Functional analysis of human nima related kinase 8 (nek8)

FUNCTIONAL ANALYSIS OF HUMAN NIMA-RELATED KINASE (Nek8) LIU JUN NATIONAL UNIVERSITY OF SINGAPORE 2010 FUNCTIONAL ANALYSIS OF HUMAN NIMA-RELATED KINASE (Nek8) LIU JUN (B. Sc.) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2010 ACKNOWLEDGEMENTS First of all, I would like to express my deepest gratitude and appreciation to my supervisor Dr. Liou Yih-Cherng, who guides me into the research area of molecular cell biology. His stimulating suggestions, criticisms and encouragements during my entire postgraduate study period have been a great value for me. I am deeply grateful to Dr. Deng Lih Wen, for her valuable technical advices on cell cycle assays. I would like to thank Lai Cheng Yu for his help of recombinant protein purification. I wish to extend my warmest thanks to all other current and past members of Dr. Liou Yih-Cherng’s laboratory, for their constant assistance and support through these years. They are: Zhou Wei, Xia Yun, Zhao Liqun, Tan Wee Wee, Wang Yu, Yang Qiaoyun, Ye Fan, Song Pei Chee, Pheobe and Tan Pei Ling, Shirley. I would like to acknowledge the National University of Singapore for awarding me the research scholarship. This thesis is dedicated to my beloved family: my parents and my brother, whose love, encouragement, and support have always been my greatest inspiration. Liu Jun 2010 I TABLE OF CONTENTS ACKNOLEGEMENTS……………………………………… I TABLE OF CONTENTS…………………………………… II SUMMARY………………………………………………….VII LIST OF FIGURES………………………………………… IX LIST OF TABLES………… .XI LIST OF ABBREVIATIONS………………………………XII CHAPTER INTRODUCTION……………………………… 1.1 Introduction of the NIMA-related kinase family………………….2 1.1.1 NIMA-related kinases (Nrks) in different biological systems…… 1.1.2 Human NIMA-related kinases (Neks) and their roles in cellular regulation……………………………………………………… . 1.2 NIMA related kinase 8(Nek8)……………………………………. 13 1.2.1 Sequence and molecular structure of Nek8………………………. 14 1.2.2 Functions in cellular regulation and diseases of Nek8……… 14 1.3 Involvment of Nek8 in cellular processes……………………… . 16 1.3.1 Signaling network related to RCC1-like domain and Ran……… 17 1.3.1.1 RCC1 and RCC1 superfamily……………………………… 17 II 1.3.2 Ran network…………………………………………………… . 21 1.3.2.1 Ran-dependent nuclear transport…………………………… 22 1.3.2.2 RanGTP and spindle assembly…………………………… . 25 1.3.2.3 RanGTP and nuclear envelope assembly………………… . 27 1.3.3 Signaling network in ER-stress induced apoptosis……………… 28 1.3.3.1 Activation of the unfolded protein response (UPR)……… . 29 1.3.3.2 ER-associated cell death and survival……………………… 32 1.3.3.3 ER stress and diseases……………………………………… 33 1.4 Hypothesis and objectives………………………………………… 35 CHAPTER MATERIALS AND METHODS………………. 37 2.1 Total RNA isolation and first strand cDNA synthesis………… .38 2.2 DNA amplification and cloning………………………………… . 38 2.3 Site-directed mutagenesis……… .41 2.4 Plasmid DNA isolation, restriction enzyme digestion and sequencing analysis……………………………………………………. 42 2.5 Mammalian cell culture………………………………………… . 45 2.6 Cell-cycle synchronization in mammalian cells………………….45 2.7 Mammalian cell transfection, lysis and immunoprecipitation…. 45 2.8 SDS-PAGE gel electrophoresis and Western blot analysis…… .47 III 2.9 Recombinant protein production in E. coli……………………… 48 2.10 GST-fusion protein binding assays…………………………… . 49 2.11 Immunostaining and confocal flurorescence microscopy…… . 50 2.12 FACS (Fluorescence Activated Cell Sorting)………………… . 51 2.13 FRAP (Fluorescence Recovery After Photobleaching)……… . 51 2.14 Antibodies and reagents…………………………………………. 52 CHAPTER RESULTS………………………………………. 53 3.1 Identification of hNek8……………………………………………. 54 3.1.1 Isolation and sequence verification of human Nek8………… . 54 3.1.2 Bioinfomatics analysis of hNek8……………………………… . 56 3.2 Characterization of hNek8……………………………………… .58 3.2.1 Protein expression profile of Nek8 in mammalian cell lines…… .58 3.2.2 Nek8 in cell cycle regulation…………………………………… . 59 3.2.3 Loss of function of Nek8 using siRNA………………………… . 65 3. Investigation of Nek8 potential binding partners………………. 