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• W2076359094 abstract "MYC levels are tightly regulated in cells, and deregulation is associated with many cancers. In this report, we describe the existence of a MYC-protein kinase A (PKA)-polo-like kinase 1 (PLK1) signaling loop in cells. We report that sequential MYC phosphorylation by PKA and PLK1 protects MYC from proteasome-mediated degradation. Interestingly, short term pan-PKA inhibition diminishes MYC level, whereas prolonged PKA catalytic subunit α (PKACα) knockdown, but not PKA catalytic subunit β (PKACβ) knockdown, increases MYC. We show that the short term effect of pan-PKA inhibition on MYC is post-translational and the PKACα-specific long term effect on MYC is transcriptional. These data also reveal distinct functional roles among PKA catalytic isoforms in MYC regulation. We attribute this effect to differential phosphorylation selectivity among PKA catalytic subunits, which we demonstrate for multiple substrates. Further, we also show that MYC up-regulates PKACβ, transcriptionally forming a proximate positive feedback loop. These results establish PKA as a regulator of MYC and highlight the distinct biological roles of the different PKA catalytic subunits. MYC levels are tightly regulated in cells, and deregulation is associated with many cancers. In this report, we describe the existence of a MYC-protein kinase A (PKA)-polo-like kinase 1 (PLK1) signaling loop in cells. We report that sequential MYC phosphorylation by PKA and PLK1 protects MYC from proteasome-mediated degradation. Interestingly, short term pan-PKA inhibition diminishes MYC level, whereas prolonged PKA catalytic subunit α (PKACα) knockdown, but not PKA catalytic subunit β (PKACβ) knockdown, increases MYC. We show that the short term effect of pan-PKA inhibition on MYC is post-translational and the PKACα-specific long term effect on MYC is transcriptional. These data also reveal distinct functional roles among PKA catalytic isoforms in MYC regulation. We attribute this effect to differential phosphorylation selectivity among PKA catalytic subunits, which we demonstrate for multiple substrates. Further, we also show that MYC up-regulates PKACβ, transcriptionally forming a proximate positive feedback loop. These results establish PKA as a regulator of MYC and highlight the distinct biological roles of the different PKA catalytic subunits. MYC is a basic helix-loop-helix leucine zipper transcription factor that regulates a large number of target genes important in cell growth, metabolism, differentiation, proliferation, and apoptosis (1Bernard S. Eilers M. Control of cell proliferation and growth by Myc proteins.Results Probl. Cell Differ. 2006; 42: 329-342Crossref PubMed Scopus (40) Google Scholar, 2Dang C.V. c-Myc target genes involved in cell growth, apoptosis, and metabolism.Mol. Cell. Biol. 1999; 19: 1-11Crossref PubMed Scopus (1383) Google Scholar, 3Hoffman B. Liebermann D.A. Apoptotic signaling by c-MYC.Oncogene. 2008; 27: 6462-6472Crossref PubMed Scopus (374) Google Scholar, 4Jiang G. Albihn A. Tang T. Tian Z. Henriksson M. Role of Myc in differentiation and apoptosis in HL60 cells after exposure to arsenic trioxide or all-trans-retinoic acid.Leuk. Res. 2008; 32: 297-307Crossref PubMed Scopus (19) Google Scholar, 5Gao P. Tchernyshyov I. Chang T.C. Lee Y.S. Kita K. Ochi T. Zeller K.I. De Marzo A.M. Van Eyk J.E. Mendell J.T. Dang C.V. c-Myc suppression of miR-23a/b enhances mitochondrial glutaminase expression and glutamine metabolism.Nature. 2009; 458: 762-765Crossref PubMed Scopus (1581) Google Scholar, 6Schmidt E.V. The role of c-myc in cellular growth control.Oncogene. 