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

The Carboxyl-terminal Tail of a Heterotrimeric Kinesin 2 Motor Subunit Directly Binds to β2-tubulin

  • 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)
  • 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)
  • 투고 : 2018.10.01
  • 심사 : 2018.10.17
  • 발행 : 2019.03.30

초록

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.

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.

키워드

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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