AKT1, LKB1, and YAP1 Revealed as MYC Interactors with NanoLuc-Based Protein-Fragment Complementation Assay

The c-Myc (MYC) transcription factor is a major cancer driver and a well-validated therapeutic target. However, directly targeting MYC has been challenging. Thus, identifying proteins that interact with and regulate MYC may provide alternative strategies to inhibit its oncogenic activity. In this study, we report the development of a NanoLuc-based protein-fragment complementation assay (NanoPCA) and mapping of the MYC protein interaction hub in live mammalian cells. The NanoPCA system was configured to enable detection of protein–protein interactions (PPI) at the endogenous level, as shown with PRAS40 dimerization, and detection of weak interactions, such as PINCH1-NCK2. Importantly, NanoPCA allows the study of PPI dynamics with reversible interactions. To demonstrate its utility for large-scale PPI detection in mammalian intracellular environment, we have used NanoPCA to examine MYC interaction with 83 cancer-associated proteins in live cancer cell lines. Our new MYC PPI data confirmed known MYC-interacting proteins, such as MAX, GSK3A, and SMARCA4, and revealed a panel of novel MYC interaction partners, such as RAC-α serine/threonine-protein kinase (AKT)1, liver kinase B (LKB)1, and Yes-associated protein (YAP)1. The MYC interactions with AKT1, LKB1, and YAP1 were confirmed by coimmunoprecipitation of endogenous proteins. Importantly, AKT1, LKB1, and YAP1 were able to activate MYC in a transcriptional reporter assay. Thus, these vital growth control proteins may represent promising MYC regulators, suggesting new mechanisms that couple energetic and metabolic pathways and developmental signaling to MYC-regulated cellular programs.

[1]  Brock F. Binkowski,et al.  NanoLuc Complementation Reporter Optimized for Accurate Measurement of Protein Interactions in Cells. , 2016, ACS chemical biology.

[2]  G. Hannon,et al.  p15INK4B is a potential effector of TGF-beta-induced cell cycle arrest. , 1994, Nature.

[3]  Bridget E. Begg,et al.  A Proteome-Scale Map of the Human Interactome Network , 2014, Cell.

[4]  Cliff I. Stains,et al.  Self-Assembling NanoLuc Luciferase Fragments as Probes for Protein Aggregation in Living Cells. , 2016, ACS chemical biology.

[5]  S S Gambhir,et al.  Noninvasive imaging of protein–protein interactions in living subjects by using reporter protein complementation and reconstitution strategies , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[6]  M. Moran,et al.  Large-scale mapping of human protein–protein interactions by mass spectrometry , 2007, Molecular systems biology.

[7]  Brock F. Binkowski,et al.  Engineered Luciferase Reporter from a Deep Sea Shrimp Utilizing a Novel Imidazopyrazinone Substrate , 2012, ACS chemical biology.

[8]  R. Weissleder,et al.  Codon-optimized Gaussia luciferase cDNA for mammalian gene expression in culture and in vivo. , 2005, Molecular therapy : the journal of the American Society of Gene Therapy.

[9]  Yuhong Du,et al.  A time-resolved fluorescence resonance energy transfer assay for high-throughput screening of 14-3-3 protein-protein interaction inhibitors. , 2013, Assay and drug development technologies.

[10]  R. Medema,et al.  Myc‐induced proliferation and transformation require Akt‐mediated phosphorylation of FoxO proteins , 2004, The EMBO journal.

[11]  A. Kimchi,et al.  Discovering protein-protein interactions within the programmed cell death network using a protein-fragment complementation screen. , 2014, Cell reports.

[12]  A. Vallée-Bélisle,et al.  Detection of protein-protein interactions by protein fragment complementation strategies. , 2000, Methods in enzymology.

[13]  S. Masters,et al.  14-3-3 Proteins Mediate an Essential Anti-apoptotic Signal* , 2001, The Journal of Biological Chemistry.

[14]  I. Jurisica,et al.  Myc and its interactors take shape. , 2015, Biochimica et biophysica acta.

[15]  Jiandie D. Lin,et al.  TEAD mediates YAP-dependent gene induction and growth control. , 2008, Genes & development.

[16]  J. Qin,et al.  Structure of an ultraweak protein-protein complex and its crucial role in regulation of cell morphology and motility. , 2005, Molecular cell.

[17]  P. L. DaviesFebruary.  THE ONE-WAY ANALYSIS OF VARIANCE , 1997 .

[18]  Yang Zhang,et al.  The I-TASSER Suite: protein structure and function prediction , 2014, Nature Methods.

[19]  Yukiko Matsuoka,et al.  Adding Protein Context to the Human Protein-Protein Interaction Network to Reveal Meaningful Interactions , 2013, PLoS Comput. Biol..

[20]  Gregory J. Hannon,et al.  pl5INK4B is a potentia| effector of TGF-β-induced cell cycle arrest , 1994, Nature.

[21]  Y Taya,et al.  Multiple Ras-dependent phosphorylation pathways regulate Myc protein stability. , 2000, Genes & development.