1999; 18: 2988-2996Crossref PubMed Scopus (313) Google Scholar). Complete loss of MYC function results in embryonic lethality, whereas its overexpression predisposes cells to malignant transformation (7Davis A.C. Wims M. Spotts G.D. Hann S.R. Bradley A. A null c-myc mutation causes lethality before 10.5 days of gestation in homozygotes and reduced fertility in heterozygous female mice.Genes Dev. 1993; 7: 671-682Crossref PubMed Scopus (431) Google Scholar, 8Lee C.M. Reddy E.P. The v-myc oncogene.Oncogene. 1999; 18: 2997-3003Crossref PubMed Scopus (39) Google Scholar, 9Ramsay G.M. Moscovici G. Moscovici C. Bishop J.M. Neoplastic transformation and tumorigenesis by the human protooncogene MYC.Proc. Natl. Acad. Sci. U.S.A. 1990; 87: 2102-2106Crossref PubMed Scopus (20) Google Scholar). MYC overexpression is an early and consistent feature of many human malignancies, where it is suspected to regulate key events in tumorigenesis. Because MYC drives the transcription of genes important in multiple cellular processes, precise temporal regulation of MYC is required. Tight regulation of MYC is achieved through multiple mechanisms at the transcriptional, post-transcriptional, translational, and post-translational levels (10Dani C. Blanchard J.M. Piechaczyk M. El Sabouty S. Marty L. Jeanteur P. Extreme instability of myc mRNA in normal and transformed human cells.Proc. Natl. Acad. Sci. U.S.A. 1984; 81: 7046-7050Crossref PubMed Scopus (334) Google Scholar, 11Sears R.C. The life cycle of c-Myc: from synthesis to degradation.Cell Cycle. 2004; 3: 1133-1137Crossref PubMed Scopus (283) Google Scholar, 12Lemm I. Ross J. Regulation of c-myc mRNA decay by translational pausing in a coding region instability determinant.Mol. Cell. Biol. 2002; 22: 3959-3969Crossref PubMed Scopus (122) Google Scholar, 13Wall M. Poortinga G. Hannan K.M. Pearson R.B. Hannan R.D. McArthur G.A. Translational control of c-MYC by rapamycin promotes terminal myeloid differentiation.Blood. 2008; 112: 2305-2317Crossref PubMed Scopus (78) Google Scholar, 14Malempati S. Tibbitts D. Cunningham M. Akkari Y. Olson S. Fan G. Sears R.C. Aberrant stabilization of c-Myc protein in some lymphoblastic leukemias.Leukemia. 2006; 20: 1572-1581Crossref PubMed Scopus (73) Google Scholar). Once translated, MYC is rapidly turned over in normal cells. The MYC steady-state level is regulated at the post-translational level through a series of exquisitely orchestrated phosphorylation events. Ras-mediated activation of MAPK stabilizes MYC by phosphorylation at Ser-62 within the evolutionarily conserved MYC box I region (11Sears R.C. The life cycle of c-Myc: from synthesis to degradation.Cell Cycle. 2004; 3: 1133-1137Crossref PubMed Scopus (283) Google Scholar). Ser-62-phosphorylated MYC is recognized by GSK3β, which phosphorylates Ser-62-primed MYC at Thr-58. Peptidylprolyl isomerase 1 then catalyzes a cis to trans isomerization of the bond preceding Ser-62, thereby allowing the trans-specific protein phosphatase 2A to remove the stabilizing phosphorylation at Ser-62 (15Yeh E. Cunningham M. Arnold H. Chasse D. Monteith T. Ivaldi G. Hahn W.C. Stukenberg P.T. Shenolikar S. Uchida T. Counter C.M. Nevins J.R. Means A.R. Sears R. A signalling pathway controlling c-Myc degradation that impacts oncogenic transformation of human cells.Nat. Cell Biol. 2004; 6: 308-318Crossref PubMed Scopus (621) Google Scholar, 16Arnold H.K. Sears R.C. Protein phosphatase 2A regulatory subunit B56α associates with c-myc and negatively regulates c-myc accumulation.Mol. Cell. Biol. 2006; 26: 2832-2844Crossref PubMed Scopus (200) Google Scholar). MYC phosphorylated at Thr-58, but not Ser-62, is recognized by the E3 ligase Fbw7, which ubiquitinates MYC at the N terminus and targets it for proteasome-dependent degradation (11Sears R.C. The life cycle of c-Myc: from synthesis to degradation.Cell Cycle. 