increases Ser 62 phosphoryla-<lb/>tion <ref type="biblio">11</ref> . Here, Ad-Myc-infected cells were treated with the proteasome<lb/> inhibitor lactacystin for 6 h and ubiquitinated proteins were<lb/> immunoprecipitated from cell lysates with an anti-ubiquitin anti-<lb/>body. The immunoprecipitates, along with 10% of the original lysate<lb/> and control immunoprecipitates without the ubiquitin antibody,<lb/> were analysed in triplicate by SDS–PAGE and immunoblotting with<lb/> either a pan-Myc antibody, a phospho-Thr 58 antibody or a phos-<lb/>pho-Ser 62 antibody. A substantial amount of ubiquitinated c-Myc<lb/> accumulates after 6 h of proteasome inhibition <ref type="figure">(Fig. 1, lane 4)</ref>.<lb/> Ubiquitinated c-Myc runs with a slightly higher relative molecular<lb/> mass than c-Myc produced in the absence of proteasome inhibition<lb/> <ref type="figure">(Fig. 1, compare lanes 2 and 4)</ref>. In addition, several higher-molecu-<lb/>lar-weight species are detected with the pan-Myc antibody that we<lb/> assume represent alternate or additional ubiquitinated species of<lb/> c-Myc. Ubiquitinated c-Myc, which accumulates in quiescent cells<lb/> after proteasome inhibition, is primarily (if not exclusively) phospho-<lb/>rylated on Thr 58 <ref type="figure">(Fig. 1, lane 7)</ref>, as there was little or no Ser 62-<lb/>phosphorylated c-Myc immunoprecipitated by the ubiquitin<lb/> antibody <ref type="figure">(Fig. 1, lane 10)</ref>.<lb/></p> <head>A role for PP2A in controlling c-Myc degradation<lb/></head> <p>The observations that GSK-3β-mediated phosphorylation at Thr 58<lb/> requires earlier phosphorylation of c-Myc at Ser 62 <ref type="biblio">(refs 11–14)</ref> and<lb/> that ubiquitinated c-Myc is only phosphorylated at Thr 58 suggest that<lb/> the Ser 62 phosphate is removed in the process of c-Myc ubiquitina-<lb/>tion. Assays using various cell-permeable inhibitors specific for com-<lb/>mon serine/threonine phosphatases indicated a possible role for PP2A<lb/> in the control of c-Myc stability. The level of c-Myc present in quiescent<lb/> cells was increased by okadaic acid <ref type="figure" >(Fig. 2a, compare lanes 1 and 2)</ref>,<lb/> which is a potent inhibitor of PP2A with some activity against PP1, but<lb/> was not affected by cyclosporin A <ref type="figure">(Fig. 2a, lane 3)</ref>, which specifically<lb/> inhibits PP2B. This data suggest that PP2A, or possibly PP1, could be<lb/> involved in controlling c-Myc protein accumulation.<lb/></p> <p>As a more specific test to examine the role of PP2A in controlling<lb/> accumulation of c-Myc, we used a recombinant adenovirus that<lb/> expresses SV40 small T antigen, a highly selective inhibitor of PP2A.<lb/> Small T antigen functions by displacing specific regulatory B subunits<lb/> that normally incorporate into cellular PP2A complexes and thereby<lb/> reduces PP2A activity against certain endogenous substrates 20 .<lb/> Increasing doses of the Ad-small T antigen virus had a marked effect on<lb/> c-Myc protein levels, resulting in a substantial increase in the accumu-<lb/>lation of c-Myc in quiescent fibroblasts <ref type="figure">(Fig. 2b)</ref>. The effects of<lb/> expressing small T antigen on accumulation of endogenous c-Myc in<lb/> quiescent or asynchronous REF52 fibroblasts were also examined.<lb/> Endogenous c-Myc accumulation was increased substantially by inhi-<lb/>bition of PP2A with small T antigen in either serum-starved or asyn-<lb/>chronously growing cells <ref type="figure">(Fig. 2c)</ref>.<lb/></p> <p>To test whether the increase in c-Myc accumulation observed after<lb/> PP2A inhibition is caused by protein stabilization, and to rule out<lb/> effects of viral infection on the interpretation of our results, the effect<lb/> of okadaic acid on the half-life of endogenous c-Myc protein was<lb/> examined by pulse-chase analysis in quiescent REF52 fibroblasts. c-<lb/>Myc protein produced in growth-arrested cells was short-lived, with a<lb/> half-life of 13 min. Importantly, this was true for endogenous c-Myc in<lb/> uninfected cells <ref type="figure">(Fig. 2d, top)</ref>, as well as for c-Myc produced by aden-<lb/>ovirus <ref type="figure">(Fig. 2d, bottom)</ref>. In contrast, endogenous c-Myc produced in<lb/> uninfected quiescent cells treated with okadaic acid was significantly<lb/> more stable, with a half-life of 62 min. A similar stabilization of c-Myc<lb/> was observed in cells infected with the Ad-small T antigen virus (see<lb/></p> <figure>Untreated<lb/> cell lysate<lb/> In p u t<lb/> A n t i-U b IP<lb/> C o n t r o l IP<lb/> In p u t<lb/> A n t i-U b IP<lb/> C o n t r o l IP<lb/> In p u t<lb/> A n t i -U b IP<lb/> C o n t r o l IP<lb/> 105 -<lb/>-105<lb/> -75<lb/> -50<lb/> 75 -<lb/>50 -<lb/>M r (K)<lb/> Ubi−Myc<lb/> IgG<lb/> c-Myc -<lb/>1<lb/> 2<lb/> 3<lb/> 4<lb/> 5<lb/> 6<lb/> 7<lb/> 8<lb/> 9<lb/> 1 0<lb/> 1 1<lb/> c-Myc<lb/> c-Myc<lb/> c-Myc (P-T58)<lb/> c-Myc (P-S62)<lb/> Blot: anti-<lb/>Figure 1 Ubiquitinated c-Myc protein is phosphorylated on Thr 58, but not<lb/> on Ser 62. Quiescent REF 52 fibroblasts were infected with Ad-c-Myc (MOI<lb/> = 50). At 16 h after infection, cells maintained in low-serum medium were<lb/> treated with 10 µM lactacystin for 6 h, except for the samples shown in<lb/> lanes 1 and 2, which represent 10 and 40 µg of untreated Ad-c-Myc-<lb/>infected cell lysate, respectively. Lactacystin-treated cells were harvested<lb/> and lysates were divided for immunoprecipitation (IP) with an anti-ubiquitin<lb/> antibody or control protein A + G beads. The input lysates (10% volume),<lb/> anti-ubiquitin IP, and control IP were then analysed in triplicate by<lb/> SDS–PAGE and western blotting with an anti-c-Myc monoclonal antibody<lb/> (C-33/sc-42), or one of the two phospho-specific c-Myc antibodies 11 . The<lb/> c-Myc and ubiquitinated c-Myc proteins are indicated. The 50K band visible<lb/> in lanes 4 and 10 is the IgG heavy chain, which is recognized by the anti-<lb/>mouse secondary antibody. This band is not visible in lane 7 and only faintly<lb/> visible in lane 10 (longer exposure), where anti-rabbit secondary antibodies<lb/> were used.<lb/></figure> <p><ref type="figure">Supplementary Information, Fig. S1</ref>).<lb/></p> <p>In vitro phosphatase assays were then performed to examine the role<lb/> of PP2A in targeting the Ser 62 phosphate. c-Myc protein was<lb/> immunoprecipitated from Ad-Myc-infected quiescent REF52 fibrob-<lb/>lasts that were co-infected with Ad-Ras to maximize phosphorylation<lb/> of c-Myc <ref type="biblio">10</ref> . Immunoprecipitated c-Myc was incubated with either<lb/> buffer only (−), or with purified PP1 or PP2A. Ser 62 and Thr 58 phos-<lb/>phorylation were assessed by western blotting with the Ser 62 or Thr 58<lb/> phospho-specific antibodies. PP2A, but not PP1, removed Ser 62 phos-<lb/>phate from c-Myc without significantly reducing Thr 58 phosphate<lb/> <ref type="figure">(Fig. 3a)</ref>. The ability of PP2A to dephosphorylate the stable c-Myc<lb/> mutant, c-Myc T58A , was also evaluated. This mutant has enhanced Ser<lb/></p> <figure>a<lb/> d<lb/> b<lb/> c<lb/> −<lb/> −<lb/> −<lb/> O k a d a ic<lb/> a c id C y c lo s p o r in<lb/> A<lb/> c-Myc<lb/> c-Myc<lb/> c-Myc -<lb/>c-Myc -<lb/>Ad-c-Myc -<lb/>c-Myc -<lb/>+ OA<lb/> c-Myc -<lb/>N.S.