초록 ▼

연구목표
1) human KIF4 단백질의 특성을 규명한다.
2) huma KIF4단백질과 결합하는 세포단백질을 확인하여 KIF4의 기능을 밝힌다.
연구내용
1) KIF4의 Subcellular localization을 조사했을 때 대부분의 KIF4가 핵에 소재함을 알아내었다. 핵 내에서는 nuclear matrix에 결합되어 있었다. KIF4가 핵단백질이므로 nuclear localization signal(NLS)이 있을 것이므로 여러 가지 실험을 통하여 hexapeptide PKLRRR이 NLS임을 밝혔다

연구목표
1) human KIF4 단백질의 특성을 규명한다.
2) huma KIF4단백질과 결합하는 세포단백질을 확인하여 KIF4의 기능을 밝힌다.
연구내용
1) KIF4의 Subcellular localization을 조사했을 때 대부분의 KIF4가 핵에 소재함을 알아내었다. 핵 내에서는 nuclear matrix에 결합되어 있었다. KIF4가 핵단백질이므로 nuclear localization signal(NLS)이 있을 것이므로 여러 가지 실험을 통하여 hexapeptide PKLRRR이 NLS임을 밝혔다. 특히 miitosis시 KIF4는 chromosome과 결합하는 것으로 보아 KIF4는 microtubule-based mitotic motor로서 DNA를 실어나르는 것으로 추정된다.
2) 대부분의 KIF4는 interphase시 nuclear matrix에 결합해 있으면서 mitosis때에는 chromosome과 결합하는 것으로 보아 적어도 두 가지 이상의 기능을 수행할 것으로 짐작된다. 이는 결합하는 단백질의 종류가 여러 가지임을 시사한다, KIF4와 결합하는 단백질을 확인하기 위하여 yeast two hybrid system을 이용하여 후보단백질을 골라낸 후 co-immunoprecipitation, and co-fractionation 방법으로 화인하였다. 그 단백질은 BRCA2-associated factor 35 (BRAF35)로서 nonspecific DNA binding high-mobility-group domain과 kinesin-likecoiled-coil domain을 가진 단백질이다. 두 단백질의 결합은 각각의 alpha-helical coiled-coil domain에서 일어나는 듯 하다. co-fractionation 실험결과 KIF4와 BRAF35는 약 540 kD의 복합체로 존재한다.
연구성과
두 편의 논문 발표
1) Lee, Y. M., and Kim, W.(2003) Association of human kinesin superfamily protein member 4 with BRCA2-associated factor 35. Biochem J. 374:497-503
2) Lee, Y. M., Lee, S., Lee, E., Shin, H., Hahn, H., Choi, W., and Kim, W. (2001) Human kinesin family member 4 protein dominantly localized in the nuclear matrix is ssociated with Chromosomes during mitosis. Biochem. J. 360:549-556.

Abstract ▼

Purpose of Research
1) Characterization of human KIF4 protein
2) Identification of cellular protein(s) interacting with human KIF4 protein
Contents of Research
1) Subcellular localization of KIF4 was examined by using immunocytochemical and subcellular fractionation methods, and it was f

Purpose of Research
1) Characterization of human KIF4 protein
2) Identification of cellular protein(s) interacting with human KIF4 protein
Contents of Research
1) Subcellular localization of KIF4 was examined by using immunocytochemical and subcellular fractionation methods, and it was found that most KIF4 was localized in the nucleus. Subsequent subnuclear fractionation revealed predominant association of KIF4 with the nuclear matrix. In further confirmation of this conclusion, the hexapeptide of PKLRRR (amino acid 773-778) in the molecule was found to function as a nuclear localization signal. During mitoticphase of cell cycle human KIF4 was associated with the chromosomes, suggesting that human KIF4 might be a microtubule-based mitotic motor with DNA as its cargo.
2) A large portion of human kinesin superfamily protein member 4 (KIF4) is associated with the nuclear matrix during the interphase, while a small portion in the cytoplasm. During the mitosis it is associated with chromosomes throughout the entire process. In the present study, we identified a protein that interacts with KIF4 by use of yeast two hybrid system, co-immunoprecipitation, and co-fractionation. It is BRCA2-associated factor 35 (BRAF35) containing a nonspecific DNA binding high-mobility-group domain and a kinesin-likecoiled-coil domain. It appeared that the interaction between the two proteins occurs through their respective -helical coiled-coil domains. The co-fractionation experiment revealed that KIF4 and BRAF35 were present in a complex of approximately 540 kD. The compositionand biological significance of that complex should be further studied.
Effectiveness of Research
Two research papers were published.
1) Lee, Y. M., and Kim, W.(2003) Association of human kinesin superfamily protein member 4 with BRCA2-associated factor 35. Biochem J. 374:497-503
2) Lee, Y. M., Lee, S., Lee, E., Shin, H., Hahn, H., Choi, W., and Kim, W. (2001) Human kinesin family member 4 protein dominantly localized in the nuclear matrix is ssociated with Chromosomes during mitosis. Biochem. J. 360:549-556.

목차 Contents

• Ⅰ. 연구계획 요약문 ...3
• 1. 국문요약문 ...3
• Ⅱ. 연구결과 요약문 ...4
• 1. 국문요약문 ...4
• 2. 영문요약문 ...5
• Ⅲ. 연구내용 ...6
• 1. 서론 ...6
• 2. 연구방법 및 이론 ...7
• 3. 결과 및 고찰 ...9
• 4. 결론 ...14
• 5. 인용문헌 ...15