The functional motifs for nucleocytoplasmic shuttling of C-terminal fragment of c-Met : A novel "pH-dependent' nuclear localization signal
- 발행기관 아주대학교 Ajou University School of Medicine, The Graduate School, Ajou University
- 지도교수 Jae-Ho Lee
- 발행년도 2015
- 제출일 2014-12-19
- 학위수여년월 2015. 2
- 학위명 박사
- 학과 일반대학원 의생명과학과
- 실제URI http://www.dcollection.net/handler/ajou/000000019619
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
The C-terminal fragment of the c-Met receptor tyrosine kinase is present in the nuclei of various cells irrespective of ligand stimulation, but neither the specific amino acid motif nor the responsible nuclear localization signal (NLS) has not been previously reported. Here, we report that two histidine residues separated by a 10-amino-acid spacer (H1068-H1079) located in the juxtamembrane region of c-Met function as a putative novel NLS. Immunocytochemistry and cellular fractionation assays revealed that deletion of these sequences significantly abolished the nuclear translocation of c-Met in HeLa cells, as did substitution of the histidines with alanines. This substitution also decreased the nuclear translocation of the c-Met fragment and its association with importin β, the carrier protein. The putative NLS of c-Met is unique in that it relies on histidines, whose positive charge changes depending on pH, rather than the lysines or arginines, commonly found in classical bipartite NLSs, suggesting the possible ‘pH-dependency’ of this NLS. Indeed, decreasing the cytosolic pH either with nigericin, a Na+/H+ exchanger or low pH KRB (pH=6.5) buffer significantly increased the level of nuclear c-Met and the interaction of the c-Met fragment with importin β, indicating that low cytosolic pH itself enhanced nuclear translocation of cytosolic fragment of c-Met. Consistent with this, nigericin treatment also enhanced the nuclear accumulation of endogenous c-Met in HeLa cells. Moreover, replacement of histidines either with lysines or arginines abolished the “pH-dependency” at the same time. To the best of our knowledge, the putative aberrant bipartite NLS of c-Met seems to be the first example of what we call a “pH-dependent” NLS. Furthermore, we also report here a putative leucine-rich nuclear export signal (NES) in c-Met which plays a decisive role for nucleocytoplasmic shuttling of the receptor. Taken together, these results suggest that the shuttling of the C-terminal fragment of c-Met between the cytosol and nucleus appears to be due to the presence of both a putative NLS and an NES in the juxtamembrane region.
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ABSTRACT i
TABLE OF CONTENTS iii
LIST OF TABLES v
LIST OF ABBREVIATIONS vi
I. INTRODUCTION 1
II. MATERIALS AND METHODS 19
A. Reagent and antibody 19
B. Plasmid construction 19
C. Cell culture and transfection 20
D. Cell fractionation 20
E. Western bloting 20
F. Immunocytochemistry 21
G. Decrease of cytosolic pH 21
H. Cytosolic pH measurement 21
I. Importin β binding assay 22
J. Time-lapse analysis 22
K. Leptomycin B treatment 23
L. ATP depletion 23
M. Statistical analysis 23
III. RESULTS 25
Part. I. Nuclear translocation of C-terminal fragment of c-Met 25
A. The c-Met fragment localizes to the nuclei of HeLa cells 25
B. Serial deletion of juxtamembrane domain shows that c-Met contains an NLS at H1068-H1079 29
C. Two histidine residues are important for the nuclear translocation of c-Met fragment 34
D. The NLS-directed nuclear accumulation of the c-Met fragment is pH-dependent 41
E. Low pH KRB buffer solution enhances the nuclear accumulation of c-Met fragment 48
F. Decrease of cytosolic pH enhances nuclear accumulation of ~ 60 kDa Met. 51
Part. II. Nuclear export of C-terminal fragment of c-Met 53
A. c-Met harbors a putative leucine-rich nuclear export signal (LR-NES) 53
IV. DISCUSSION 59
V. CONCLUSION 64
VI. REFFERENCES 65
- 국문요약 - 73