2004; 3: 1133-1137Crossref PubMed Scopus (283) Google Scholar, 17Welcker M. Orian A. Jin J. Grim J.E. Grim J.A. Harper J.W. Eisenman R.N. Clurman B.E. The Fbw7 tumor suppressor regulates glycogen synthase kinase 3 phosphorylation-dependent c-Myc protein degradation.Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 9085-9090Crossref PubMed Scopus (670) Google Scholar). Recently, another E3 ligase, βtrcp, was shown to interact through a previously unknown phospho-degron and oppose the Fbw7-mediated ubiquitination at the N terminus. PLK1 3The abbreviations used are: PLK1polo-like kinase 1MBP-1MYC promoter-binding protein 1qRTquantitative RTRIKAreverse in-gel kinase assayTBPTATA-binding protein. phosphorylation within the phospho-degron was reported to be critical for βtrcp binding (18Popov N. Schülein C. Jaenicke L.A. Eilers M. Ubiquitylation of the amino terminus of Myc by SCF(β-TrCP) antagonizes SCF(Fbw7)-mediated turnover.Nat. Cell Biol. 2010; 12: 973-981Crossref PubMed Scopus (112) Google Scholar). polo-like kinase 1 MYC promoter-binding protein 1 quantitative RT reverse in-gel kinase assay TATA-binding protein. In this study, we demonstrate the existence of a MYC-PKA-PLK1 signaling loop and show that MYC is regulated both through transcriptional and post-translational mechanisms by PKA, in a PKA catalytic subunit isoform-specific manner. This work also highlights both the promise and potential pitfalls of global kinase inhibition and emphasizes the need to develop next generation therapeutic strategies capable of disrupting specific kinase-substrate interactions. Antibodies were as follows: c-MYC (N-term) (Epitomics, 1472-1); pan-PKA antibody (BD Biosciences, 610980); Thr-197 phospho-PKA (Cell Signaling, 4781S); HA antibody (Roche Applied Science, 11867423001); donkey anti-rabbit, donkey anti-mouse, and goat anti-rat HRP-linked (Jackson Laboratories). Reagents were as follows: H89 Insolution (Calbiochem), BI2536 (Selleck Chemicals, S1109), myristoylated PKI (14Malempati S. Tibbitts D. Cunningham M. Akkari Y. Olson S. Fan G. Sears R.C. Aberrant stabilization of c-Myc protein in some lymphoblastic leukemias.Leukemia. 2006; 20: 1572-1581Crossref PubMed Scopus (73) Google Scholar, 15Yeh E. Cunningham M. Arnold H. Chasse D. Monteith T. Ivaldi G. Hahn W.C. Stukenberg P.T. Shenolikar S. Uchida T. Counter C.M. Nevins J.R. Means A.R. Sears R. A signalling pathway controlling c-Myc degradation that impacts oncogenic transformation of human cells.Nat. Cell Biol. 2004; 6: 308-318Crossref PubMed Scopus (621) Google Scholar, 16Arnold H.K. Sears R.C. Protein phosphatase 2A regulatory subunit B56α associates with c-myc and negatively regulates c-myc accumulation.Mol. Cell. Biol. 2006; 26: 2832-2844Crossref PubMed Scopus (200) Google Scholar, 17Welcker M. Orian A. Jin J. Grim J.E. Grim J.A. Harper J.W. Eisenman R.N. Clurman B.E. The Fbw7 tumor suppressor regulates glycogen synthase kinase 3 phosphorylation-dependent c-Myc protein degradation.Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 9085-9090Crossref PubMed Scopus (670) Google Scholar, 18Popov N. Schülein C. Jaenicke L.A. Eilers M. Ubiquitylation of the amino terminus of Myc by SCF(β-TrCP) antagonizes SCF(Fbw7)-mediated turnover.Nat. Cell Biol. 2010; 12: 973-981Crossref PubMed Scopus (112) Google Scholar, 19Liu J. Schuff-Werner P. Steiner M. Double transfection improves small interfering RNA-induced thrombin receptor (PAR-1) gene silencing in DU 145 prostate cancer cells.FEBS Lett. 2004; 577: 175-180Crossref PubMed Scopus (25) Google Scholar, 20Amarzguioui M. Improved siRNA-mediated silencing in refractory adherent cell lines by detachment and transfection in suspension.BioTechniques. 2004; 36 (770): 766-768Crossref PubMed Google Scholar, 21Padmanabhan A. Gosc E.B. Bieberich C.J. Stabilization of the prostate-specific tumor suppressor NKX3.1 by the oncogenic protein kinase Pim-1 in prostate cancer cells.J. Cell. Biochem. 