Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
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JSAP1 Interacts with Kinesin Light Chain 1 through Conserved Binding Segments

JSAP1과 Kinesin Light Chain 1의 결합 및 결합부위 규명

  • Kim, Sang-Jin (Departments of Biochemistry, Neurology, Inje University) ;
  • Lee, Chul-Hee (Department of Neorosurgery, College of Medicine, Gyeongsang National University) ;
  • Park, Hye-Young (Departments of Biochemistry, Inje University) ;
  • Yea, Sung-Su (Departments of Biochemistry, Inje University) ;
  • Jang, Won-Hee (Departments of Biochemistry, Inje University) ;
  • Lee, Sang-Kyeong (Departments of Biochemistry, Psychiatry, Inje University) ;
  • Park, Yeong-Hong (Departments of Biochemistry, Inje University) ;
  • Cha, Ok-Soo (Department of Physical Education, Jinju National University of Education) ;
  • Moon, Il-Soo (Departments of Anatomy, College of Medicine, Dongguk University) ;
  • Seog, Dae-Hyun (Departments of Biochemistry, Inje University)
  • 김진상 (인제대학교 의과대학 생화학교실, 신경과학교실) ;
  • 이철희 (경상대학교 의과대학 신경외과학교실) ;
  • 박혜영 (인제대학교 의과대학 생화학교실) ;
  • 예성수 (인제대학교 의과대학 생화학교실) ;
  • 장원희 (인제대학교 의과대학 생화학교실) ;
  • 이상경 (인제대학교 의과대학 생화학교실, 정신과학교실) ;
  • 박영홍 (인제대학교 의과대학 생화학교실) ;
  • 차옥수 (진주교육대학교 체육교육학과) ;
  • 문일수 (동국대학교 의과대학 해부학교실) ;
  • 석대현 (인제대학교 의과대학 생화학교실)
  • Published : 2007.07.30
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Abstract

A conventional kinesin, KIF5/kinesin-I, is composed of two kinesin heavy chains (KHCs) and two kinesin light chains (KLCs) and binds directly to microtubules. KIF5 motor mediates the transport of various membranous organelles, but the mechanism how they recognize and bind to a specific cargo has not yet been completely elucidated. Here, we used the yeast two-hybrid system to identify the neuronal protein(s) that interacts with the tetratricopeptide repeats (TRP) of KLCI and found a specific interaction with JNK/stress-activated protein kinase-associated protein 1 (JSAP1/JIPP3). The yeast two-hybrid assay demonstrated that the TRP 1,2 domain-containing region of KLCI mediated binding to the leucine zipper domain of JSAP1. JSAP1 also bound to the TRP region of lac2 but not to neuronal KIF5A, KIF5C and ubiquitous KIF5B in the yeast two-hybrid assay. In addition, these proteins showed specific interactions in the GST pull-down assay and by co-immunoprecipitation. KLCI and KIF5B interacted with GST-ISAP1 fusion proteins, but not with GST alone. An antibody to JSAPI specifically co-immunoprecipitated KIF5s associated with JSAP1 from mouse brain extracts. These results suggest that JSAP1, as KLC1 receptor, is involved in the KIF5 mediated transport.

KIF5는 2분자의 kinesin heavy chain (KHC)과 2분자의 kinesin light cham (KLC)으로 구성되며 미세소관과 직접 결합한다. KIF5는 여러가지 세포 내 소기관을 이동시키나 KIF5가 이동시키는 운반체가 어떻게 특이적으로 결합하는지는 아직 밝혀지지 못하였다. 본 연구에서 효모 two-hybrid system을 사용하여 KLC1의 tetratricopeptide repeats (TRP) 부위와 결합하는 세포 내의 단백질을 분리하였다. 결과 KLC1와 특이적으로 결합하는JNK/stress-activated protein kinase-associated protein 1 (JSAP1/JIP3)을 분리하였다. 이러한 결합은 KLC1의 TRP1, 2 영역과 JSAP1의 leucine zipper 영역이 결합에 관여하며, 또한 효모 two-hybrid assay에서 JSAP1은 KLC2와 결합하지만 신경세포에서 발현하는 KIF5A, KIF5C 그리고 모든 세포에서 발현하는 KIF5B와는 결합하지 않았다. 단백질간의 결합을 pull-down assay로 확인한 결과 KLC1은 glutathione S-transferase (GST)와는 결합하지 않으나 GST결한 JSAP1과는 결합하였다. 또한 생쥐의 뇌 파쇄 액으로부터 JSAP1 항체로 면역침강을 행한 결과 KLC1은 JSAP1과 같이 침강하였다. 이러한 결과들은 KLC1은 JSAP1과 결합하며, JSAP1은 KLC1의 수용체로세포 내 KIF5의 수송의 매개 단백질로 작용함을 시사한다.

Keywords

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