UNIVERSITETET I TROMSØ UIT THE FACULTY OF HEALTH SCIENCES, UNIVERSITY OF TROMSØ , NORWAY . Layout/design: Rod Wolstenholme, Faculty of Health Sciences, University of Tromsø, Norway Ph.D. School in Molecular and Structural Biology Phosphorylation of Transforming growth factor β Inducible Early response Gene 1 by atypical Protein Kinase Cs affects both DNA binding and transactivation Endalkachew A. Alemu, Eva Sjøttem, Kenneth B. Larsen, Heidi Outzen, Turid Holm, Geir Bjørkøy, and Terje Johansen PhD school in Molecular and Structural Biology, Molecular Cancer Research Group, Department of Biochemistry, Institute of Medical Biology, Faculty of Health Sciences, University of Tromsø, N-9037 Tromsø, Norway Transforming growth factor β Inducible Early response Gene 1 (TIEG1) is an early response gene which is expressed in response to TGF-β. TIEG1 belongs to a family of Sp1-like (krüppel-like) transcription factors that are characterized by the pres- ence of three highly conserved C-terminal C 2 H 2 zinc inger mot ifs, which constitute the DNA binding domain (DBD) (Fig.2). he pro-apoptotic role of TIEG1 has been widely reported and more interestingly, TIEG1 has been shown to have stage spe- ci ic express ion in human breast cancer and has also been implicated in cardiac hypertrophy. Introduction he Protein Kinase C (PKC) family of serine/t hreonine kinases consists of ten dif- ferent isoforms that can be grouped into three classes denoted classical-, novel- and atypical PKCs (Fig.1). PKCζ and PKCλ/ι constitute the atypical PKCs (aPKCs), which serve important roles during development and in processes subverted in cancer such as cell-and tissue polarity, cell proliferation, di ferentiation and apoptosis. Our research group studies these kinases and their interaction networks. 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Biochem. 133, 9-16 Tachibana, I., Imoto, M., Adjei, P.N., Gores, G.G.J., Subramaniam, M., Spelsberg, T.C., & Urrutia, R. (1997) J. Clin. Invest. 99, 2365-74. Wang, Z., Peter, B., Klussmann, S., Bender, H., Herb, A., & Krieglstein, K. (2004) Gene 325, 25-34. Aim To identify and characterize novel interaction partners for atypical PKCs. Conclusion • here is a direct interaction between aPKCs and TIEG1 • TIEG1 is an in vitro substrate of aPKCs • Phosphorylated or TIEG1(S384E) loses its DNA binding ability • TIEG1 (T445E) showed enhanced transactivation potential Figure 1. Schematic representation of the diferent PKC subfamilies. C2, C2 domain; PB1, PB1 dimerization domain; ZF, zinc-inger. PB1 ZF Kinase Atypical PKCζ PKCλ /ι Kinase ZF ZF C2 Classical PKCα PKCβ I PKCβ II PKCγ Kinase ZF ZF C2 Novel PKCδ PKCε PKCη PKCθ Results In an efort to identify novel interaction partners for aPKCs, a yeast two-hybrid screening of a HeLa cell cDNA library was carried out using the regulatory domain of PKCλ/ι as bait. his screen yielded t hree independent clones that encode the C-terminal part of TIEG1 as a putative interaction partner for PKCλ/ι (Fig.2). We have conirmed this interaction both in vitro and in vivo (Fig.2). aPKCs phos- phorylate the DNA binding domain (DBD) of TIEG1 on two critical residues (Fig. 3). Besides, aPKCs-mediated phosphorylation of TIEG1 a fects its DNA binding activ- ity (Fig. 4), subnuclear localization and transactivation potential (Fig. 5). Figure 2. TIEG1 interacts with aPKCs. ( A) Schematic representation of the domain structure of TIEG1. The region of TIEG1 isolated in the yeast two-hybrid (aa 349-480) is indicated below the schematic. (B) and (C) , GST pull-down assay showing the interaction between TIEG1 and in vitro translated PKCλ/ι (B) or PKCζ and PKCλ/ι from HeLa cell extracts (C) . (D) Co-immunoprecipitation assay showing the interaction between over-expressed EGFP-TIEG1 and endogenous PKCλ/ι from U2OS cells. (E) Confocal microscopic images of EGFP-TIEG1 and mCherry-PKCλ/ι over-expressed in HeLa cells. 