<lb/> Ponceau S<lb/> 1<lb/> 2<lb/> 3<lb/> 1<lb/> 2<lb/> 3<lb/> 1<lb/> 2<lb/> 3<lb/> Starved<lb/> Asynchronous<lb/> 1<lb/> 2<lb/> 1<lb/> 2<lb/> 3<lb/> 4<lb/> + Ad-small T<lb/> + A d -s m a ll T<lb/> Chase time (min)<lb/> 0<lb/> 1 5<lb/> 3 0<lb/> Chase time (min)<lb/> Chase time (min)<lb/> 0<lb/> 1 0<lb/> 2 0<lb/> 35 S-c-Myc remaining (percentage)<lb/> 100<lb/> 50<lb/> 10<lb/> 0<lb/> 1 0<lb/> 2 0<lb/> 3 0<lb/> Endogenous control<lb/> (t 1/2 = 12.6 min)<lb/> Endogenous + OA<lb/> (t 1/2 = 61.8 min)<lb/> Ectopic control<lb/> (t 1/2 = 12.5 min)<lb/> Figure 2 Accumulation of c-Myc is regulated by PP2A. (a) Okadaic acid (OA)<lb/> increases accumulation of c-Myc. Quiescent REF52 fibroblasts were<lb/> infected with Ad-c-Myc (MOI = 25) and maintained in medium with 0.25%<lb/> serum for an additional 20 h. At 4 h before harvesting, infected cells were<lb/> either treated with ethanol (−), 100 nM OA or 5 µM cyclosporin A. Extracts<lb/> from an equal number of cells were subjected to western blot analysis with<lb/> the c-Myc C-33 antibody. Equal protein loading was verified by Ponceau S<lb/> staining. (b) SV40 small T antigen increases Ad-c-Myc protein levels.<lb/> Quiescent REF52 cells were infected with Ad-c-Myc (MOI = 25), together<lb/> with a control virus (−), Ad-GFP (MOI = 200), or a recombinant adenovirus<lb/> expressing SV40 small T antigen, Ad-small T (MOIs = 100, 200 and 400 in<lb/> lanes 2–4), and maintained in medium containing 0.25% serum. At 20 h<lb/> after infection, equal cell counts were harvested for western blot analysis<lb/> with the C-33 c-Myc antibody. Equal protein loading was verified by<lb/> Ponceau S staining of the membrane and the presence of a non-specific<lb/> (N.S.) band on the blot. (c) SV40 small T antigen increases endogenous c-<lb/>Myc protein levels. Quiescent or asynchronous REF52 cells were infected<lb/> with either control Ad-GFP virus (−) or Ad small T antigen, both at<lb/> MOI = 200, and maintained in growth medium (asynchronous) or starvation<lb/> medium (starved) for an additional 20 h. Equal cell numbers were harvested<lb/> and immunoprecipitations were performed to concentrate endogenous<lb/> c-Myc, before western blot analysis with the c-Myc C-33 monoclonal<lb/> antibody. (d) Okadaic acid stabilizes endogenous c-Myc protein. Quiescent<lb/> REF52 fibroblasts were either infected with Ad-c-Myc (MOI = 50) and<lb/> maintained in medium with 0.25% serum for 18 h, or not infected. Non-<lb/>infected cells were either mock treated with ethanol (control) or treated with<lb/> 100 nM okadaic acid (OA) for 6 h. Cells were pulse-labelled with<lb/> 35 S-methionine/cysteine and chased with medium containing excess<lb/> unlabelled methionine and cysteine in the continued presence of ethanol or<lb/> okadaic acid. 35 S-labelled c-Myc was immunoprecipitated, separated by<lb/> SDS–PAGE and quantified with a phosphorimager. Best-fit exponential lines<lb/> are shown for endogenous c-Myc control, endogenous c-Myc + OA and<lb/> ectopic c-Myc control. Calculated half-lives are also shown.<lb/></figure> <p>62 phosphorylation <ref type="biblio">11,14</ref> . Whereas PP2A effectively dephosphorylated<lb/> Ser 62 in wild-type c-Myc <ref type="figure">(Fig. 3b, left)</ref>, the c-Myc T58A mutant was not<lb/> dephosphorylated by PP2A <ref type="figure">(Fig. 3b, right)</ref>. These results suggest a role<lb/> for Thr 58 phosphorylation in facilitating removal of Ser 62 phosphate<lb/> by PP2A.<lb/></p> <p>In addition, accumulation of c-Myc in vivo was assessed using<lb/> RNA interference (RNAi) to inhibit endogenous PP2A activity.<lb/> Small-interfering RNA (siRNA) directed against the catalytic sub-<lb/>unit (C) of PP2A reduced the level of endogenous C subunit, and<lb/> coincident with this reduction there was an increase in the level of<lb/> c-Myc protein <ref type="figure">(Fig. 3c)</ref>. In contrast, a scrambled sequence siRNA or<lb/> an siRNA targeting luciferase had no effect on the level of the C sub-<lb/>unit and did not alter c-Myc levels, although the luciferase siRNA did<lb/> inhibit luciferase activity in this experiment (see <ref type="figure" >Supplementary<lb/> Information, Fig. S2</ref>). Taken together, these results suggest that<lb/> PP2A contributes to degradation of c-Myc by dephosphorylating Ser<lb/> 62 and imply that this degradation is dependent on Thr 58<lb/> phsophorylation.<lb/></p> <figure>a<lb/> b<lb/> c<lb/> Anti-c-Myc-S62-P<lb/> c-Myc (P-S62)<lb/> c-Myc (P-S62)<lb/> c-Myc<lb/> c-Myc<lb/> Anti-c-Myc-T58-P<lb/> Anti-c-Myc<lb/> c-Myc<lb/> PP2A C<lb/> −<lb/> PP1c<lb/> PP2A<lb/> PP2A:<lb/> −<lb/> +<lb/> +<lb/> +<lb/> c-Myc -<lb/>c-Myc<lb/> PP2A:<lb/> −<lb/> +<lb/> +<lb/> +<lb/> c-Myc T58A<lb/> IgG -<lb/>c-Myc -<lb/>IgG -<lb/>Time<lb/> (min)<lb/> Time<lb/> (min)<lb/> 20<lb/> 0<lb/> 10<lb/> 1<lb/> 2<lb/> 3<lb/> 20<lb/> 20<lb/> 0<lb/> 10<lb/> 20<lb/> C o n t r o l R N A i<lb/> L u c if e r a s e R N A i<lb/> P P 2 A C<lb/> R N A i<lb/> Blot: anti-<lb/>Blot: anti-<lb/>Blot: anti-<lb/> Figure 3 Ser 62 phosphate is a substrate for PP2A. (a) PP2A<lb/> dephosphorylates c-Myc at Ser 62. Quiescent REF52 cells were infected<lb/> with Ad-c-Myc (MOI = 50) together with Ad-Ras Q61L (MOI = 200),<lb/> maintained in starvation medium and harvested 20 h after infection. c-Myc<lb/> was immunoprecipitated from cell lysates and incubated with either control<lb/> buffer (−), 1 µg of purified PP1 (human recombinant) or 0.1 µg of PP2A<lb/> (bovine kidney), as indicated, at 30 °C for 30 min. Samples were run in<lb/> triplicate and immunoblotted with the phospho-Ser 62 antibody, the<lb/> phospho-Thr 58 antibody, or the C-33 c-Myc antibody, as indicated. (b) Thr<lb/> 58 phosphorylation is necessary for PP2A-mediated dephosphorylation of<lb/> Ser 62. Quiescent REF52 fibroblasts were infected with wild-type Ad-c-Myc<lb/> or Ad-c-Myc T58A virus (MOI = 200) and maintained in low-serum medium<lb/> overnight. At 4 h before harvesting, cells were treated with 10 µM MG132<lb/> and 100 nM okadaic acid. c-Myc was immunoprecipitated from the cell<lb/> extracts using the C-33 c-Myc monoclonal antibody. Immunoprecipitated<lb/> c-Myc samples were incubated with purified PP2A for the indicated times.