2013; 114: 1050-1057Crossref PubMed Scopus (20) Google Scholar, 22Guan B. Pungaliya P. Li X. Uquillas C. Mutton L.N. Rubin E.H. Bieberich C.J. Ubiquitination by TOPORS regulates the prostate tumor suppressor NKX3.1.J. Biol. Chem. 2008; 283: 4834-4840Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar) amide (Biomol, P-210), and MG132 (Sigma). For vector constructs, all proteins purified from Escherichia coli were expressed using pQE80 vectors (Qiagen). The pCDNA3.1 vector (Invitrogen) was used for mammalian cell transfections. All proteins were cloned with an N-terminal His6 tag and expressed in either E. coli or mammalian cells (COS cells or PC3 cells). Recombinant proteins were purified under native conditions using Ni-affinity chromatography. The purified proteins were further enriched by ion-exchange chromatography whenever necessary. The final buffer condition for all proteins used in in vitro kinase assay is 50 mm NaH2PO4, 300 mm NaCl, 250 mm imidazole, and 0.1% Tween 20. PC3, LNCaP, and 22RV1 cells (Invitrogen) were cultured in RPMI 1640 medium containing l-glutamine and supplemented with 10% heat-inactivated fetal bovine serum in 5% CO2 at 37 °C. Cells were transfected using LipoD293 (SignaGen) according to the manufacturer's instructions. Lipofectamine 2000 (Invitrogen) was used to transfect siRNA as described previously (19Liu J. Schuff-Werner P. Steiner M. Double transfection improves small interfering RNA-induced thrombin receptor (PAR-1) gene silencing in DU 145 prostate cancer cells.FEBS Lett. 2004; 577: 175-180Crossref PubMed Scopus (25) Google Scholar, 20Amarzguioui M. Improved siRNA-mediated silencing in refractory adherent cell lines by detachment and transfection in suspension.BioTechniques. 2004; 36 (770): 766-768Crossref PubMed Google Scholar). The siRNA sequences used for PKACα knockdown are 5′-AAGCUCCCUUCAUACCAAAGU-3′ and 5′-ACUUUGGUAUGAAGGGAGCUU-3′. The siRNA sequences used for PKACβ knockdown are 5′-AAGGUCCGAUUCCCAUCCCAC-3′ and 5′-GUGGGAUGGGAAUCGGACCUU-3′. For Western blot analysis, mammalian cells were harvested 24–48 h post-transfection and lysed using 150 mm NaCl, 50 mm Tris (pH 7.5), 0.5% Nonidet P-40, and Complete protease inhibitor (Roche Applied Science). Western blotting was performed as described previously (21Padmanabhan A. Gosc E.B. Bieberich C.J. Stabilization of the prostate-specific tumor suppressor NKX3.1 by the oncogenic protein kinase Pim-1 in prostate cancer cells.J. Cell. Biochem. 2013; 114: 1050-1057Crossref PubMed Scopus (20) Google Scholar). Total protein from mouse prostate was extracted during RNA extraction using the protocol described in the RNeasy Kit (Qiagen). Western blotting was performed as described (22Guan B. Pungaliya P. Li X. Uquillas C. Mutton L.N. Rubin E.H. Bieberich C.J. Ubiquitination by TOPORS regulates the prostate tumor suppressor NKX3.1.J. Biol. Chem. 2008; 283: 4834-4840Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). PKACβ2 protein containing the N-terminal HA and His6 tag was purified from transiently transfected COS and PC3 cells either by immunoprecipitation using an antibody against the HA tag or by Ni-affinity chromatography (further enriched by anion-exchange chromatography). Total RNA from mammalian cells was extracted using the RNeasy Kit (Qiagen) and reverse-transcribed using the Superscript cDNA synthesis kit (Bio-Rad). qRT-PCR was performed as per the instruction in the iScript qRT-PCR kit (Bio-Rad). -Fold difference in gene expression was determined after normalizing against GAPDH or TATA-binding protein (TBP) (as mentioned in the legends). Primers used for qRT-PCR are: MYC (134 bp), 5′-GCTCTCCTCGACGGAGTCC-3′ and 