35 S-PKCλ Autoradiograph GST GST-TIEG1(DBD) GST-TIEG1(1-362) 5% of Input 25 48 62 Coomassie B MWM 50 60 80 2% of Input IP PKCλ IP : α GFP WB: α PKCλ + - + - - + - + EGFP EGFP-TIEG1 IgG hc 80 100 60 50 IP + - - + IP : α GFP WB: α PKCλ D 50 60 80 GST GST-TIEG1 Input (1%) PKCζ PKCλ MWM WB: α PKCζ (c20) C mCherry-PKCλ Merged EGFP-TIEG1 HeLa HeLa HeLa E Proline-rich ZF ZF ZF 480 1 TIEG1 ZF ZF ZF 480 349 A Figure 3. TIEG1 is an in vitro substrate of aPKCs. (A) and (B) Autoradiographs after in vitro kinase assay showing phosphorylation of TIEG1 by aPKCs. This aPKCs-mediated phosphorylation of TIEG1 is within the linear range up to 90 minutes of incubation. (C) Amino acid sequence align- ment of the DBDs of some members of the krüppel-like proteins. The three zinc inger (ZF) domains are underlined and marked ZF1, ZF2 and ZF3. The amino acids within the recognition helix that are proposed to contact the DNA bases are indicated by lines and serine 384 and threo- nine 445 in zinc ingers 1 and 3, respectively, are marked in red. (D) Autoradiograph showing lack of phosphorylation of TIEG1 on the irst 362 amino acids. (E) Phosphorylation of the DBD of TIEG1 is almost abolished following mutation of serine 384 and threonine 445 to alanine. MBP-TIEG1 MBP pPKCζ pTIEG1 A MBP-TIEG1 MBP pPKCλ pTIEG1 5 15 30 45 60 90 pTIEG1 pPKCζ Time in minutes B ZF1 ZF2 ZF3 -KSSHLKAHVRTH- -RSDHLTKHARRH- GGG-3' 5'-GGG (-1) (-1) (3) (3) (6) ZF1 Recognition helix ZF3 Recognition helix C GST pPKCζ pTIEG1(DBD) D GST-TI EG1(1-362) GST-TIEG1(DBD) WT GST-TIEG1(DBD) GST S384A T445A pPKCζ pTIEG1(DBD) Coomasie blue E Figure 4. The DNA binding activity of TIEG1 is impaired by PKCζ-mediated phosphorylation of serine 384. (A) Gel mobility shift assay (GMSA) demonstrating impairment of the DNA binding ability of TIEG1 due to substitution of serine 384 by glutamate, or (B) due to PKCζ-mediated phosphorylation of TIEG1 (DBD). WT (wild type); EE (S384E/T445E); AA (S384A/T445A). GST-TIEG1(DBD) GST WT TIEG1-DNA Complex Free Probe - + - + - + - + - + - + - + + -- ++ -- ++ -- ++ ++ - ATP PKCζ EE AA B dH 2 O GST WT S384A T445A AA WT S384E T445E EE WT GST-TIEG1(DBD) TIEG1-DNA Complex Free Probe A Figure 5. The phospho-mimicking mutant of TIEG1 showed diferent subnuclear localization but similar mobility as compared with its wild type counter part. (A) Confocal microscopic images of U2OS and HeLa cells transfected with either EGFP-TIEG1 (WT) or EGFP –TIEG1 (S384E/ T445E). (B) Fluorescence Recovery After Photobleaching (FRAP) curve showing the mobility of the wild type and the phospho-mimicking mutant of EGFP-tagged TIEG1 transfected into HeLa cells. (C) Reporter gene assay showing enhanced transactivation potential of the TIEG1 (T445E) mutant transfected into U2OS cells in the presence of the p21-luciferase reporter. HeLa WT S384E/T445E WT EGFP-TIEG1 A U2OS HeLa B 0 0.2 0.4 0.6 0.8 1.0 1.2 0 20 40 60 80 Time (s) Relative Fluorescence (S384E/T445E WT 5 10 15 20 25 30 100ng 250ng 0 Empty Vector WT S384A S384E T445A T445E Fold Induction Luciferase p21 promoter C F1000 Poster Ba ht protected. F1000 Poster Bank. Copyright ank. Copyright protected. F1000 Poster Bank. Copyright protected. 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