Kinesin Superfamily KIF1Bα Protein Binds to the PDZ Domain of MALS-3

김상진(Sang-Jin Kim),이철희(Chul-Hee Lee),박혜영(Hye-Young Park),예성수(Sung Su Yea),장원희(Won Hee Jang),이상경(Sang Kyeong Lee),박영홍(Yeong-Hong Park),정용욱(Yongwook Jung),석대현(Dae-Hyun Seog) 대한해부학회 2006 Anatomy & Cell Biology Vol.39 No.5

분자 motor로서 큰 superfamily를 형성하는 Kinesin 단백질은 분비소포, 단백질 복합체, 세포 내 각 소기관을 운반한다. KIF1Bα는 단량체의 motor 단백질로서 세포 내에서 미토콘드리아를 세포 말단으로 이동시키는 역할이 밝혀졌다. 본 연구에서 효모 two-hybrid system을 사용하여 KIF1Bα와 결합하는 세포 내의 단백질을 분리하였다. 결과 KIF1Bα와 특이적으로 결합하는 mammalian LIN-7 (MALS)-3/vertebrate homology of LIN-7(Veri)와 synaptic scaffolding molecule (S-SCAM)을 분리하였다. MALS-3는 KIF1Bα의 C-말단 영역과 결합에 관여하며 KIF1Bα의 C-말단에 존재하는 “T-X-V”아미노산 배열이 MALS-3와의 결합에 중요하게 관여하였다. 또한 효모 two-hybrid assay에서 MALS-3는 KIF1Bα와 결합하지만 다른 종류의 KIFs와는 결합하지 않았다. 단백질간의 결합을 pull-down assay로 확인한 결과 MALS-3는 glutathione S-transferase (GST)와는 결합하지 않으나 GST결합 KIF1Bα와는 결합하였다. 또한 생쥐의 뇌 파쇄 액에 MALS-3 항체로 면역침강을 행하여 KIF1Bα를 확인한 결과 MALS-3와 같이 침강하였다. 이러한 결과들은 KIF1Bα는 MALS-3와 결합하며, MALS-3는 KIF1Bα의 수용체로 세포 내 KIF1Bα의 수송의 매개 단백질로 작용함을 시사한다. The Kinesin superfamily proteins (KIFs) make up a large superfamily of molecular motors that transport cargo such as vesicles, protein complexes, and organelles. KIF1Bα is a monomeric motor that conveys mitochondria and plays an important role in cellular function. Here, we used the yeast two-hybrid system to identify the proteins that interacts with KIF1Bα and found a specific interaction with the mammalian LIN-7 (MALS)- 3/vertebrate homology of LIN-7 (Veri) and synaptic scaffolding molecule (S-SCAM). MALS-3 protein bound to the tail region of KIF1Bα but not to other kinesin family members in the yeast two-hybrid assay. The “T-X-V” motif at the C-terminal end of KIF1Bα is essential for interaction with MALS-3. In addition, this protein showed specific interactions in the Glutathione S-transferase (GST) pull-down assay. An antibody to MALS-3 specifically coimmunoprecipitated KIF1Bα associated with MALS-3 from mouse brain extracts. These results suggest that MALS-3, as KIF1Bα receptor, is involved in the KIF1Bα-mediated transport.

Ferritin, an Iron Storage Protein, Associates with Kinesin 1 through the Cargo-binding Region of Kinesin Heavy Chains (KHCs)

Won Hee Jang(장원희),Young Joo Jeong(정영주),Won Hee Lee(이원희),Mooseong Kim(김무성),Sang-Jin Kim(김상진),Sang-Hwa Urm(엄상화),Il Soo Moon(문일수),Dae-Hyun Seog(석대현) 한국생명과학회 2016 생명과학회지 Vol.26 No.6