68 3.3.1 Ran is a potential binding partner of Nek8 based on the domain analysis…………………………………………………… 68 IV 3.3.2 Nek8 can interact with Ran in vitro and in vivo………………… 70 3.3.3 Nek8 prefers to bind to RanGDP…………………………………. 73 3. Investigation of Nek8 functions in nuclear transport………… .77 3.4.1 Cellular localization of Nek8 and functional domains……… 77 3.4.2 Localization changes of Nek8 in the presence of Ran…………….79 3.4.3 Investigation of the effects of Ran on Nek8 in nuclear transport……………………………………………………… . 82 3. Investigation of Nek8’s functions in ER stress………………… 84 3.5.1 Cellular localization of Nek8 in ER region under stress…………. 84 3.5.2 Investigation of Nek8’s changes under the ER stress…………… 87 3.5.3 Nek8 can delay cell preceding to apoptosis under ER stress…… 87 3.5.4 Hsp70 is revealed as a Nek8’s binding partner based on the pull down assay…………………………………………………………… 92 3. Investigation of Nek8’s biological functions linked with human diseases………………………………………………………… 95 CHAPTER DISCUSSION AND CONCLUSIONS…………98 4.1 Significance of the Nek8 domain architecture and functional V characterization…………………………………… 99 4.2 The roles of Nek8 in cell cycle regulation………………………. 102 4.3 The roles of Nek8 in nuclear transport…………………………. 104 4.4 The roles of Nek8 in ER-stress induced apoptosis…………… . 108 4.5 Implication of Nek8 related to diseases………………………… 112 4.6 Conclusions and perspectives…………………………………… 114 REFERENCES………………………………………………… 118 VI SUMMARY As a member of NIMA-related kinase family (Nrk), Nek8 has an N-terminal serine/threonine kinase domain highly homologous with that of NIMA. Most of the NIMA-related kinase family members are involved in cell cycle regulation. Roles of Nek8 in cell cycle and other cellular regulation are very little known. Nek8 is only shown to be related to kidney disease. Therefore, the main purpose of the studies in this thesis was to explore Nek8’s functions in cellular regulation. Nek8 contains an N-terminal catalytic domain and a C-terminal RCC1-like domain. We are the first to identify a small GTPase Ran and a chaperone protein hsp70 as binding partners of Nek8. We demonstrate that Nek8 is not an essential cell cycle regulator as most of its family members, in that overexpression of Nek8 has little effect on changing cell cycle progression. We also found that Ran can interact with Nek8 and facilitate translocation of Nek8 between cytoplasm and nucleus, suggesting Nek8 may be involved in the process of nuclear transport. Furthermore, the cellular localization of Nek8 in the endoplasmic reticulum (ER) and its role in delaying apoptosis induced by the ER stress drugs indicate that Nek8 could be responsible for protecting cells from ER stress-induced apoptosis. We found that Nek8 could interact with Hsp70. Hsp70 can enhance cell survival under several lethal conditions and has been revealed to negatively regulate the mitochondrial pathway of apoptosis. Thus it is possible that protection of cells from ER stress-induced apoptosis by Nek8 is due to the association of Nek8 with Hsp70. VII ER stress has been associated with a wide range of diseases, including neurodegeneration, cardiac disease, cancer and diabetes. Nek8 itself has been known to be linked with some diseases, such as cancer and kidney disease. Defects in Nek8 gene can cause polycystic kidney disease (PKD). Nek8 was reported to regulate cilia formation. The mutations in Nek8 may affect its ciliary localization. The observation that jck mice have longer cilia than wild-type mice indicates that Nek8 may control the cilia length. However the detailed mechanism under this disease is not clear. 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Eur J Cell Biol 81(11): 623-33. 133 [...]... 3.12 Nek8 binds to Ran in vivo 72 Figure 3.13 Nek8 prefers to bind to RanGDP 75 Figure 3.14 Cellular Localization of Nek8 domains 78 Figure 3.15 Nek8 localization changes in the presence of Ran 80 Figure 3.16 Nek8R2 localization changes in the presence of Ran 81 Figure 3.17 Cellular dynamics of Nek8 in the presence of Ran 83 Figure 3. 