<lb/> Western blot analysis was performed with the phospho-Ser 62 antibody or<lb/> the C-33 c-Myc antibody. The lower band in the anti-c-Myc blot is IgG. (c)<lb/> Specific inhibition of PP2A by siRNA results in accumulation of c-Myc.<lb/> 293a cells were transfected with 500 ng CMV-c-Myc and 100 ng CMV-β-gal,<lb/> along with 100nM scrambled siRNA (lane 1), siRNA specific for luciferase<lb/> (lane 2), or pooled siRNAs consisting of four different siRNAs specific for<lb/> the PP2A C subunit (lane 3). Cells transfected with scrambled siRNA and<lb/> luciferase siRNA also received 250 ng pGL3-luciferase plasmid. Transfected<lb/> cells were serum-starved for 48 h and harvested. β-Galactosidase activity<lb/> was measured to adjust protein load volumes for transfection efficiency. The<lb/> top half of the western blot was probed with the c-Myc antibody (N262) and<lb/> the bottom half with the PP2A C subunit antibody. Luciferase assays from<lb/> lane 1 and 2 extracts are presented in Supplementary Information, Fig. S2.<lb/></figure> <head>The Pin1 prolyl isomerase interacts with c-Myc<lb/></head> <p>PP2A may be a conformation-sensitive protein phosphatase, prefer-<lb/>ring the trans configuration of a proline residue adjacent to the phos-<lb/>pho-serine or phospho-threonine in substrates such as Cdc25 and<lb/> Tau <ref type="biblio">18</ref> . In addition, the phosphorylation-directed prolyl isomerase<lb/> Pin1 may catalyse the isomerization of proline residues in such phos-<lb/>pho-proteins to promote their dephosphorylation by PP2A <ref type="biblio">19</ref> .<lb/> Furthermore, the presence of doubly phosphorylated serine/threo-<lb/>nine-proline residues, as found in the motifs that comprise the car-<lb/>boxy-terminal domain of RNA polymerase II, increase the binding<lb/> affinity and catalytic efficacy of Pin1 towards these substrates through<lb/> recognition of one of the two phosphorylated residues by the Pin1-<lb/>WW domain <ref type="biblio">21,22</ref> . These findings led us to test whether c-Myc could<lb/> bind to Pin1. As an alternative approach, we have used quiescent<lb/> REF52 fibroblasts infected with Ad-Myc, made extracts from these cells<lb/> and incubated them with glutathione S-transferase (GST) alone,<lb/> GST–Pin1 or GST–Pin1 bearing point mutations in either the WW<lb/> domain or the catalytic domain <ref type="biblio" >30</ref> immobilized on glutathione–agarose<lb/> beads. c-Myc bound preferentially to wild-type GST–Pin1 beads, com-<lb/>pared with GST alone <ref type="figure">(Fig. 4a, compare lanes 2 and 3)</ref>. c-Myc inter-<lb/>acted poorly with the Pin1 W33A WW mutant <ref type="figure">(Fig. 4a, )lane 4</ref>, but<lb/> bound the Pin1 C109A catalytic mutant with similar efficacy to wild-type<lb/> Pin1 <ref type="figure">(Fig. 4a, compare lanes 3 and 5)</ref>.<lb/></p> <p>To investigate the role of Thr 58 and Ser 62 phosphorylation in the<lb/> Pin1–Myc interaction, the ability of wild-type c-Myc, c-Myc T58A or<lb/> c-Myc S62A mutants <ref type="biblio">11</ref> to interact with Pin1 in vivo was examined. 293a<lb/> cells were transfected with vectors to express His 6 -tagged wild-type or<lb/> mutant c-Myc proteins. Cells were lysed and His 6 –Myc proteins were<lb/> selected on nickel–NTA beads. Bound material was eluted with imidi-<lb/>zole and then analysed by western blotting. Pin1 was recovered in asso-<lb/>ciation with wild-type c-Myc <ref type="figure">(Fig. 4b, compare lanes 1 and 2)</ref>, but a<lb/> reduced interaction was detected with either c-Myc T58A or c-Myc S62A<lb/> <ref type="figure">(Fig. 4b, lanes 3 and 4)</ref>. As c-Myc T58A remains highly phosphorylated<lb/> on Ser 62, whereas c-Myc S62A lacks phosphorylation at either Thr 58 or<lb/> Ser 62 <ref type="biblio">(refs 11–14)</ref>, these results suggest that the phosphorylation of<lb/> c-Myc at Thr 58 is important for Pin1 binding.<lb/></p> <head>Pin1 facilitates c-Myc protein degradation<lb/></head> <p>We next examined whether Pin1 expression affected the level of c-Myc<lb/> protein by utilizing primary mouse embryo fibroblasts (MEFs) derived<lb/> from Pin1 −/− embryos. Heterozygous Pin1 mice, maintained in an iso-<lb/>genic C57BL/6J background, were bred, and sibling 13.5-day embryos<lb/> were used to prepare primary MEFs with either +/+ or −/− genotypes <ref type="biblio">23</ref> .<lb/> Substantially more c-Myc protein accumulated in Pin1 −/− cells, com-<lb/>pared with wild-type MEFs after Ad-Myc infection <ref type="figure">(Fig. 5a, compare<lb/> )lanes 1 and 2</ref>. This increase in c-Myc levels was caused by the absence<lb/> of Pin1, as re-introduction of Pin1 (using Ad-Pin1) into the Pin1 −/−<lb/> MEFs reduced c-Myc levels to those detected in wild-type MEFs <ref type="figure">(Fig.<lb/> 5a, compare lanes 1 and 3)</ref>. Furthermore, a pulse-chase experiment<lb/> demonstrated that the increased accumulation of c-Myc in the absence<lb/> of Pin1 was associated with an increased half-life of c-Myc in Pin1 −/−<lb/> MEFs <ref type="figure">(Fig. 5b)</ref>. Moreover, additional pulse-chase assays demonstrated<lb/> that re-introduction of Pin1 into Pin −/− cells reduced the half-life of<lb/> c-Myc in these cells (see <ref type="figure">Supplementary Information, Fig. S3</ref>).<lb/></p> <p>The accumulation of c-Myc observed in the absence of Pin1 was not<lb/> significantly affected by inhibition of PP2A through expression of<lb/> small T antigen <ref type="figure">(Fig. 5c, lanes 2 and 4)</ref>, when compared with the effects<lb/> of small T antigen expression on c-Myc in Pin1 +/+ cells <ref type="figure">(Fig. 5c, lanes 1<lb/> and 3)</ref>. This result suggests a similar role for the actions of Pin1 and<lb/> PP2A on c-Myc. Moreover, the enhancement of wild-type c-Myc accu-<lb/>mulation in the absence of Pin1 <ref type="figure">(Fig. 5d, top)</ref> was not observed for the<lb/> c-Myc T58A mutant <ref type="figure">(Fig. 5d, bottom)</ref>, consistent with the observation<lb/> that this mutant shows reduced binding to Pin1 and displays increased<lb/> Ser 62 phosphorylation <ref type="biblio">11</ref> . Taken together, these results suggest a com-<lb/>plementary role for Pin1 and PP2A in controlling c-Myc turnover.