[22]  J. Havel,et al.  Nuclear PRAS40 couples the Akt/mTORC1 signaling axis to the RPL11-HDM2-p53 nucleolar stress response pathway , 2014, Oncogene.

[23]  H. Blau,et al.  Monitoring protein-protein interactions in intact eukaryotic cells by beta-galactosidase complementation. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[24]  S. Michnick,et al.  Exploring protein interactions by interaction-induced folding of proteins from complementary peptide fragments. , 2001, Current opinion in structural biology.

[25]  S. Michnick,et al.  An infrared reporter to detect spatiotemporal dynamics of protein-protein interactions , 2014, Nature Methods.

[26]  Roger Y. Tsien,et al.  Creating new fluorescent probes for cell biology , 2003, Nature Reviews Molecular Cell Biology.

[27]  Lee A. D. Cooper,et al.  The OncoPPi network of cancer-focused protein–protein interactions to inform biological insights and therapeutic strategies , 2017, Nature Communications.

[28]  Frederick J. Gravetter,et al.  Statistics for the Behavioral Sciences [6th ed.] , 2004 .

[29]  S. Michnick,et al.  A highly sensitive protein-protein interaction assay based on Gaussia luciferase , 2006, Nature Methods.

[30]  B. Tannous,et al.  Gaussia luciferase reporter assay for monitoring biological processes in culture and in vivo , 2009, Nature Protocols.

[31]  J. Blenis,et al.  Activation of PI3K/Akt and MAPK pathways regulates Myc-mediated transcription by phosphorylating and promoting the degradation of Mad1 , 2008, Proceedings of the National Academy of Sciences.

[32]  S S Gambhir,et al.  Monitoring protein-protein interactions using split synthetic renilla luciferase protein-fragment-assisted complementation. , 2003, Analytical chemistry.

[33]  Rina Su,et al.  Enabling systematic interrogation of protein-protein interactions in live cells with a versatile ultra-high-throughput biosensor platform. , 2016, Journal of molecular cell biology.

[34]  Amber L. Couzens,et al.  The MYC-Associated Protein CDCA7 Is Phosphorylated by AKT To Regulate MYC-Dependent Apoptosis and Transformation , 2012, Molecular and Cellular Biology.

[35]  Chang‐Deng Hu,et al.  Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation. , 2002, Molecular cell.

[36]  A. Barabasi,et al.  Network medicine : a network-based approach to human disease , 2010 .

[37]  Chris Sander,et al.  MYC Cooperates with AKT in Prostate Tumorigenesis and Alters Sensitivity to mTOR Inhibitors , 2011, PloS one.

[38]  M. DePamphilis,et al.  TEAD/TEF transcription factors utilize the activation domain of YAP65, a Src/Yes-associated protein localized in the cytoplasm. , 2001, Genes & development.

[39]  J. Sedivy,et al.  Proteomic profiling of Myc-associated proteins , 2010, Cell cycle.

[40]  B. Vennstrom,et al.  Isolation and characterization of c-myc, a cellular homolog of the oncogene (v-myc) of avian myelocytomatosis virus strain 29 , 1982, Journal of virology.

[41]  Swneke D. Bailey,et al.  BioID identifies novel c-MYC interacting partners in cultured cells and xenograft tumors. , 2015, Journal of proteomics.

[42]  L. Tsai,et al.  The MZF1/c-MYC axis mediates lung adenocarcinoma progression caused by wild-type lkb1 loss , 2014, Oncogene.

[43]  Qing Xu,et al.  Overexpression of the LKB1 gene inhibits lung carcinoma cell proliferation partly through degradation of c-myc protein. , 2009, Oncology reports.

[44]  Q. Pan,et al.  Mutual interaction between YAP and c-Myc is critical for carcinogenesis in liver cancer. , 2013, Biochemical and biophysical research communications.

[45]  Thomas J Magliery,et al.  Detecting protein-protein interactions with a green fluorescent protein fragment reassembly trap: scope and mechanism. , 2005, Journal of the American Chemical Society.

[46]  L. Penn,et al.  Reflecting on 25 years with MYC , 2008, Nature Reviews Cancer.

[47]  S. Masters,et al.  14-3-3 proteins: structure, function, and regulation. , 2000, Annual review of pharmacology and toxicology.

[48]  Stephen W. Michnick,et al.  Universal strategies in research and drug discovery based on protein-fragment complementation assays , 2007, Nature Reviews Drug Discovery.

[49]  T. Mäkelä,et al.  Suppression of oncogenic properties of c-Myc by LKB1-controlled epithelial organization , 2007, Proceedings of the National Academy of Sciences.

[50]  Yutaka Kodama,et al.  Bimolecular fluorescence complementation (BiFC): a 5-year update and future perspectives. , 2012, BioTechniques.

[51]  P. Vogt,et al.  Inhibitor of MYC identified in a Kröhnke pyridine library , 2014, Proceedings of the National Academy of Sciences.

[52]  H. Fu,et al.  BRET: NanoLuc-Based Bioluminescence Resonance Energy Transfer Platform to Monitor Protein-Protein Interactions in Live Cells. , 2016, Methods in molecular biology.