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Activation of the transcription of Gal4-regulated genes by Physarum 14-3-3 in yeast is related to dimer-binding motif-2 and three phosphorylation sites

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Abstract

The roles of 14-3-3 proteins in the lower eukaryotes are still elusive. We isolated a cDNA encoding the 14-3-3 protein (P14-3-3) from the lower eukaryote Physarum polycephalum. This P14-3-3 gene was then inserted downstream of the Gal4 DNA-binding domain in the yeast expression vector pGBKT7. The recombinant vector was transformed into auxotrophic AH109 and Y187 yeast cells to detect the activation of Gal4-regulated gene expression mediated by P14-3-3. The results showed that three reporter genes (ADE2, HIS3, and lacZ) could be normally expressed, indicating that the transcriptional activation function of P14-3-3 was retained. We subsequently used a truncated P14-3-3 peptides and mutant peptides to study the activation of the Gal4-regulated genes ADE2, HIS3, and lacZ. We found that deletion of the N-terminal second dimer-binding motif (DBM2) sequence or the C-terminal coil sequence led to the loss of P14-3-3’s transcriptional activation function. Specifically, any mutation at the potential phosphorylation sites (Ser62 and Ser67) on DBM2 or at the C-terminal potential phosphorylation site (235ThrSer236) led to the loss of the transcriptional activation function of P14-3-3. Taken together, these observations suggest that the transcriptional activation function of P14-3-3 in lower eukaryotes is related to DBM2 and the C-terminal coil structures.

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References

  • Aitken A (2006) 14-3-3 proteins: a historic overview. Semin Cancer Biol 16:162–172

    Article  CAS  PubMed  Google Scholar 

  • Aitken A, Howell S, Jones D, Madrazo J, Patel Y (1995) 14-3-3 alpha and delta are the phosphorylated forms of raf-activating 14-3-3 beta and zeta. In vivo stoichiometric phosphorylation in brain at a Ser-Pro-Glu-Lys MOTIF. J Biol Chem 270:5706–5709

    Article  CAS  PubMed  Google Scholar 

  • Aitken A, Baxter H, Dubois T, Clokie S, Mackie S, Mitchell K, Peden A, Zemlickova E (2002) Specificity of 14-3-3 isoform dimer interactions and phosphorylation. Biochem Soc Trans 30:351–360

    Article  CAS  PubMed  Google Scholar 

  • Arnold K, Bordoli L, Kopp J, Schwede T (2006) The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics 22:195–201

    Article  CAS  PubMed  Google Scholar 

  • Benzinger A, Popowicz GM, Joy JK, Majumdar S, Holak TA, Hermeking H (2005) The crystal structure of the non-liganded 14-3-3 sigma protein: insights into determinants of isoform specific ligand binding and dimerization. Cell Res 15:219–227

    Article  CAS  PubMed  Google Scholar 

  • Chaudhri M, Scarabel M, Aitken A (2003) Mammalian and yeast 14-3-3 isoforms form distinct patterns of dimers in vivo. Biochem Biophys Res Commun 300:679–685

    Article  CAS  PubMed  Google Scholar 

  • Clokie SJ, Cheung KY, Mackie S, Marquez R, Peden AH, Aitken A (2005) BCR kinase phosphorylates 14-3-3 Tau on residue 233. FEBS J 272:3767–3776

    Article  CAS  PubMed  Google Scholar 

  • Coblitz B, Wu M, Shikano S, Li M (2006) C-terminal binding: an expanded repertoire and function of 14-3-3 proteins. FEBS Lett 580:1531–1535

    Article  CAS  PubMed  Google Scholar 

  • Daniel JW, Baldwin HH (1964) Methods of culture of plasmodial myxomycetes. In: Prescott DM (ed) Methods in cell physiol, vol. 1, pp. 9–41. Academic Press, New York