5′-CCACAGAAACAACATCGATTTCTT-3′; PKACβ(240 bp), 5′-TGGTGGGCATTAGGAG-3′ and 5′-CTTGTGAGTTTTTATATCACTGAC-3′; PKACβ2 (191 bp), 5′-AACCACCTTGTAACCAGTAT-3′ and 5′-TTTGGCTAGAAACTCTTTCA-3′; TBP (100 bp), 5′-GCCAGCTTCGGAGAGTTCTG-3′ and 5′-GCACGAAGTGCAATGGTCTTT-3′; GAPDH (226 bp), 5′-GAAGGTGAAGGTCGGAGT-3′ and 5′-GAAGATGGTGATGGGATTTC-3′. In vitro kinase assay was performed using purified recombinant proteins as described previously (21Padmanabhan A. Gosc E.B. Bieberich C.J. Stabilization of the prostate-specific tumor suppressor NKX3.1 by the oncogenic protein kinase Pim-1 in prostate cancer cells.J. Cell. Biochem. 2013; 114: 1050-1057Crossref PubMed Scopus (20) Google Scholar, 23Li X. Guan B. Srivastava M.K. Padmanabhan A. Hampton B.S. Bieberich C.J. The reverse in-gel kinase assay to profile physiological kinase substrates.Nat. Methods. 2007; 4: 957-962Crossref PubMed Scopus (14) Google Scholar). Kinase assay was carried out for 2 h at room temperature and stopped using 2× Laemmli buffer. After in vitro kinase assay, the proteins were analyzed by SDS-PAGE, transferred to a PVDF membrane, and analyzed by autoradiography. The PVDF membrane was also stained with Coomassie Brilliant Blue to reveal protein loading. 50 mg/ml PKACα in 8 m urea, 50 mm NaH2PO4, 300 mm NaCl, 250 mm imidazole, and 0.1% Tween 20 was co-polymerized in the gel, and RIKA was performed as described previously (23Li X. Guan B. Srivastava M.K. Padmanabhan A. Hampton B.S. Bieberich C.J. The reverse in-gel kinase assay to profile physiological kinase substrates.Nat. Methods. 2007; 4: 957-962Crossref PubMed Scopus (14) Google Scholar). RIKA was performed on a PVDF membrane for PLK1-priming experiments (on-membrane kinase assay). Purified recombinant MYC and MYC mutants were resolved by SDS-PAGE and transferred onto a PVDF membrane. The proteins on the membrane were refolded using the buffer conditions used in RIKA. The membrane was incubated with purified active recombinant PLK1 (PLK1T210D) for 2 h in a buffer containing 20 mm Tris (pH 7.6), 20 mm MgCl2, 50 mm DTT) and [γ-32P]ATP. The membrane was washed with water overnight to remove nonspecifically bound ATP. Phosphorylation of MYC by PLK1 was detected by autoradiography. In-gel tryptic digestion was performed as described previously (23Li X. Guan B. Srivastava M.K. Padmanabhan A. Hampton B.S. Bieberich C.J. The reverse in-gel kinase assay to profile physiological kinase substrates.Nat. Methods. 2007; 4: 957-962Crossref PubMed Scopus (14) Google Scholar). Desalted tryptic peptides were analyzed by nano-LC-tandem MS on a linear ion-trap mass spectrometer (LTQ; Thermo Fisher). Acquired data were searched against a Homo sapiens protein database or phosphoproteome database using the TurboSEQUEST algorithm (Thermo Fisher). Cells were plated on poly-l-lysine-treated glass coverslips in 6-well plates. PC3 cells were grown on coverslips and transfected using lipoD293T (Signagen) according to the manufacturer's instructions. Transfected cells were fixed using 2% paraformaldehyde in PBS. Immunofluorescence analysis was performed as described previously (22Guan B. Pungaliya P. Li X. Uquillas C. Mutton L.N. Rubin E.H. Bieberich C.J. Ubiquitination by TOPORS regulates the prostate tumor suppressor NKX3.1.J. Biol. Chem. 2008; 283: 4834-4840Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). During our efforts to identify PKA substrates in prostate cancer cells, we identified α-enolase as a PKACα substrate (Table 1). An alternative in-frame internal translation initiating at +291 of the α-enolase mRNA yields an alternate product termed MYC promoter-binding protein 1 (MBP1) (24Feo S. Arcuri D. Piddini E. Passantino R. Giallongo A. ENO1 gene product binds to the c-myc promoter and acts as a transcriptional repressor: relationship with Myc promoter-binding protein 1 (MBP-1).FEBS Lett. 2000; 473: 47-52Crossref PubMed Scopus (227) Google Scholar). We confirmed both ENO1 and MBP1 to be substrates of PKACα by in vitro kinase assay (Fig. 4E). Because MBP1 is a known transcriptional repressor of MYC, we sought to determine whether phosphorylation of MBP1 by PKA has an effect on MBP1 function and steady-state MYC level (25Chaudhary D. Miller D.M. The c-myc promoter-binding protein (MBP-1) and TBP bind simultaneously in the minor groove of the c-myc P2 promoter.Biochemistry. 