<lb/></p> <head>Pin1 ensures transient accumulation of c-Myc after growth<lb/> stimulation<lb/></head> <p> Given the role of Pin1 in the degradation of c-Myc, we investigated the<lb/> contribution of Pin1 to the normal regulation of c-Myc levels after<lb/> growth stimulation. Wild-type or Pin1 −/− primary MEFs were density<lb/> arrested and then plated into low-serum medium for 24 h. The quies-<lb/>cent MEFs were then stimulated with 20% foetal calf serum and<lb/> endogenous c-Myc protein levels were determined by western blotting<lb/> at 0, 3, 6 and 24 h after serum addition. Endogenous c-Myc in wild-type<lb/></p> <figure>a<lb/> b<lb/> In p u t<lb/> G S T<lb/> G S T − P in 1<lb/> G S T − P in 1 -W W<lb/> H is − c -M y c T 5 8 A<lb/> H is − c -M y c S 6 2 A<lb/> G S T − P in 1<lb/> (c a t a ly t ic<lb/> m u t a n t )<lb/> c-Myc<lb/> 1<lb/> 2<lb/> 3<lb/> 4<lb/> 1<lb/> 2<lb/> 3<lb/> 4<lb/> 5<lb/> Blot: anti-c-Myc<lb/> c-Myc<lb/> Pin1<lb/> −<lb/> H is − c -M y c W T<lb/> Blot: anti-<lb/>Figure 4 Pin1 interacts with c-Myc. (a) Interaction between c-Myc and Pin1<lb/> requires the Pin1 WW domain. Quiescent REF52 fibroblasts were infected<lb/> with Ad-c-Myc (MOI = 25 ). Infected cells were maintained in medium with<lb/> 0.25% serum for 20 h and then harvested in low-stringency buffer. Cell<lb/> lysates from equal cell counts were incubated with either GST, or GST–Pin1<lb/> wild-type or mutant proteins bound to glutathione–sepharose beads, as<lb/> indicated. Precipitated proteins were washed four times in low-stringency<lb/> buffer and then released with SDS sample buffer. Samples plus a 10%<lb/> volume of input lysate were subjected to western blot analysis with the C-33<lb/> c-Myc antibody. (b) c-Myc and Pin1 interact in vivo. 293a cells were<lb/> transfected with 2 µg pD40-His-c-Myc, pD40-His-c-Myc T58A , pD40-His-c-<lb/>Myc S62A or empty pD40-His vector (−), as indicated, plus 2 µg Ad-Trk-Pin1<lb/> and 0.5 µg CMV-β-gal. Cells were serum-starved for 48 h and then<lb/> harvested by lysis in low-stringency co-immunoprecipitation buffer.<lb/> Supernatants were normalized for β-galactosidase levels and incubated with<lb/> nickel–NTA agarose beads at 4 °C for 3–4 h to precipitate His 6 -tagged<lb/> c-Myc proteins. Precipitated proteins were washed with co-<lb/>immunoprecipitation buffer containing 20 mM imidazole and eluted with<lb/> co-immunoprecipitation buffer containing 250 mM imidazole. Eluted<lb/> proteins were analysed by western blotting. The upper half of the western<lb/> blot was probed with anti-Myc (N262) and the bottom half with anti-Pin1,<lb/> as indicated.<lb/></figure> <p>MEFs exhibits a typical temporal pattern <ref type="biblio">11</ref> : that is, a low level in quies-<lb/>cent cells <ref type="figure">(Fig. 6, lane 1)</ref>, a peak accumulation at 3 h <ref type="figure">(Fig. 6, lane 2)</ref>, a<lb/> decline at 6 h <ref type="figure">(Fig. 6, lane 3)</ref> and a further decrease to a basal steady-<lb/>state level by 24 h <ref type="figure">(Fig. 6, lane 4)</ref>. Quiescent MEFs lacking Pin1 also<lb/> showed undetectable levels of c-Myc protein before serum stimulation,<lb/> because of very low levels of c-myc gene transcription in growth-<lb/>arrested cells <ref type="figure">(Fig. 6, lane 5)</ref>. Endogenous c-Myc was induced at 3 h<lb/> after serum stimulation in Pin1 −/− cells <ref type="figure" >(Fig. 6, lane 6)</ref>. In contrast to<lb/> wild-type MEFs, however, c-Myc levels increased continuously with<lb/> time, reaching maximal levels at 24 h after serum stimulation <ref type="figure">(Fig. 6,<lb/> lane 8)</ref>. This result is very similar to that resulting from inhibition of<lb/> GSK-3β activity <ref type="biblio">11</ref> and is consistent with stabilization of c-Myc in the<lb/> Pin1 −/− cells. In addition, the increased levels of c-Myc in the Pin1 −/−<lb/> cells correlated with enhanced Ser 62 phosphorylation <ref type="figure">(Fig. 6, second<lb/> panel)</ref>. This is particularly notable at the 6-h time point, when total lev-<lb/>els of c-Myc are largely equivalent between wild-type and Pin1 −/−<lb/> MEFs, but when phospho-Ser 62 levels are much higher in the Pin1 −/−<lb/> MEFs <ref type="figure">(Fig. 6, compare lanes 3 and 7)</ref>. This result was also supported by<lb/> treatment of wild-type MEFs with okadaic acid, which also increased<lb/> Ser 62 phosphorylation and prolonged c-Myc expression in response<lb/> to growth stimulation (data not shown). Examination of Pin1 levels in<lb/> the same samples demonstrates that Pin1 expression in wild-type<lb/> MEFs is not affected by serum stimulation <ref type="figure">(Fig. 6)</ref>. These results sug-<lb/>gest that one way in which Pin1 promotes a decrease in c-Myc levels is<lb/> through enhancing Ser 62 dephosphorylation.<lb/></p> <head>Control of c-Myc phosphorylation affects c-Myc transactivation<lb/> capacity<lb/></head> <p>A primary function of c-Myc is to activate transcription of a large<lb/> number of target genes encoding proteins important for cell growth. To<lb/> assess the functional significance of the regulation of c-Myc phospho-<lb/>rylation and turnover by PP2A, the transcriptional activation capacity<lb/> of c-Myc was assessed in the absence or presence of small T antigen.<lb/> Previously, we demonstrated a role for c-Myc in activation of the E2F2<lb/> promoter that is dependent on the presence of c-Myc-binding sites<lb/> (E-box elements) in the promoter <ref type="biblio">24</ref> . To examine how controlling<lb/> c-Myc degradation affects its function, we reduced the level of Ad-Myc<lb/> sufficiently to minimally activate the E2F2 promoter (<ref type="figure" >Fig. 7</ref>; E2F2-Luc,<lb/> compare control with c-Myc). We also observed only minimal effects<lb/> on the activity of the E2F2 promoter as a result of expressing small T<lb/></p> <figure>WT<lb/> Pin1 −/−<lb/> Pin1 −/−<lb/> Pin1 +/+<lb/> Pin1<lb/> +/+<lb/> Pin1<lb/> +/+<lb/> Pin1<lb/> −/−<lb/> Pin1<lb/> −/−<lb/> Pin1<lb/> +/+<lb/> Pin1<lb/> −/−<lb/> Pin1 +/ ++<lb/> (t 1/2 = 9.2 min)<lb/> Pin1 −/−<lb/> (t 1/2 =137.7 min)<lb/> + C o n t r o l<lb/> + C o n t r o l<lb/> + P in 1<lb/> c-Myc<lb/> c-Myc<lb/> c-Myc<lb/> c-Myc<lb/> c-Myc<lb/> c-Myc T58A<lb/> Ponceau S<lb/> 1<lb/> 0<lb/> 1<lb/> 2<lb/> 3<lb/> 4<lb/> 1<lb/> 2<lb/> 3<lb/> 4<lb/> 7<lb/> 15<lb/> 30<lb/> 2<lb/> 3<lb/> Chase time (min)<lb/> Chase time (min)<lb/> a<lb/> c<lb/> d<lb/> b<lb/> 100<lb/> 1<lb/> 0<lb/> 1 0<lb/> 3 0<lb/> 20<lb/> 10<lb/> Ad-small T antigen<lb/> 35 S-c-Myc remaining (percentage)<lb/> Figure 5 Accumulation of c-Myc is regulated by Pin1. (a) c-Myc levels<lb/> are elevated in Pin1 −/− cells. Passage-3 primary MEFs from siblings with<lb/> either wild-type (WT) or Pin1 −/− genotypes were made quiescent by<lb/> serum starvation for 48 h and then infected with Ad-c-Myc (MOI = 100)<lb/> together with either Ad-GFP (control) or Ad-Pin1 (both at MOI = 400).<lb/> Infected cells were maintained in medium with 0.25% serum for 20 h<lb/> and extracts prepared from an equal number of cells for each condition<lb/> were subjected to western blot analysis with the C-33 c-Myc antibody.