    Google Scholar 

  • de Vetten NC, Lu G, Feri RJ (1992) A maize protein associated with the G-box binding complex has homology to brain regulatory proteins. Plant Cell 4:1295–1307

    Article  PubMed  Google Scholar 

  • Dubois T, Rommel C, Howell S, Steinhussen U, Soneji Y, Morrice N, Moelling K, Aitken A (1997) 14-3-3 is phosphorylated by casein kinase I on residue 233. Phosphorylation at this site in vivo regulates Raf/14-3-3 interaction. J Biol Chem 272:28882–28888

    Article  CAS  PubMed  Google Scholar 

  • Fu H, Subramanian RR, Masters SC (2000) 14-3-3 proteins: structure, function, and regulation. Annu Rev Pharmacol Toxicol 40:617–647

    Article  CAS  PubMed  Google Scholar 

  • Gardino AK, Smerdon SJ, Yaffe MB (2006) Structural determinants of 14-3-3 binding specificities and regulation of subcellular localization of 14-3-3-ligand complexes: a comparison of the X-ray crystal structures of all human 14-3-3 isoforms. Semin Cancer Biol 16:173–182

    Article  CAS  PubMed  Google Scholar 

  • Gietz RD, Schiestl RH, Willems AR, Woods RA (1995) Studies on the transformation of intact yeast cells by the LiAc/SS-DNA/PEG procedure. Yeast 11:355–360

    Article  CAS  PubMed  Google Scholar 

  • Gu YM, Jin YH, Choi JK, Baek KH, Yeo CY, Lee KY (2006) Protein kinase A phosphorylates and regulates dimerization of 14-3-3 epsilon. FEBS Lett 580:305–310

    Article  CAS  PubMed  Google Scholar 

  • Guex N, Peitsch MC (1997) SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modelling. Electrophoresis 18:2714–2723

    Article  CAS  PubMed  Google Scholar 

  • Ho SN, Hunt HD, Horton RM, Pullen JK, Pease LR (1989) Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77:51–59

    Article  CAS  PubMed  Google Scholar 

  • Jones DH, Ley S, Aitken A (1995a) Isoforms of 14-3-3 protein can form homo- and heterodimers in vivo and in vitro: implications for function as adapter proteins. FEBS Lett 368:55–58

    Article  CAS  PubMed  Google Scholar 

  • Jones DH, Martin H, Madrazo J, Robinson KA, Nielsen P, Roseboom PH, Patel Y, Howell SA, Aitken A (1995b) Expression and structural analysis of 14-3-3 proteins. J Mol Biol 245:375–384

    Article  CAS  PubMed  Google Scholar 

  • Liu D, Bienkowska J, Petosa C, Collier RJ, Fu H, Liddington R (1995) Crystal structure of the zeta isoform of the 14-3-3 protein. Nature 376:191–194

    Article  CAS  PubMed  Google Scholar 

  • Mhawech P (2005) 14-3-3 proteins-an update. Cell Res 15:228–236

    Article  CAS  PubMed  Google Scholar 

  • Obsil T, Ghirlando R, Klein DC, Ganguly S, Dyda F (2001) Crystal structure of the 14-3-3zeta: serotonin N-acetyltransferase complex, a role for scaffolding in enzyme regulation. Cell 105:257–267

    Article  CAS  PubMed  Google Scholar 

  • Pan S, Sehnke PC, Ferl RJ, Gurley WB (1999) Specific interactions with TBP and TFIIB in vitro suggest that 14-3-3 proteins may participate in the regulation of transcription when part of a DNA binding complex. Plant Cell 11:1591–1602

    Article  CAS  PubMed  Google Scholar 

  • Pozuelo Rubio M, Geraghty KM, Wong BH, Wood NT, Campbell DG, Morrice N, Mackintosh C (2004) 14-3-3-affinity purification of over 200 human phosphoproteins reveals new links to regulation of cellular metabolism, proliferation, and trafficking. Biochem J 379:395–408