1995; 34: 3438-3445Crossref PubMed Scopus (46) Google Scholar). We monitored changes in endogenous MYC level in prostate cancer cells treated with the PKA-selective small molecule inhibitor H89 by Western blotting. Pan-PKA inhibition using H89 resulted in decreased MYC accumulation in prostate cancer cells (Fig. 1A). This was found to be consistent in HeLa and K562 cells as well (Fig. 1B). Inhibition of PKA using the PKA-specific peptide inhibitor PKI also destabilized MYC (Fig. 1C). Conversely, activation of PKA using forskolin increased MYC levels (Fig. 1D). Although these results were consistent with our initial hypothesis, the rapid kinetics suggested that the effect could be the result of a direct interaction between PKA and MYC. To determine whether the effect of PKA on MYC is transcriptional, we evaluated changes in MYC mRNA levels upon PKA inhibition by qRT-PCR. The MYC mRNA level did not change upon H89 treatment, suggesting the effect of PKA inhibition on MYC to be post-transcriptional (Fig. 1E). Because MYC is degraded in cells via the 26S proteasome, we determined whether inhibition of proteasome activity rescues the effect of PKA inhibition on MYC accumulation. MG132 rescued the effect of PKA inhibition, thereby demonstrating that PKA protects MYC from 26S proteasome-mediated degradation (Fig. 1F).TABLE 1PKACα substrates identified by mass spectrometryProtein nameAccession numberRankMascot scorePeptides matchedSequence coveragePredicted mass kDa/pIKnown PKA substrate?%ATP synthase subunit β (ATPB)P065761124143856.5/5.2NoActin, cytoplasmic 1 (β-actin)P6070915872441.7/5.3YesTriose phosphate isomerase I (TPI I)P601741153126326.6/6.4NoElongation factor 1δP296921131103831.1/4.9NoHetrogeneous nuclear ribonucleoprotein H (hnRNP H)P3194329092149.1/5.8No40S ribosomal protein SA (p40)P0886517093832.8/4.7NoThyroid receptor-interacting protein 13 (TRIP-13)Q15645151123148.5/5.7NoUbiquitin carboxyl-terminal hydrolase isozyme L5 (UCHL5)Q9Y5K5159195237.5/5.2NoComplement component 1Q subcomponent-binding proteinQ0702116563831.3/4.7NoUbiquitin carboxyl-terminal Hydrolase isozyme L3 (UCHL3)P15374112595126.1/4.8No78-kDa glucose-regulated protein precursor (GRP 78)P110211155152672.2/5.0NoT-complex protein 1 subunit θP509901144253459.5/5.4Noα-Enolase (ENO1)P06733181245447.1/7.0NoHistone-binding protein (RBBP4)Q0902817471947.6/4.7NoPeptidyl-prolyl cis-trans isomerase B precursorP232841148125222.7/9.3NoGlucosidase 2 subunit β PrecursorP14314172102159.3/4.3NoMethyl-CpG-binding domain protein 4Q95243152202766/9.1NoHistone H2BCAB025451NAaNA, not applicable.15NAaNA, not applicable.13.9/10.3YesFamily with sequence similarity 82EAW91638185103531/6.21Noa NA, not applicable. Open table in a new tab FIGURE 1PKA stabilizes MYC in cells. A and B, PKA inhibition using H89 (10 mm, 1 h) decreased the MYC steady-state level in cells. C, PKA-specific peptide inhibitor PKI (20 mm, 1 h) destabilized MYC. D, PKA activation by forskolin (1 h) results in MYC accumulation. E, MYC mRNA level does not change upon H89 treatment (n = 3). F, PKA protects MYC from proteasome-mediated degradation. MG132 rescued the effect of PKA inhibition on MYC stability. DMSO, dimethyl sulfoxide.