18 The cellular localization of Nek8 under LMB treatment 85 Figure... mechanisms of Nek8 in these diseases are far from clear Functional studies on Nek8 are necessary and important 1.3 Involvement of Nek8 in cellular processes To date, no binding partner of Nek8 has been identified except polycystin-2 Signaling pathways that Nek8 is involved are yet to be discovered Bioinformatics analysis of Nek8’s molecular structure and the available information of NIMA- related 16... primary human colon cDNA library, whose ORF (Open Reading Frame) encodes a 692 amino-acid protein with a predicted molecular weight of 75kDa (Bowers and Boylan 2004) Sequence similarity between human Nek8 (hNek8) and murine Nek8 (mNek8) is as high as 99% Thus, the function information of Nek8 in mice can most likely be applied to Nek8 in human The sequence of Nek8 is highly homologous to many known kinases... functions in cell regulation, not limited in the area of cell cycle The divergent cellular functions of Neks are possibly due to their various domains in the C terminus, in addition to the homologous kinase domain in the N-terminus 1.2 NIMA related kinase 8 (Nek8) 13 Chapter 1 Introduction 1.2.1 Sequence and molecular structure of Nek8 Most members of Nrk kinase family were identified only recently, their... 3.5 Localization of Nek8 in different cell cycle stages 60 Figure 3.6 Endogenous Nek8 level in different cell cycle stages 61 Figure 3.7 Cell cycle profiles of cells with Nek8 or Nek8 mutants 63 Figure 3 .8 Endogenous Nek8 protein level after siRNA treatment 66 Figure 3.9 Nek8 protein level is very stable in cells 67 Figure 3.10 Cellular localization of Nek8 and Ran 69 Figure 3.11 Nek8 binds to Ran in... homologs of NIMA found in mouse (Ben-David et al 1991; Letwin et al 1992; Schultz et al 1994; Chen et al 1999) Figure 1.1 shows the schematic map of the NIMA related kinase family (O'Connell et al 2003) These Nrk family members share an N-terminal catalytic domain which is highly similar to the kinase domain of NIMA In addition, in term of function, most of the family members are cell cycle -related kinases... far, at least twelve NIMA- related protein kinases, named as Neks (Nek1-Nek12), have been identified in human genome (O'Connell et al 2003) Among these human NIMA- related kinases, Nek2 is closest to NIMA with 44% identical sequence in the kinase domain (Fry 2002) Nek2 has two spliced isoforms: Nek2A and Nek2B Their molecular structures are shown in Figure 1.1 Nek2A has an N-terminal kinase domain, a leucine... contains all of the required motifs for a functional kinase (Bowers and Boylan 2004), but the downstream substrates of Nek8 have not yet been identified Studies on kinase activity using β-casein as a general substrate showed no activity, suggesting that Nek8 may have specific substrates 1.2.2 Functions in cellular regulation and diseases of Nek8 Murine Nek8 was first identified because of a mutation of this... turned to focus on this kinase family 1.1.1 NIMA- related kinases (Nrks) in different biological systems The NIMA- related protein family is named from the protein NIMA (Never In Mitosis A), which is a serine/theronine kinase first identified in Aspergillus nidulans encoded by nimA gene Mutation in nimA gene can arrest cells in G2 and excess nimA can initiate mitosis, suggesting that the NIMA protein has a... several reports on the functional study of Nrks More evidences found in mammalian Nrks will be demonstrated in chapter 1.1.2 5 Chapter 1 Introduction Figure 1.1 Molecular strucutures of NIMA- related kinase (Nrk) family members 6 Chapter 1 Introduction 1.1.2 Human NIMA- related kinases (Neks) and their roles in cellular regulation Researchers paid more attention on the mammalian NIMA family members recently . FUNCTIONAL ANALYSIS OF HUMAN NIMA- RELATED KINASE 8 (Nek8) LIU JUN NATIONAL UNIVERSITY OF SINGAPORE 2010 FUNCTIONAL ANALYSIS OF HUMAN NIMA- RELATED. REFERENCES………………………………………………… 1 18 VII SUMMARY As a member of NIMA- related kinase family (Nrk), Nek8 has an N-terminal serine/threonine kinase domain highly homologous with that of NIMA. Most of the NIMA- related. NIMA related kinase 8( Nek8)……………………………………. 13 1.2.1 Sequence and molecular structure of Nek8………………………. 14 1.2.2 Functions in cellular regulation and diseases of Nek8……… 14 1.3 Involvment of