<lb/> (b) c-Myc half-life is increased in the absence of Pin1. Quiescent<lb/> primary Pin1 +/+ or Pin1 −/− MEFs were infected with Ad-c-Myc<lb/> (MOI = 100) and maintained in low-serum medium. At 18 h after<lb/> infection, cells were labelled with 35 S-methionine/cysteine for 30 min<lb/> and chased in medium with 0.20% serum containing excess unlabelled<lb/> methionine and cysteine for the indicated times. Labelled c-Myc was<lb/> immunoprecipitated from equal cell numbers at each time, analysed by<lb/> SDS–PAGE and then quantified with a phosphorimager. Best-fit<lb/> exponential lines are shown for c-Myc expressed in Pin1 +/+ and Pin1 −/−<lb/> MEFs. (c) c-Myc protein levels in Pin1 −/− MEFS are not affected by small<lb/> T antigen. Quiescent primary Pin1 +/+ or Pin1 −/− MEFs were infected with<lb/> Ad-c-Myc (MOI = 100) and either Ad-GFP (control; MOI = 400) or Ad-<lb/>small T (MOI = 400). Infected cells were treated as described in a.<lb/> Equal protein loading was confirmed by Ponceau S staining and<lb/> measurement of a reference band. (d) The c-Myc T58A mutant is not<lb/> affected by deletion of Pin1. Quiescent primary Pin1 +/+ or Pin1 −/− MEFs<lb/> (passage 3) were infected with Ad-c-Myc or Ad-c-Myc T58A (MOI = 100)<lb/> and treated as described in a.<lb/></figure> <p>antigen. In contrast, co-expression of small T antigen and c-Myc<lb/> resulted in a substantial increase in E2F2 promoter activity, and this<lb/> activation was dependent on the presence of intact c-Myc-binding sites<lb/> in the promoter (<ref type="figure" >Fig. 7</ref>; E2F2(-Ebox)-Luc, c-Myc + tAg). We conclude<lb/> that the control of c-Myc phosphorylation not only regulates the stabil-<lb/>ity of c-Myc, but also has a direct impact on c-Myc function.<lb/></p> <head>Stabilized c-Myc can replace small T antigen in transformation<lb/> of human cells<lb/></head> <p>Human fibroblasts can be transformed by a combination of Ras,<lb/> telomerase and SV40 large T and small T antigens <ref type="biblio">25</ref> . Given the role of<lb/> small T antigen in regulating PP2A, together with the role for PP2A in<lb/> mediating the destabilization of c-Myc, we evaluated whether a stabi-<lb/>lized form of c-Myc could replace small T antigen as a necessary<lb/> requirement for transformation and tumorigenesis. Human embry-<lb/>onic kidney (HEK) cells expressing large T antigen, the catalytic sub-<lb/>unit of human telomerase (hTERT) and Ras G12V (hereby referred to as<lb/> HEK-TER cells) cannot grow in soft agar (a measure of transformed<lb/> cell growth) or form tumours in mice, unless small T antigen is intro-<lb/>duced <ref type="biblio">26</ref> . We ectopically expressed the stable c-Myc T58A mutant in the<lb/> HEK-TER cells or independently expressed small T antigen (as a posi-<lb/>tive control), wild-type c-Myc or c-Myc S62A (which cannot be phos-<lb/>phorylated and consequently is rapidly degraded 11 ) as negative<lb/> controls, and developed cell populations that expressed each of the four<lb/> proteins. These cell populations were then assayed for anchorage-inde-<lb/>pendent growth in soft agar, the best in vitro corollary to tumorigenic<lb/> growth in vivo. Addition of small T antigen promoted aggressive trans-<lb/>formed growth, as previously reported <ref type="biblio">26</ref> , whereas cells expressing wild-<lb/>type c-Myc or c-Myc S62A failed to grow in an anchorage-independent<lb/> manner <ref type="figure">(Fig. 8a)</ref>. However, the cells expressing stabilized c-Myc T58A<lb/> protein grew in soft agar identically to cells expressing small T antigen<lb/> <ref type="figure">(Fig. 8a)</ref>. Similar transformation trends for the c-Myc S62A and<lb/> c-Myc T58A mutants have been reported previously in rodent cells <ref type="biblio">12,13</ref> .<lb/> To confirm our result, mixed populations were examined, as well as<lb/> clonally isolated cell lines expressing c-Myc T58A . All these cell lines<lb/> exhibited growth in soft agar, whereas none of the wild-type c-Myc<lb/> populations were able to grow (see <ref type="figure">Supplementary Information, Fig.<lb/> S4</ref>). Finally, c-Myc T58A was tested in a second human cell type — BJ<lb/> fibroblasts also containing small T antigen, hTERT and Ras G12V — and<lb/> compared with small T antigen as a positive control or vector alone as a<lb/> negative control. c-Myc T58A -expressing cells grew as well as the small-<lb/>T-antigen -positive control cells <ref type="figure">(Fig. 8a)</ref>. Thus, a stable, and therefore<lb/> oncogenic, c-Myc can functionally replace small T antigen in trans-<lb/>formed cell growth of human cells expressing a defined set of genes.<lb/></p> <p>We then questioned whether c-Myc T58A could also replace small T<lb/> antigen in the most stringent test of human oncogenesis: in vivo tumour<lb/> growth. The four HEK-TER cell populations described above were<lb/> assayed for tumour growth in immuno-compromised mice. As previ-<lb/>ously observed, small T antigen was essential for primary human cells<lb/> expressing large T antigen, hTERT and Ras G12V to form tumours <ref type="figure">(Fig.<lb/> 8b)</ref>. Consistent with soft agar assays, mice injected with cells expressing<lb/> wild-type c-Myc or the c-Myc S62A mutant completely failed to exhibit<lb/> tumour growth for up to 35 days, which is three times longer than it<lb/> takes positive control cells expressing small T antigen to form tumours.<lb/> Strikingly, cells expressing c-Myc T58A formed tumours in an almost<lb/> identical manner to cells expressing small T antigen <ref type="figure">(Fig. 8b)</ref>. On the<lb/> basis of these results, we conclude that the oncogenic transformation of<lb/> human cells can be accomplished by the combined action of large T<lb/> antigen, Ras, telomerase, and an activated, stabilized, c-Myc. Thus, at<lb/> least one functional consequence of c-Myc stabilization in human cells<lb/> is to sensitize the cells to transformation and tumorigenesis.<lb/></p> <head>DISCUSSION<lb/></head> <p>Although an increase in protein levels could be achieved in principle<lb/></p> <figure>WT<lb/> Pin −/−<lb/> 0<lb/> 3<lb/> 6<lb/> 2 4<lb/> 0<lb/> 3<lb/> 6<lb/> 2 4<lb/> c-Myc<lb/> c-Myc-P-S62<lb/> α-tubulin<lb/> Pin1<lb/> 1<lb/> 2<lb/> 3<lb/> 4<lb/> 5<lb/> 6<lb/> 7<lb/> 8<lb/> (h)<lb/> Time<lb/> Figure 6 Pin1 is essential for normal control of c-Myc accumulation.<lb/> Accumulation of endogenous c-Myc is prolonged in Pin1 −/− fibroblasts and<lb/> shows increased Ser 62 phosphorylation. Primary Pin1 +/+ or Pin1 −/− MEFs<lb/> (passage 2 ) were density growth-arrested, plated at low density into medium<lb/> with 0.25% serum and incubated for 24 h. These quiescent cells were then<lb/> stimulated by the addition of 20% FCS for the indicated times. Equal cell<lb/> counts from serum-starved (0 h) or stimulated (3, 6, and 24 h) cells were<lb/> harvested and subjected to western blot analysis with the mouse monoclonal<lb/> C33 c-Myc antibody (top). The blot was stripped and re-probed with the<lb/> rabbit polyclonal phosphoSer62 antibody (second panel) or the α-tubulin<lb/> antibody (third panel). Samples were then re-run and probed with the anti-<lb/>Pin1 antibody (bottom).