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To determine whether PKA can phosphorylate MYC, we performed in vitro kinase reactions using recombinant PKACα and MYC in the presence of [γ-32P]ATP. PKA efficiently phosphorylated MYC, and the phosphorylation was inhibited by H89 (Fig. 2, A and C). LC-MS2 analysis identified Ser-279 as a PKA phospho-acceptor site on MYC (Fig. 2B and supplemental Tables S1 and S2). The ability of PKA to phosphorylate a MYCS279A mutant in vitro was nearly extinguished compared with WT-MYC (MYCWT), demonstrating Ser-279 as the major PKA phospho-acceptor site on MYC (Fig. 2C). Ser-279 is a predicted PKA phosphorylation site and is situated within an evolutionarily conserved region between the N-terminal transactivation and C-terminal DNA binding domains in MYC (Fig. 2, D and E). Furthermore, the region spanning Ser-279 is predicted to be solvent-exposed and intrinsically disordered (data not shown). Such intrinsically disordered domains have been implicated in the regulation of protein stability (26Dyson H.J. Wright P.E. Intrinsically unstructured proteins and their functions.Nat. Rev. Mol. Cell Biol. 2005; 6: 197-208Crossref PubMed Scopus (3035) Google Scholar). To determine whether the site phosphorylated by PKA on MYC is accessible and able to be phosphorylated in vivo, we transfected His6-3×HA-tagged MYC in PC3 cells. The cells were harvested 36 h post-transfection, lysed (using 150 mm NaCl, 50 mm Tris (pH 7.5), 0.5% Nonidet P-40 and Complete protease inhibitor), and MYC was purified using Ni-affinity chromatography. The purified MYC was divided in half. One half was completely dephosphorylated by hydrogen fluoride (HF) treatment as described (27Li X. Rao V. Jin J. Guan B. Anderes K.L. Bieberich C.J. Identification and validation of inhibitor-responsive kinase substrates using a new paradigm to measure kinase-specific protein phosphorylation index.J. Proteome Res. 2012; 11: 3637-3649Crossref PubMed Scopus (5) Google Scholar). The remaining half was left untreated and contained the pool of MYC molecules that retained their in vivo phosphorylation status. We performed a RIKA with PKA in the gel on the HF-treated and untreated purified MYC samples. If MYC is phosphorylated in vivo at the site phosphorylated by PKA in vitro (in a RIKA), then we would expect an increase in phosphorylation signal in the HF-treated samples (because these sites would be dephosphorylated upon HF treatment and become available to be phosphorylated during the RIKA). Total levels of MYC loaded in these samples were quantified by anti-HA Western blotting, and the change in phosphorylation signal was calculated after normalization as described previously (27Li X. Rao V. Jin J. Guan B. Anderes K.L. Bieberich C.J. Identification and validation of inhibitor-responsive kinase substrates using a new paradigm to measure kinase-specific protein phosphorylation index.J. Proteome Res. 2012; 11: 3637-3649Crossref PubMed Scopus (5) Google Scholar). This process is illustrated in Fig. 3A. We observed ∼50% increase in MYC phosphorylation upon HF treatment, suggesting that 50% of the in vivo MYC population in the cells was phosphorylated at the PKA site under the given culture conditions (Fig. 3B). Importantly, endogenous PKA and MYC co-localized to the nucleus (Fig. 3C). To ascertain whether MYC phosphorylation at Ser-279 by PKA affected the MYC steady-state level, HA-tagged MYCWT and MYCS279A were transfected into PC3 cells, and the effect of PKA inhibition on their accumulation was analyzed. Whereas MYCWT was destabilized upon H89 treatment, the steady-state level of MYCS279A was unresponsive to PKA inhibition (Fig. 3D). Further, the MYCS279A steady-state level was considerably lower compared with MYCWT (Fig. 3D). These data demonstrate that phosphorylation at Ser-279 by PKA