세포내소기관과 단백질 복합체의 운반은 kinesin superfamily proteins (KIFs)에 의해 매개된다. 처음으로 밝혀진 kinesin인 kinesin 1은 motor단백질로서 세포 내에서 미세소관을 따라 이동하며, 다양한 세포내 소기관이나 단백질복합체를 운반한다. Kinesin 1은 장쇄(KHC, 또한 KIF5s로도 통칭) 2분자와 단쇄(KLCs) 2분자로 구성된 4합체(tetramer) 구조를 가진다. KIF5s의 운반체 결합영역을 포함하는 말단영역은 다수의 운반체와 결합하지만, 결합운반체에 관하여 아직 충분히 밝혀지지 않았다. 본 연구에서 KIF5A의 결합 단백질을 동정하기 위하여 효모 two-hybrid screening을 수행하였고 철 저장 및 해독 기능을 하는 단백질인 ferritin heavy chain (Frt-h)을 찾아내었다. Frt-h은 KIF5A의 아미노산 800번과 940번 사이의 부위와 결합하며, 다른 KIF5s와도 결합함을 효모 two-hybrid assay로 확인하였다. 또한 Frt-h의 coiled-coil 도메인이 KIF5A와의 결합에 필수영역임을 밝혔다. 한편, ferritin light chain (Frt-l) 또한 KIF5s와 결합함을 효모 two-hybrid assay로 확인하였다. 이러한 단백질간의 결합을 glutathione S-transferase (GST) pull-down assay를 통하여 검증하였다. 추가적으로 생쥐의 뇌 파쇄액을 항 KHC 항체로 면역침강을 행한 결과, KLC1뿐만 아니라 Frt-h와 Frt-l도 같이 침강하였다. 이러한 결과들은 세포 내에서 kinesin 1이 ferritin 복합체를 운반함을 시사한다. The intracellular transport of organelles and protein complexes is mediated by kinesin superfamily proteins (KIFs). The first kinesin, kinesin 1, was identified as a molecular motor protein that moves various organelles and protein complexes along the microtubule rails in cells. Kinesin 1 is a tetramer of two heavy chains (KHCs, also called KIF5s) and two kinesin light chains (KLCs). KIF5s interact with many different proteins through their tail region, but their binding proteins have not yet been fully identified. To identify the interaction proteins for KIF5A, we performed yeast two-hybrid screening and found a specific interaction with ferritin heavy chain (Frt-h), which has a role in iron storage and detoxification. Frt-h bound to the amino acid residues between 800 and 940 of KIF5A and to other KIF5s in the yeast two-hybrid assay. The coiled-coil domain of Frt-h is essential for interaction with KIF5A. In addition, ferritin light chain (Frt-l) interacted with KIF5s in the yeast two-hybrid assay. In addition, these proteins showed specific interactions in the glutathione S-transferase (GST) pull-down assay. An antibody to KHC specifically co-immunoprecipitated Frt-h and Frt-l from mouse brain extracts. These results suggest the kinesin 1 motor protein may transport the ferritin complex in cells.

APP Tail 1 (PAT1) Interacts with Kinesin Light Chains (KLCs) through the Tetratricopeptide Repeat (TPR) Domain

Won Hee Jang(장원희),Sang-Jin Kim(김상진),Young Joo Jeong(정영주),Hee Jae Jun(전희재),Il Soo Moon(문일수),Dae-Hyun Seog(석대현) 한국생명과학회 2012 생명과학회지 Vol.22 No.12

KIF5/Kinesin-I는 경쇄(light chain)를 통하여 결합함으로써 다양한 운반체들을 미세소관을 따라 운반한다. Kinesin light chains (KLCs)은 tetratricopeptide repeat (TPR) 영역을 매개로 운반체와 결합한다. 현재까지 KLCs와 결합하는 많은 운반체들이 확인되었으나 KLCs가 어떻게 특정운반체를 인식하여 결합하는지는 아직 확실히 밝혀지지 않았다. 본 연구에서 KLC1의 TPR 영역과 결합하는 단백질을 분리하기 위하여 효모 two-hybrid system을 이용하여 탐색한 결과 amyloid precursor protein (APP)과 결합하는 것으로 보고된 protein interacting with APP tail 1 (PAT1)을 분리하였다. KLC1은 PAT1의 C-말단 부위와 결합하며, PAT1은 KLC1의 TPR 영역을 포함한 부위와 결합함을 효모 two-hybrid assay로 확인하였다. 또한 PAT1는 KLC2와도 결합하였지만 kinesin heavy chains (KHCs)인 KIF5A, KIF5B, KIF5C와는 결합하지 않았다. 단백질간 결합은 glutathione S-transferase (GST) pull-down assay와 공동면역침강으로도 확인하였다. 생쥐의 뇌 파쇄액을 PAT1 항체와 APP 항체로 면역침강을 행한 결과 KLC와 KHCs가 같이 침강하였다. 이러한 결과들은 PAT1이 Kinesin-I와 APP 포함 소포간의 상호작용을 매개한다는 것을 시사한다. A conventional kinesin, KIF5/Kinesin-I, transports various cargoes along the microtubule through interaction between its light chain subunit and the cargoes. Kinesin light chains (KLCs) interact with many different cargoes using their tetratricopeptide repeat (TPR) domain, but the mechanism underlying recognition and binding of a specific cargo has not yet been completely elucidated. We used the yeast two-hybrid assay to identify proteins that interact with the TPR domain of KLC1. We found an interaction between the TPR domain of KLC1 and an amyloid precursor protein (APP)-binding protein PAT1 (protein interacting with APP tail 1). The yeast two-hybrid assay demonstrated that the TPR domain-containing region of KLC1 mediated binding to the C-terminal tail region of PAT1. PAT1 also bound to KLC2 but not to kinesin heavy chains (KIF5A, KIF5B, and KIF5C) in the yeast two-hybrid assay. These protein-protein interactions were also observed in the glutathione S-transferase (GST) pull-down assay and by co-immunoprecipitation. Anti-PAT1 antibody as well as anti-APP antibody co-immunoprecipitated KLC and KHCs associated with PAT1 from mouse brain extracts. These results suggest that PAT1 could mediate interactions between Kinesin-I and APP containing vesicles.

