Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
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The Carboxyl-terminal Tail of a Heterotrimeric Kinesin 2 Motor Subunit Directly Binds to β2-tubulin

Heterotrimeric Kinesin 2 모터 단백질의 Carboxyl-말단과 β2-tubulin의 결합

  • Jeong, Young Joo (Departments of Biochemistry, Inje University College of Medicine) ;
  • Park, Sung Woo (Departments of Convergence Biochmedical Science, Inje University College of Medicine) ;
  • Kim, Sang-Jin (Departments of Neurology, Inje University College of Medicine) ;
  • Lee, Won Hee (Department of Neurosurgery, Inje University College of Medicine) ;
  • Kim, Mooseong (Department of Neurosurgery, Inje University College of Medicine) ;
  • Urm, Sang-Hwa (Department of Preventive Medicine, Inje University College of Medicine) ;
  • Seog, Dae-Hyun (Departments of Biochemistry, Inje University College of Medicine)
  • 정영주 (인제대학교 의과대학 생화학교실) ;
  • 박성우 (인제대학교 의과대학 생의학융합교실) ;
  • 김상진 (인제대학교 의과대학 신경과학교실) ;
  • 이원희 (인제대학교 의과대학 신경외과학교실) ;
  • 김무성 (인제대학교 의과대학 신경외과학교실) ;
  • 엄상화 (인제대학교 의과대학 예방의학교실) ;
  • 석대현 (인제대학교 의과대학 생화학교실)
  • Received : 2018.10.01
  • Accepted : 2018.10.17
  • Published : 2019.03.30
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Abstract

Microtubules form through the polymerization of ${\alpha}-$ and ${\beta}-tubulin$, and tubulin transport plays an important role in defining the rate of microtubule growth inside cellular appendages, such as the cilia and flagella. Heterotrimeric kinesin 2 is a molecular motor member of the kinesin superfamily (KIF) that moves along the microtubules to transport multiple cargoes. It consists of two motor subunits (KIF3A and KIF3B) and a kinesin-associated protein 3 (KAP3), forming a heterotrimeric complex. Heterotrimeric kinesin 2 interacts with many different binding proteins through the cargo-binding domains of the KIF3s, but these binding proteins have not yet been specified. To identify these proteins for KIF3A, we performed yeast two-hybrid (Y2H) screening and found a specific interaction with ${\beta}2-tubulin$ (Tubb2), a microtubule component. Tubb2 was found to bind to the cargo-binding domain of KIF3A but did not interact with KIF3B, KIF5B, or kinesin light chain 1 in the Y2H assay. The carboxyl-terminal region of Tubb2 is essential for interaction with KIF3A. Other Tubb isoforms, including Tubb1, Tubb3, Tubb4, and Tubb5, also interacted with KIF3A in the Y2H screening. However, ${\alpha}1-tubulin$ (Tuba1) did not interact with KIF3A. In addition, an antibody to KIF3A specifically co-immunoprecipitated the KIF3B and KAP3 associated with Tubb2 from mouse brain extracts. In combination, these results suggest that a heterotrimeric kinesin 2 motor protein is capable of binding to tubulin and may transport it in cells.

미세소관은 알파- 와 베타-tubulin의 이량체가 종합되어 형성되며, 또한 세포내에서 tubulin 수송은 섬모나 편모와 같은 세포 부착 기관의 성장에 중요한 역할을 한다. Kinesin 2는 kinesin superfamily (KIF)의 분자 모터 단백질의 한 종류로 미세소관를 따라 다양한 운반체를 운반하며, 2종류의 다른 모터 단백질(KIF3A, KIF3B)과 kinesin-associated protein 3 (KAP3)로 구성되어 있다. Kinesin 2는 KIF3A의 cargo binding domain을 통하여 다양한 단백질과의 결합이 알려져 있지만, 아직 결합단백질의 다수는 아직 밝혀지지 않았다. 본 연구에서 KIF3A와 결합하는 단백질을 분리하기 위하여 효모 two-hybrid system을 사용하여 탐색한 결과 미세소관의 단위체의 한 종류인 ${\beta}2-tubulin$ type (Tubb2)을 분리하였다. Tubb2는 KIF3A와 결합하지만, KIF3B, KIF5B와 kinesin light chain 1 (KLC1)과는 결합하지 않았다. Tubb2의 C-말단은 KIF3A와의 결합에 필요하며, 다른 KIF3A는 Tubb의 isoforms인 Tubb1, Tubb2, Tubb3, Tubb4, Tubb5와도 결합하였다. 그러나 Tuba1은 KIF3A와 결합하지 않았다. 생쥐의 뇌 파쇄액을 KIF3A 항체로 면역침강한 결과 Tubb2는 heterotrimeric kinesin 2의 구성단백질들과 같이 침강하였다. 이러한 결과들은 heterotrimeric kinesin 2는 tubulin과 결합하여 세포 내에서 tubulins을 운반하는 것을 시사한다.

Keywords

SMGHBM_2019_v29n3_369_f0001.png 이미지

Fig. 1. Identification of the proteins interacted with KIF3A by yeast two-hybrid screening.

SMGHBM_2019_v29n3_369_f0002.png 이미지

Fig. 2. Minimal Tubb2 binding region in KIF5A.

SMGHBM_2019_v29n3_369_f0003.png 이미지

Fig. 3. Association of heterotrimeric kinesin 2 with Tubb2 in co-immunoprecipitation.

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