plays a role in stabilizing MYC in cells. No difference in subcellular localization between the endogenous MYC, HA-tagged MYCWT and HA-tagged MYCS279A mutant was observed (data not shown). A recent report showed the region spanning Ser-279 to constitute a phospho-degron (Fig. 4A). PLK1-mediated βtrcp binding within this region was shown to stabilize MYC (18Popov N. Schülein C. Jaenicke L.A. Eilers M. Ubiquitylation of the amino terminus of Myc by SCF(β-TrCP) antagonizes SCF(Fbw7)-mediated turnover.Nat. Cell Biol. 2010; 12: 973-981Crossref PubMed Scopus (112) Google Scholar). However, neither the events preceding PLK1 phosphorylation nor the PLK1 phospho-acceptor site(s) on MYC is known. Because both PLK1 and PKA phosphorylate residues within this phospho-degron, we sought to clarify their roles. We found that the effect of combined inhibition of PKA and PLK1 on MYC stability is more profound compared with inhibiting either kinase alone (Fig. 4B). We hypothesized three possible scenarios: (i) PKA and PLK1 phosphorylate the same residue on MYC (i.e. Ser-279) and hence elicit a similar response; (ii) PKA and PLK1 phosphorylate mutually independent residues on MYC within the phospho-degron; and (iii) PKA phosphorylates MYC at Ser-279, which primes PLK1 to phosphorylate an adjacent residue within the phospho-degron by relieving the inhibitory effect of the polo-box domain or by making an adjacent site a more favorable PLK1 phospho-acceptor (Fig. 4C). To distinguish among these models, we performed an in vitro kinase reaction using recombinant PLK1T210D and recombinant MYC, which was either prephosphorylated by PKA in vitro using nonradiolabeled ATP (primed) or not prephosphorylated by PKA (unprimed). His6-tagged MYC purified from E. coli was prephosphorylated by in vitro kinase assay using recombinant PKACα and nonradiolabeled ATP. Nonphosphorylated MYC served as the unprimed control. Subsequently, H89 (30 μm), was added to all reactions to inhibit PKA activity. The absence of radiolabeling upon further incubation (1 h at room temperature) of the reactions containing MYC, PKA and H89 with [γ-32P]ATP confirmed complete inactivation of PKA by H89. Recombinant PLK1T210D and [γ-32P]ATP was then added to these tubes containing either primed or unprimed MYC. The in vitro kinase reaction was allowed to proceed for 1 h at room temperature. Radioactive labeling of MYC by PLK1T210D was assessed by running the reaction on a SDS-polyacrylamide gel, transferring to a PVDF membrane followed by autoradiography. We used the PLK1T210D mutant version of PLK1 because it was previously shown to be constitutively active. The mutation at Thr-210 mimics the phosphorylation of PLK1 at this site within the activation loop, and phosphorylation at Thr-210 is necessary for PLK1 activation (28Smits V.A. Klompmaker R. Arnaud L. Rijksen G. Nigg E.A. Medema R.H. Polo-like kinase-1 is a target of the DNA damage checkpoint.Nat. Cell Biol. 2000; 2: 672-676Crossref PubMed Scopus (396) Google Scholar). We observed that the phosphorylation signal of MYC by PLK1T210D (using [γ-32P]ATP) was higher when MYC was primed by PKA (using nonradiolabeled ATP) compared with unprimed MYC (Fig. 4D). This result suggested that PKA phosphorylation at Ser-279 on MYC primes subseq" @default.
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• W2076359094 date "2013-05-01" @default.
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• W2076359094 title "Protein Kinase A Regulates MYC Protein through Transcriptional and Post-translational Mechanisms in a Catalytic Subunit Isoform-specific Manner" @default.
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• W2076359094 doi "https://doi.org/10.1074/jbc.m112.432377" @default.
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