Detection of Mitotic Centromere-Associated Kinesin (MCAK) During Cell- Cycle Progression of Human Jurkat T Cells Using Polyclonal Antibody Raised Against Its N-Terminal Region Overexpressed in E. coli

( Do Youn Jun ),( Seok Woo Rue ),( Byung Woo Kim ),( Young Ho Kim ) 한국미생물생명공학회 2003 Journal of microbiology and biotechnology Vol.13 No.6

Sorting Nexin 17 Interacts Directly with Kinesin Superfamily KIF1B<FONT FACE= HCI Tulip >Ղ Protein

Dae-Hyun Seog,Jin Han 대한생리학회-대한약리학회 2008 The Korean Journal of Physiology & Pharmacology Vol.12 No.4

KIF1BՂ is a member of the Kinesin superfamily proteins (KIFs), which are microtubule-dependent molecular motors that are involved in various intracellular organellar transport processes. KIF1BՂ is not restricted to neuronal systems, however, is widely expressed in other tissues, even though the function of KIF1BՂ is still unclear. To elucidate the KIF1BՂ-binding proteins in non-neuronal cells, we used the yeast two-hybrid system, and found a specific interaction of KIF1BՂ and the sorting nexin (SNX) 17. The C-terminal region of SNX17 is required for the binding with KIF1BՂ. SNX17 protein bound to the specific region of KIF1BՂ (813-916. aa), but not to other kinesin family members. In addition, this specific interaction was also observed in the Glutathione S-transferase pull-down assay. An antibody to SNX17 specifically co-immunoprecipitated KIF1BՂ associated with SNX17 from mouse brain extracts. These results suggest that SNX17 might be involved in the KIF1BՂ-mediated transport as a KIF1BՂ adaptor protein.

Sorting Nexin 17 Interacts Directly with Kinesin Superfamily KIF1B${\beta}$ Protein

Seog, Dae-Hyun,Han, Jin The Korean Society of Pharmacology 2008 The Korean Journal of Physiology & Pharmacology Vol.12 No.4

KIF1B${\beta}$ is a member of the Kinesin superfamily proteins (KIFs), which are microtubule-dependent molecular motors that are involved in various intracellular organellar transport processes. KIF1B${\beta}$ is not restricted to neuronal systems, however, is widely expressed in other tissues, even though the function of KIF1B${\beta}$ is still unclear. To elucidate the KIF1B${\beta}$-binding proteins in non-neuronal cells, we used the yeast two-hybrid system, and found a specific interaction of KIF1B${\beta}$ and the sorting nexin (SNX) 17. The C-terminal region of SNX17 is required for the binding with KIF1B${\beta}$. SNX17 protein bound to the specific region of KIF1Bf3 (813-916. aa), but not to other kinesin family members. In addition, this specific interaction was also observed in the Glutathione S-transferase pull-down assay. An antibody to SNX17 specifically co-immunoprecipitated KIF1B${\beta}$ associated with SNX17 from mouse brain extracts. These results suggest that SNX17 might be involved in the KIF1B${\beta}$-mediated transport as a KIF1B${\beta}$ adaptor protein.

Sorting Nexin 17 Interacts Directly with Kinesin Superfamily KIF1Bβ Protein

석대현,한진 대한약리학회 2008 The Korean Journal of Physiology & Pharmacology Vol.12 No.4

KIF1Bβ is a member of the Kinesin superfamily proteins (KIFs), which are microtubule-dependent molecular motors that are involved in various intracellular organellar transport processes. KIF1Bβ is not restricted to neuronal systems, however, is widely expressed in other tissues, even though the function of KIF1Bβ is still unclear. To elucidate the KIF1Bβ-binding proteins in non-neuronal cells, we used the yeast two-hybrid system, and found a specific interaction of KIF1Bβ and the sorting nexin (SNX) 17. The C-terminal region of SNX17 is required for the binding with KIF1Bβ. SNX17 protein bound to the specific region of KIF1Bβ (813-916. aa), but not to other kinesin family members. In addition, this specific interaction was also observed in the Glutathione S-transferase pull-down assay. An antibody to SNX17 specifically co-immunoprecipitated KIF1Bβ associated with SNX17 from mouse brain extracts. These results suggest that SNX17 might be involved in the KIF1Bβ-mediated transport as a KIF1Bβ adaptor protein.