    Article  PubMed  Google Scholar 

  • Rusch HP, Sachsenmaier W, Behrens K, Gruter V (1966) Synchronization of mitosis by the fusion of the plasmodia of Physarum polycephalum. J Cell Biol 31:204–209

    Article  CAS  PubMed  Google Scholar 

  • Schultz TF, Medina J, Hill A, Quatrano RS (1998) 14-3-3 proteins are part of an abscisic acid VIVIPAROUS1 (VP1) response complex in the Em promoter and interact with VP1 and EmBP1. Plant Cell 10:837–847

    Article  CAS  PubMed  Google Scholar 

  • Schwede T, Kopp J, Guex N, Peitsch MC (2003) SWISS-MODEL: an automated protein homology- modeling server. Nucleic Acids Res 31:3381–3385

    Article  CAS  PubMed  Google Scholar 

  • Toker A, Sellers LA, Amess B, Patel Y, Harris A, Aitken A (1992) Multiple isoforms of a protein kinase C inhibitor (KCIP-1/14-3-3) from sheep brain. Amino acid sequence of phosphorylated forms. Eur J Biochem 206:453–461

    Article  CAS  PubMed  Google Scholar 

  • Woodcock JM, Murphy J, Stomski FC, Berndt MC, Lopez AF (2003) The dimeric versus monomeric status of 14-3-3zeta is controlled by phosphorylation of Ser58 at the dimer interface. J Biol Chem 278:36323–36327

    Article  CAS  PubMed  Google Scholar 

  • Xiao B, Smerdon SJ, Jones DH, Dodson GG, Soneji Y, Aitken A, Gamblin SJ (1995) Structure of a 14-3-3 protein and implications for coordination of multiple signalling pathways. Nature 376:188–191

    Article  CAS  PubMed  Google Scholar 

  • Yaffe MB, Rittinger K, Volinia S, Caron PR, Aitken A, Leffers H, Gamblin SJ, Smerdon SJ, Cantley LC (1997) The structural basis for 14-3-3: phosphopeptide binding specificity. Cell 9:961–971

    Article  Google Scholar 

  • Yang X, Lee WH, Sobott F, Papagrigoriou E, Robinson CV, Grossmann JG, Sundström M, Doyle DA, Elkins JM (2006) Structural basis for protein-protein interactions in the 14-3-3 protein family. Proc Natl Acad Sci USA 103:17237–17242

    Article  CAS  PubMed  Google Scholar 

  • Zhang JH, Liu SD, Xing M (2005) Construction of a cDNA expression library of Physarum polycephalum at G2-phase. J Shenzhen Univ Sci Eng 22:50–56

    Google Scholar 

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Acknowledgments

This work was supported by grants from the National Natural Science Foundation of China (No. 30470113), the Guangdong Natural Science Foundation (No. 04011314), and the Shenzhen Science & Technology Foundation (No. 200442).

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Authors and Affiliations

  1. Shenzhen Key Laboratory of Microbial and Genetic Engineering, College of Life Science, Shenzhen University, 518060, Shenzhen, China

    Shide Liu, Minghua Li, Jianhua Zhang, Kang Kang, Shengli Tian, Yisi Wang & Miao Xing

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  1. Shide Liu
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  2. Minghua Li
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  3. Jianhua Zhang
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  4. Kang Kang
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  5. Shengli Tian
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  7. Miao Xing
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Correspondence to Miao Xing.

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Communicated by Erko Stackebrandt.

Shide Liu and Minghua Li contributed equally to this work.

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Liu, S., Li, M., Zhang, J. et al. Activation of the transcription of Gal4-regulated genes by Physarum 14-3-3 in yeast is related to dimer-binding motif-2 and three phosphorylation sites. Arch Microbiol 192, 33–40 (2010). https://doi.org/10.1007/s00203-009-0526-3

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  • DOI: https://doi.org/10.1007/s00203-009-0526-3

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