<lb/></figure> <p>through transcriptional activation coupled with a short half-life of the<lb/> protein, the action of Ras provides a mechanism to amplify c-Myc<lb/> accumulation by stabilizing this otherwise short-lived protein. We sug-<lb/>gest that the role of the inter-related phosphorylation and dephospho-<lb/>rylation events is to ensure that Ras-mediated amplification of c-Myc<lb/> protein levels is indeed transient and self-limited. The Raf–MEK–ERK<lb/> pathway stabilizes c-Myc by enhancing Ser 62 phosphorylation, and<lb/> the PI(3)K/Akt pathway prevents the subsequent phosphorylation of c-<lb/>Myc at Thr 58 by inhibiting GSK-3β. However, as Ras activity declines<lb/> after cessation of the growth stimulus, PI(3)K and Akt activities also<lb/> decline, resulting in reactivation of GSK-3β and phosphorylation of c-<lb/>Myc on Thr 58. Our data suggest that phosphorylation of Thr 58 is<lb/> important for recognition of c-Myc by the Pin1 prolyl isomerase, the<lb/> actions of which facilitate c-Myc dephosphorylation at Ser 62 by PP2A,<lb/> which then promotes c-Myc turnover by the ubiquitin-proteasome<lb/> pathway. Thus, the very mechanism that stabilizes and amplifies c-Myc<lb/> accumulation — c-Myc phosphorylation at Ser 62 — also triggers the<lb/> subsequent phosphorylation at Thr 58 and the series of events that cul-<lb/>minate in degradation of c-Myc.<lb/></p> <p>Considerable evidence suggests that Thr 58 phosphorylation is criti-<lb/>cal for ensuring the transient and timely degradation of c-Myc. All v-<lb/>myc genes recovered in transforming retroviruses harbour a mutation<lb/> at Thr 58 <ref type="biblio">(ref. 27)</ref>. Similarly, a large number of c-myc genes amplified<lb/> in Burkitt's lymphoma carry a mutation at Thr 58, as well as in other<lb/> residues between amino acids 57 and 63 <ref type="biblio">(refs 12, 13, 16, 17 and 28)</ref>.<lb/> Assay of these mutants generally demonstrates their increased onco-<lb/>genic potential in both cell transformation assays and animals <ref type="biblio">12,13,29</ref> . In<lb/> conjunction with other studies, our results confirm that mutation of<lb/> c-Myc at Thr 58 increases the stability of c-Myc, highlighting a key role<lb/> for this phosphorylation site in controlling c-Myc degradation <ref type="biblio">11,16,17</ref> .<lb/> These results are consistent with a model in which the Pin1 WW<lb/> domain binds to c-Myc phosphorylated at Thr 58 in a manner that<lb/> could result in a conformational change that makes phospho-Ser 62 an<lb/> ideal site for dephosphorylation by PP2A. Our additional finding that a<lb/> stabilized c-Myc protein can completely substitute for small T antigen<lb/> by collaborating with Ras, hTERT and small T antigen in the transfor-<lb/>mation and oncogenic conversion of primary human cells, whereas<lb/> wild-type c-Myc cannot do so, establishes the importance of c-Myc sta-<lb/>bility in human cancer.<lb/></p> <head>METHODS<lb/></head> <p>Antibodies. The C-33 and N262 c-Myc antibodies are from Santa Cruz<lb/> Biotechnology (Santa Cruz, CA). The ubiquitin antibody is from Zymed (South<lb/> San Francisco, CA). The Thr 58 phosphospecific antibody is from Upstate Cell<lb/> Signaling (Waltham, MA). The Ser 62 phosphospecific antiserum was prepared<lb/> as described previously <ref type="biblio">11</ref> . The Pin1 antibody was prepared as described previ-<lb/>ously <ref type="biblio">30</ref> . The α-tubulin antibody is from Sigma (St Louis, MO). The PP2A cat-<lb/>alytic unit antibody is from BD Biosciences (San Jose, CA).<lb/></p> <p>Plasmids. CMV -Myc, CMV-β-gal, E2F2-Luc, and E2F2(-Ebox3)-Luc plasmids<lb/> have been previously described <ref type="biblio">24</ref> . His 6 -tagged c-Myc was created by PCR ampli-<lb/>fication of the coding sequence for wild-type murine c-Myc2 from the CMV-<lb/>Myc plasmid with a CACC 5′ extension added to the 5′ primer for cloning into<lb/> the TOPO entry clone from Invitrogen (Carlsbad, CA). This sequence was then<lb/> moved into the pDEST-40 mammalian expression vector using Gateway tech-<lb/>nology (Invitrogen), creating pD40-His-c-Myc. His 6 -tagged c-Myc T58A and<lb/> His 6 -tagged c-Myc S62A were constructed similarly, except that PCR amplifica-<lb/>tion was from the CMV-Myc T58A and CMV-Myc S62A plasmids previously<lb/> described <ref type="biblio">11</ref> , creating pD40-His-c-Myc(T58A) and pD40-His-c-Myc S62A . The<lb/> Ad-Trk-Pin1 plasmid was constructed as described below.<lb/></p> <p>Cells. REF52 cells were grown in DMEM containing 5% foetal calf serum (FCS)<lb/> and 5% calf serum (CS). To bring REF52 cells to quiescence, cells were plated at<lb/> ~3,500 cells cm −2 and incubated overnight. The next day the culture medium<lb/> was replaced with DMEM containing 0.25% serum (FCS/CS) and cells were<lb/> incubated for an additional 48 h. Primary MEFs were grown in DMEM contain-<lb/>ing 15% heat-inactivated FCS. MEFs were made quiescent either by plating pas-<lb/>sage-3 MEFs at a density of approximately 2,000 cells cm −2 and the next day<lb/> replacing the medium with DMEM containing 0.2% heat-inactivated FCS for<lb/> 48 h, or by allowing passage-2 MEFs to grow for 24 h after they had reached con-<lb/>fluence and then plating them directly into DMEM/0.2% serum at 4,000 cells<lb/> cm −2 for 24 h. Where indicated, cells were serum-stimulated by adding FCS<lb/> directly to the starvation media to a final concentration of 20%. 293 cells<lb/> obtained from the American Type Culture Collection were grown in DMEM<lb/> containing 10% FCS. For serum-starvation conditions, exponentially growing<lb/> 293 cells were washed with DMEM, refed with DMEM containing 2% FCS and<lb/> incubated for 48 h.<lb/></p> <p>Viruses. Stocks of purified virus were created as described previously <ref type="biblio" >31</ref> . Viral<lb/> titres (MOI) were determined in 293 cells using an indirect immunofluores-<lb/>cence microscopy assay specific for the viral 72K E2 gene product, as<lb/> described <ref type="biblio">32</ref> . Experimental comparisons demonstrate that REF52 fibroblasts are<lb/> approximately 400-fold less susceptible to infection than 293 cells, and MEFs are<lb/> approximately 1,200-fold less susceptible. Quiescent REF52 or MEFs were<lb/> infected with virus by incubation in DMEM containing 20 mM Hepes at pH 7.2<lb/> for 75 min at 37 °C at a cell-to-volume ratio of 5 × 10 5 cells ml −1 . After infec-<lb/>tion, four volumes of DMEM/0.25% (REF52) or 0.2% (MEF) serum were added<lb/> to each plate and the cells were further incubated at 37 °C for 18–20 h before<lb/> harvest and analysis. The construction of Ad-c-Myc, Ad-c-Myc T58A and Ad-c-<lb/>Myc S62A has been described 11 . Ad-GFP has been described previously <ref type="biblio">33</ref> . Ad<lb/> small T antigen was constructed by digesting the pCEP small T plasmid with<lb/> SmaI and HindIII, and ligating this fragment containing the SV40 small T anti-<lb/>gen cDNA into the HindIII/EcoRV sites in the AdTrack-CMV plasmid used to<lb/></p> <figure>E2F2-Luc<lb/> E2F2(-Ebox)-Luc<lb/> 4.00<lb/> 3.00<lb/> 2.00<lb/> 1.00<lb/> 0.00<lb/> C o n t r o l<lb/> c -M y c<lb/> t -A g<lb/> c -M y c + t -A g<lb/> C o n t r o l<lb/> c -M y c<lb/> t -A g<lb/> c -M y c + t -A g<lb/> Fold luciferase activity<lb/> Figure 7 Small T antigen enhances the transactivation function of c-Myc.