Activity-dependent synaptic localization of processing bodies and their role in dendritic structural plasticity

Oh, Jun-Young,Kwon, Ara,Jo, Anna,Kim, Hoon,Goo, Yong-Sook,Lee, Jin-A,Kim, Hyong Kyu The Company of Biologists Limited 2013 Journal of cell science Vol.126 No.9

<P>In neurons, transport of a subset of mRNAs to subcellular regions and their translation has a role in synaptic plasticity. Recent studies have suggested a control mechanism of this local translation through mRNA compartmentalization or degradation. Here we report that processing bodies (P-bodies), which are involved in mRNA degradation or storage, are transported to dendrites by conventional kinesin (KIF5A) as a motor protein. Neuronal activation induced by depolarization increased the colocalization of P-bodies with PSD-95 in dendrites. This neuronal activity increased the release of <I>Nd1</I> and <I>Arp2</I> mRNA from the P-bodies and, consequently, reversed the decrease of F-actin (induced by overexpression of Dcp1a) in the dendrites. Our data suggest that the activity-induced redistribution of P-bodies and mRNA release from P-bodies might have a role in synaptic structural plasticity by altering levels of mRNAs that are involved in the dynamics of the actin cytoskeleton in dendrites.</P>

활성 브라운 동역학 모형과 생명현상에의 응용

이공주복,박병준,안민정,최새롬 한국물리학회 2014 새물리 Vol.64 No.7

Active motion is ubiquitous in nature, ranging from macroscopic flocking of birds to the microscopic stepping motion of molecular motors, and needs processes of energy supply and of conversion to mechanical work. The active Brownian-particle model describes the motion of a particle that has an internal depot to store energy supplied from the environment and is able to convert that internal energy into mechanical work. In this article, we introduce active Brownian-particle models with several variations and discuss their dynamic properties briefly. An active model in an overdamped limit is also built and applied to study the dynamics of on molecular motor and of cargo transport by many molecular motors. Also, our recent results which were simulated by using biologically-known values of the parameters are presented. 농동적 운동은 새들의 거시적 집단운동에서 분자모터의 미시적 걸음운동에 이르기까지 자연 도처에 존재하며, 에너지 공급과정과 에너지를 역학적 일로 전환하는 과정을 필요로 한다. 활성 브라운 입자 모형은 내부 에너지저장고를 갖고 있어 외부에서 공급된 에너지를 저장하고, 내부 에너지를 역학적 일로 전환할 수 있는 입자의 운동을 기술한다. 본 논고에서는 다양한 활성 브라운 입자 모형들을 소개하고 각 모형의 동역학 성질을 간단히 논의한다. 또한, 과도감쇠영역에서의 활성 모형을 구축하여 분자모터의 동역학과 다중분자모터에 의한 카고 수송 동역학 연구에 적용하고, 실제 알려진 생물학적 변수들을 이용하여 전산 모사한 최근의 우리 결과도 제시한다.

Nucleotide-dependent control of internal strains in ring-shaped AAA+ motors.

Hwang, Wonmuk,Lang, Matthew J SPRINGER SCIENCE + BUSINESS MEDIA 2013 Cellular and molecular bioengineering Vol.6 No.1

<P>The AAA+ (ATPase Associated with various cellular Activities) machinery represents an extremely successful and widely used design plan for biological motors. Recently found crystal structures are beginning to reveal nucleotide-dependent conformational changes in the canonical hexameric rings of the AAA+ motors. However, the physical mechanism by which ATP binding on one subunit allosterically propagates across the entire ring remains to be found. Here we analyze and compare structural organization of three ring-shaped AAA+ motors, ClpX, HslU, and dynein. By constructing multimers using subunits of identical conformations, we find that individual subunits locally possess helical geometries with varying pitch, radius, chirality, and symmetry number. These results suggest that binding of an ATP to a subunit imposes conformational constraint that must be accommodated by more flexible nucleotide-free subunits to relieve mechanical strain on the ring. Local deformation of the ring contour and subsequent propagation of strains may be a general strategy that AAA+ motors adopt to generate force while achieving functional diversity.</P>