• Journal of Life Science
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• v.33 no.7
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• pp.531-537
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• 2023

Intracellular and axonal transport is mediated by microtubule-dependent motor proteins, such as kinesins and cytoplasmic dynein. Kinesin moves along the microtubule to the positive end of the microtubule, while dynein moves to the negative end of the microtubule. Kinesin-1 was first identified as a kinesin superfamily protein (KIF) that functions in the intracellular transport of various cargoes, including organelles, neurotransmitter receptors, and mRNA-protein complexes, through interactions between the carboxyl (C)-terminal domain and the cargo. It interacts with other cargoes, but the adapter/scaffold proteins that mediate between kinesin-1 and the cargo have yet to be fully identified. In this study, a yeast two-hybrid screen was used to identify adapter proteins that interact with the C-terminal region of KIF5A. We found an association between the C-terminal region of KIF5A and the cyclin-dependent kinase 2-associated protein 1 (CDK2AP1), originally identified in malignant hamster oral keratinocytes. CDK2AP1 bound to the C-terminal region of KIF5A and did not interact with KIF3A (the motor of kinesin-2), KIF5B, KIF5C, and kinesin light chain 1 (KLC1). The C-terminal region of CDK2AP1 is essential for its interaction with KIF5A. When co-expressed in HEK-293T cells, CDK2AP1 and kinesin-1 co-immunoprecipitated and co-localized in the cells. These results suggest that the KIF5A-CDK2AP1 interaction serves as an adapter protein connecting kinesin-1 and the cargo when kinesin-1 transports cargo in cells.

• Journal of Life Science
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• v.32 no.3
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• pp.189-195
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• 2022

Kinesin-2 comprises two subfamilies of the heterotrimeric or homodimeric motors found in mammalian cells. Heterotrimeric kinesin-2 consists of kinesin superfamily proteins (KIFs) 3A and 3B and kinesin-associated protein 3 (KAP3), which is a molecular motor protein that moves along microtubules. It plays diverse roles in cargo transport, including anterograde trafficking in cilia, and interacts with many different cargoes and proteins, but their binding proteins have not yet been fully identified. In this study, the yeast two-hybrid assay was used to identify the proteins that interact with the cargo-binding domain (CBD) of KIF3A, and an interaction between KIF3A and brain expressed X-linked 2 (Bex2) was found. Bex2 bound to the CBD-containing C-terminal tail region of KIF3A but did not interact with the same region of KIF3B or KIF5A (a motor protein of kinesin-1). KIF3A interacted with another isoform, Bex1, but did not interact with Bex3. In addition, glutathione S-transferase (GST) pull-downs showed that KIF3A specifically interacts with GST-Bex1 and GST-Bex2 but not with GST alone. When co-expressed in HEK-293T cells, Bex2 co-localized with KIF3A and co-immunoprecipitated with KIF3A and KIF3B but not KIF5B. In combination, these results suggest that Bex2 is capable of binding to heterotrimeric kinesin-2 and may serve as an adaptor protein that links heterotrimeric kinesin-2 with cargo.

• Journal of Life Science
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• v.33 no.11
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• pp.868-875
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• 2023

Kinesin-1 is a motor protein identified as the first member of the kinesin superfamily (KIF), which plays a role in intracellular cargo transport by acting as microtubule-dependent motor proteins within cells. Kinesin-1 consists of two heavy chains (KHCs, also known as KIF5s) and two light chains (KLCs). The 93 amino acids in the carboxyl (C)-terminal tail region of KIF5A are not homologous to the C-terminal tail region of KIF5B or the C-terminal tail region of KIF5C. In this study, we used a yeast two-hybrid screen to identify the binding proteins that interacted with the C-terminal region of KIF5A. We found an association between KIF5A and CUE domain containing 2 (CUEDC2), which is proposed to function as an adaptor protein involved in ubiquitination pathways and protein trafficking. CUEDC2 bound to the C-terminal region of KIF5A and did not interact with KIF5B (the motor of kinesin-1), KIF3A (the motor of kinesin-2), or kinesin light chain 1 (KLC1). KIF5A specifically bound to the C-terminal region of CUEDC2. Furthermore, KIF5A did not interact with another isoform: CUEDC1. In addition, glutathione S-transferase (GST) pull-downs showed that KIF5A directly bound GST-CUEDC2 but did not interact with GST-CUEDC1 and GST alone. When myc-KIF5A and EGFP-CUEDC2 were co-expressed in HEK-293T cells, CUEDC2 co-immunoprecipitated with kinesin-1, and myc-KIF5A and FLAG-CUEDC2 colocalized in the cells. These results suggest that in intracellular cargo transport by kinesin-1, CUEDC2 serves as an adaptor protein connecting kinesin-1 and cargo by binding to KIF5A.