<lb/> REF52 fibroblasts were transfected by calcium phosphate co-precipitation<lb/> with either wild-type E2F2-luciferase construct (E2F2-Luc) or mutant<lb/> E2F2-luciferase construct containing point mutations in the three E-box<lb/> c-Myc-binding sites (which eliminates c-Myc binding to the E2F2<lb/> promoter; E2F2(-Ebox)-Luc), plus CMV-β-gal as an internal control. After<lb/> 16 h, cells were placed in DMEM containing 0.25% FCS for 48 h.<lb/> Transfected cells were then infected with Ad-GFP (MOI = 200) (Ctrl), Ad-<lb/>c-Myc (MOI = 100), Ad-small T antigen (MOI = 100) or Ad-c-Myc<lb/> (MOI = 100) + Ad-small T antigen (MOI = 100), as indicated. Cells were<lb/> harvested 20 h after infection, and luciferase and β-galactosidase<lb/> activities were measured. Luciferase activity was normalized to β-<lb/>galactosidase activity, and data are presented as fold activation over<lb/> control. Transfections were performed in two separate experiments and<lb/> the mean fold induction from three separate data points per condition is<lb/> shown, with error bars indicating standard deviations.<lb/></figure> <p>construct recombinant adenoviruses as described <ref type="biblio">34</ref> . Ad-Pin was similarly con-<lb/>structed by digesting the pET28-Pin1 plasmid with BamHI and XhoI and then<lb/> ligating this fragment containing the human Pin1 cDNA into the BglII/XhoI<lb/> sites in AdTrack-CMV, creating the plasmid Ad-Trk-Pin1 used to construct the<lb/> Ad-Pin1 virus.<lb/></p> <p>Detection of ubiquitinated Myc. Cells were washed once with cold PBS and<lb/> lysed with RIPA buffer containing standard protease and phosphatase<lb/> inhibitors, as described <ref type="biblio">10</ref> , with the addition of the de-ubiquitinase inhibitor 5<lb/> mM N-ethylmaleimide (NEM; Sigma) at a cell-to-volume ratio of 1 × 10 6 cells<lb/> ml −1 . Ubiquitinated proteins were immunoprecipitated from cell lysates with an<lb/> anti-ubiquitin antibody from Zymed. After precipitation with the protein<lb/> A/protein G–agarose beads, immunoprecipitates were washed once with low-<lb/>stringency buffer containing PBS, 0.1% NP40 (Sigma) and all inhibitors used<lb/> with the RIPA buffer. Immunoprecipitated proteins were then separated by 10%<lb/> SDS–PAGE, blotted to immobilon-P membrane and detected with the indicated<lb/> c-Myc antibodies.<lb/></p> <p>Western blotting. Cells were harvested by scraping into hot 1× SDS sample<lb/> buffer at a cell-to-volume ratio of 2.5 × 10 6 cells ml −1 . Protein concentrations<lb/> were determined for normalization purposes using the BCA solutions (Sigma)<lb/> according to the manufacturer's instructions. Equal protein for each sample was<lb/> separated by SDS–PAGE and blotted to Immobilon-P membrane (Millipore,<lb/> Billerica, MA). Primary antibodies were in 5% non-fat milk, PBS, 0.1%<lb/> Tween20, except anti-phospho-Ser 62 antibody which was in 5% heat-inacti-<lb/>vated horse serum/5% BSA, PBS, 0.1% Tween20. Primary antibodies were<lb/> detected with HRP-conjugated secondary antibodies using the ECL reagents<lb/> from Amersham according to the manufacturer's instructions.<lb/></p> <p>Immunoprecipitations. Cells were harvested by washing once in cold PBS, fol-<lb/>lowed by lysis in cold c-Myc antibody lysis buffer containing protease and phos-<lb/>phatase inhibitors as previously described <ref type="biblio">10</ref> . c-Myc proteins were<lb/> immunoprecipitated from equal numbers of cells for each sample at a cell-to-<lb/>volume ratio of 1 × 10 6 cells ml −1 lysis buffer using a 1:100 dilution of the c-Myc<lb/> monoclonal antibody C-33 (Santa Cruz Biotechnology, Santa Cruz, CA).<lb/> Antibody incubation was performed at 4 °C for 14–18 h followed by immuno-<lb/>precipitation with protein A/protein G–agarose beads and washing with lysis<lb/> buffer containing protease and phosphatase inhibitors.<lb/></p> <p>Pulse-chase assays. MEFs or REFs were pre-starved for methionine and cysteine<lb/> by replacing the culture medium with DMEM lacking L-Methionine and L -<lb/>Cysteine for 15 min. Cells were labelled in vivo with <ref type="biblio">35</ref> S-methionine/cysteine<lb/> EXPRESS protein labelling mix from Perkin Elmer (Boston, MA) using 500 µCi<lb/> ml −1 at a cell-to-volume ratio of 7.5 × 10 5 cells ml −1 for 20–30 min. After<lb/> labelling, cells were immediately washed once with DMEM containing 5 mM<lb/> L-methionine, 3 mM L-cysteine and 0.2% serum, and then incubated in the<lb/> same media for the indicated chase times. Cells were harvested and labelled<lb/> c-Myc proteins were immunoprecipitated as described above. Labelled c-Myc<lb/> was visualized by autoradiography and quantified with a phosphorimager.<lb/></p> <p>Myc–GST–Pin1 interaction assays. GST and GST–Pin1 fusion proteins with<lb/> either wild-type Xenopus laevis Pin1, a X. laevis Pin1 WW-domain mutant with<lb/> substitutions at W11A and W34A, or a X. laevis Pin1 catalytic-deficient mutant<lb/> with a substitution at C109A were prepared as described <ref type="biblio">30</ref> . The GST and<lb/> GST–Pin1 recombinant proteins were bound to glutathione–agarose beads as<lb/> follows: 10 µg of recombinant protein were incubated with 25 µl of a 50% slurry<lb/> of glutathione–agarose beads in 500 µl of a low-stringency buffer containing<lb/> 1× PBS, 0.5% NP40 and 5% glycerol with standard protease and phosphatase<lb/></p> <figure>a<lb/> b<lb/> 400<lb/> 450<lb/> 400<lb/> 350<lb/> 300<lb/> 250<lb/> 200<lb/> 150<lb/> 100<lb/> 50<lb/> 0<lb/> 0<lb/> 50<lb/> 100<lb/> 150<lb/> 200<lb/> 250<lb/> 300<lb/> 350<lb/> t-Ag<lb/> c-Myc<lb/> T58A<lb/> S62A<lb/> Control<lb/> t-Ag<lb/> T58A<lb/> HEK-TER cells<lb/> BJ-TER cells<lb/> 3<lb/> 2.5<lb/> 2<lb/> 1.5<lb/> 1<lb/> 0.5<lb/> 0<lb/> 0<lb/> 3 0<lb/> 25<lb/> 20<lb/> 15<lb/> 10<lb/> 5<lb/> 3 5<lb/> Time (days)<lb/> Cell type<lb/> Tumour number<lb/> t-Ag<lb/> c-Myc WT<lb/> c-Myc T58A<lb/> c-Myc S62A<lb/> 6/6<lb/> 0/6<lb/> 6/6<lb/> 0/6<lb/> Tumour growth (cm<lb/> 3 )<lb/> Colony number<lb/> Colony number<lb/> Figure 8 Stabilized c-Myc cooperates with Ras and telomerase to transform<lb/> human fibroblasts. (a) c-Myc T58A has transforming properties. Anchorage-<lb/>independent growth of the indicated cells stably infected with retroviruses<lb/> encoding small T antigen, c-Myc, c-Myc S62A or c-Myc T58A , calculated from<lb/> the average number of colonies observed in three plates. (b) c-Myc T58A<lb/> replaces small T antigen function in tumorigenesis assays. Mean ± standard<lb/> error of tumour volumes (cm 3 ) from six mice injected with HEK cells<lb/> expressing hTERT, T-ag, Ras G12V and either small T antigen, c-Myc, c-<lb/>Myc S62A or c-Myc T58A . n = 6 for each injection.<lb/></figure> <p>inhibitors <ref type="biblio">10</ref> . Binding was performed at 4 °C for 1.5 h. GST beads were then<lb/> washed four times in 600 µl of low-stringency buffer. REF52 cells infected with<lb/> Ad-c-Myc were lysed in low-stringency buffer at a cell-to-volume ratio of<lb/> 5 × 10 5 cells ml −1 . 500 µl of cell lysate containing c-Myc was then incubated with<lb/> the various GST–Pin1 beads for 4 h at 4 °C. Samples were then washed three<lb/> times in 600 µl of low-stringency buffer and precipitated proteins were sub-<lb/>jected to western blot analysis.<lb/></p> <p>Co-precipitation of Myc and Pin1. 293 cells were transfected with expression<lb/> plasmids for wild-type or mutant His 6 -tagged c-Myc proteins, Pin1 and<lb/> β-galactosidase. Cells were lysed in co-immunoprecipitation buffer (50mM<lb/> Hepes at pH 7.2, 125 mM potassium acetate, 0.5 mM EDTA, 0.5 mM EGTA,<lb/> 0.1% Tween-20 and 12.5% glycerol) containing protease and phosphatase<lb/> inhibitors. Extracts were normalized for transfection efficiency by β-galactosi-<lb/>dase assay and incubated with nickel–NTA–agarose beads (Qiagen, Valencia,<lb/> CA) for 3–4 h at 4 °C followed by three washes in co-immunoprecipitation<lb/> buffer containing protease and phosphatase inhibitors and 20 mM imidazole.<lb/> Precipitated proteins were released with co-immunoprecipitation buffer con-<lb/>taining 250 mM imidazole and then analysed by western blotting.<lb/></p> <p>Primary wild-type and Pin1 −/− MEFs. Pin1 −/− mice were generated as previ-<lb/>ously described <ref type="biblio">35</ref> . The Pin1 gene deletion was transferred into an isogenic<lb/> C57Bl6 background using marker-assisted speed congenic breeding by Jackson<lb/> Laboratory (Bar Harbor, ME). Pin1 +/+ and Pin1 −/− embryonic fibroblasts were<lb/> isolated from the isogenic strain as detailed previously 36 and processed as<lb/> described above.<lb/></p> <p>Assay of endogenous c-Myc. Asynchronous or quiescent REF52 cells were<lb/> infected with Ad-small T antigen or control Ad-GFP, and endogenous c-Myc was<lb/> immunoprecipitated as described under Immunoprecipitations, except that cells<lb/> were lysed in antibody lysis buffer at 2 × 10 6 cells ml −1 and precipitates were not<lb/> washed, but loaded directly onto 8% gels and fractionated by SDS–PAGE.<lb/> Endogenous c-Myc from primary MEFs was assayed after cells were density<lb/> growth-arrested in DMEM containing 15% heat-inactivated FCS for 24 h after<lb/> they reached confluence, before& plating at ~4,000 cells cm −2 in DMEM con-<lb/>taining 0.2% heat-inactivated FCS. After 24 h, cells were serum-stimulated by<lb/> adding FCS to 20% volume. Samples were then scraped into hot SDS sample<lb/> buffer at the indicated times and analysed by western blotting as described above.<lb/></p> <p>Transfections. REF52 fibroblasts were transfected using calcium phosphate co-<lb/>precipitation, as previously described <ref type="biblio">24</ref> . Cells were plated the day before trans-<lb/>fection in 100-mm tissue culture dishes at ~3,500cells cm −2 . Cells were exposed<lb/> to the CaPO 4 –DNA precipitate containing 10–20 µg of total DNA for 18–20 h.<lb/> Transfected cells were washed once with DMEM and placed into starve media<lb/> (DMEM containing 0.25% FCS) for 48 h. Luciferase activity was measured<lb/> using a Promega (Madison, WI) luciferase assay system kit, as specified by the<lb/> manufacturer, and a Berthold luminometer (Bundoora, Australia). β-galactosi-<lb/>dase activity was measured as described previously <ref type="biblio" >24</ref> . 293a cells were plated at<lb/> ~5,000 cells cm −2 into tissue culture dishes and incubated overnight. Cells at<lb/> 60–80% confluency were then transfected with Perfectin (Gene Therapy<lb/> Systems, San Diego, CA) or TransIT-TKO (Mirus Co., Madison, WI) at a 4:1<lb/> ratio of lipid to DNA, in low-serum medium (2% FCS) according to the manu-<lb/>facturer's specifications. Transfected cells were maintained in DMEM contain-<lb/>ing 2% FCS and harvested 48 h after transfection.<lb/></p> <p>RNAi experiments. 293 cells were transfected with expression vectors for c-Myc,<lb/> β-galactosidase, and Luciferase, along with either pooled siRNAs directed<lb/> against the PP2A C subunit (Dharmacon, Lafayette, CO) or control siRNAs<lb/> including a scrambled siRNA or siRNA directed against luciferase from the<lb/> siSTARTER Luciferase Kit (Dharmacon) using TransIT-TKO (Mirus Co.). Cells<lb/> were collected 48 h after transfection and analysed for β-galactosidase activity as<lb/> previously described. Both luciferase activity and protein load volumes for west-<lb/>ern blot analysis were adjusted for transfection efficiency on the basis of β-galac-<lb/>tosidase activity.<lb/></p> <p>Transformation and tumorigenesis assays. The retroviral constructs containing<lb/> c-Myc, c-Myc T58A , and c-Myc S62A were created by PCR amplification of cDNA<lb/> from the pRC-CMV vector 37 using T7 and SP6 universal primers with BamHI<lb/> and EcoRI sites engineered onto the ends. The DNAs were then ligated into the<lb/> pWZL-Blast vector digested with the same enzymes to create pWZL-Blast<lb/> c-Myc, c-Myc T58A and c-Myc S62A . The HEK and BJ cell lines containing hTERT,<lb/> Ras, large T antigen and small T antigen in varying combinations were created<lb/> by retroviral transduction of parental cells as previously described <ref type="biblio">26</ref> . The subse-<lb/>quent cell lines containing the various c-Myc constructs were generated by<lb/> amphotrophic retroviral infection and polyclonal selection with 2.5 µg ml blas-<lb/>ticidin S (EMD Biosciences, San Diego, CA).<lb/></p> <p>The ability of cells to undergo anchorage-independent growth was tested as<lb/> previously described <ref type="biblio">38</ref> . Briefly, 5 × 10 4 cells were plated in 0.3% agar for 12 days<lb/> (HEK cells) or 21 days (BJ cells) and assessed for colony growth in soft agar. The<lb/> mean and standard deviation represents two independent experiments performed<lb/> in triplicate. Tumour formation in immunodeficient mice was determined by<lb/> sub-cutaneous injection of 1 × 10 7 cells per animal and was represented as the<lb/> number of tumours/number of animals (1 injection site per animal).<lb/></p> </text> </tei>