• Journal of Life Science
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• v.26 no.8
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• pp.963-969
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• 2016

Kinesin superfamily proteins (KIFs) are microtubule-dependent molecular motor proteins essential for the intracellular transport of organelles and protein complexes in cells. Kinesin 1 is a member of those KIFs that transport various cargoes, including organelles, synaptic vesicles, neurotransmitter receptors, cell signaling molecules, and mRNAs through interaction between its light chain subunit and the cargoes. Kinesin light chains (KLCs) are non-motor subunits that associate with the kinesin heavy chain (KHC) dimer. KLCs interact with many different binding proteins, but their particular binding proteins have not yet been fully identified. We used the yeast two-hybrid assay to identify proteins that interact with the tetratricopeptide repeat (TPR) domain of KLC1. We found an interaction between the TPR domain of KLC1 and Wiskott-Aldrich syndrome protein family member 1 (WAVE1), a member of the WASP/WAVE family involved in regulation of actin cytoskeleton. WAVE1 bound to the six TPR domain-containing regions of KLC1 and did not interact with KHCs (KIF5A, KIF5B, and KIF5C) in the yeast two-hybrid assay. The carboxyl (C)-terminal verprolin-cofilin-acidic (VCA) domain of WAVE1 is essential for interaction with KLC1. Also, other WAVE isoforms (WAVE2 and WAVE3) interacted with KLC1 in the yeast two-hybrid assay. When co-expressed in HEK-293T cells, WAVE1 co-localized with KLC1 and co-immunoprecipitated with KLC1 and KIF5B. These results suggest that kinesin 1 motor protein may transport WAVE complexes or WAVE-coated cargoes in cells.

• Journal of Life Science
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• v.31 no.3
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• pp.257-265
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• 2021

Heterotrimeric kinesin-2 is a molecular motor protein of the kinesin superfamily (KIF) that moves along a microtubule plus-end directed motor protein. It consists of three different motor subunits (KIF3A, KIF3B, and KIF3C) and a kinesin-associated protein 3 (KAP3) that form a heterotrimeric complex. Heterotrimeric kinesin-2 interacts with many different binding proteins through the cargo-binding domain of the KIF3s. The activity of heterotrimeric kinesin-2 is regulated to ensure that the cargo is directed to the right place at the right time. How this regulation occurs, however, remains in question. To identify the regulatory proteins for heterotrimeric kinesin-2, we performed yeast two-hybrid screening and found a specific interaction with creatine kinase B (CKB), which is the brain isoform of cytosolic creatine kinase enzyme. CKB bound to the cargo-binding domain of KIF3A but did not interact with the KIF3B, KIF5B, or KAP3 in the yeast two-hybrid assay. The carboxyl (C)-terminal region of CKB is essential for the interaction with KIF3A. Another protein kinase, CaMKIIa, interacted with KIF3A, but GSK3a did not interact with KIF3A in the yeast two-hybrid assay. KIF3A interacted with GST-CKB-C but not with GSK-CKB-N or GST alone. When co-expressed in HEK-293T cells, CKB co-localized with KIF3A and co-immunoprecipitated with KIF3A and KIF3B but not KIF5B. These results suggest that the CKB-KIF3A interaction may regulate the cargo transport of heterotrimeric kinesin-2 under energy-compromised conditions in cells.

• Journal of Life Science
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• v.29 no.3
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• pp.369-375
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• 2019

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.

• Journal of Life Science
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• v.20 no.8
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• pp.1166-1172
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• 2010

Kinesin-I exists as a tetramer of two heavy chains (KHCs, also called KIF5s), which contain the amino (N)-terminal motor domain and carboxyl (C)-terminal domain, as well as two light chains (KLCs), which bind to the KIF5s (KIF5A, KIF5B and KIF5C) stalk region. To identify the interaction proteins for KIF5A, yeast two-hybrid screening was performed and a specific interaction with the ${\beta}$ subunit of heterotrimeric G proteins ($G{\beta}$) was found. $G{\beta}$ bound to the amino acid residues between 808 and 935 of KIF5A and to other KIF5 members in the yeast two-hybrid assay. The WD40 repeat motif of $G{\beta}$ was essential for interaction with KIF5A. In addition, these proteins showed specific interactions in the glutathione S-transferase (GST) pull-down assay. An antibody to KIF5s specifically co-immunoprecipitated KIF5s associated with heterotrimeric G proteins from mouse brain extracts. These results suggest that kinesin-I motor protein transports heteroterimeric G protein attachment vesicles along microtubules in the cell.

• Journal of Life Science
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• v.33 no.1
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• pp.1-7
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• 2023

Intracellular cargo transport is mediated by molecular motor proteins, such as kinesin and cytoplasmic dynein. Kinesins make up a large subfamily of molecular motors. Kinesin-1 is a plus-end-directed molecular motor protein that moves various cargoes, such as organelles, protein complexes, and mRNAs, along a microtubule track. It consists of the kinesin superfamily protein (KIF) 5A, 5B, and 5C (also called kinesin heavy chains) and kinesin light chains (KLCs). Kinesin-1 interacts with many different binding proteins through its carboxyl (C)-terminal region of KIF5s and KLCs, but their binding proteins have not yet been fully identified. In this study, a yeast two-hybrid assay was used to identify the proteins that interact with the KIF5A specific C-terminal region. The assay revealed an interaction between KIF5A and glutamate-rich 4 (ERICH4). ERICH4 bound to the KIF5A specific the C-terminal region but did not interact with the C-terminal region of KIF5B or KIF3A (a motor protein of kinesin-2). In addition, KIF5A did not interact with another isoform, ERICH1. Glutathione S-transferase (GST) pull-downs showed that KIF5A interacts with GST-ERICH4 and GST-ERICH4-amino (N)-terminal but not with GST-ERICH4-C or GST alone. When co-expressed in HEK-293T cells, ERICH4 co-localized with KIF5A and co-immunoprecipitated with KIF5A and KLC but not KIF3B. Together, our findings suggest that ERICH4 is capable of binding to KIF5A and that it may serve as an adaptor protein that links kinesin-1 with cargo.

• Journal of Life Science
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• v.22 no.12
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• pp.1608-1613
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• 2012

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 anti-body 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.

• Journal of Life Science
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• v.24 no.1
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• pp.14-19
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• 2014

Kinesin-1 consists of two heavy chains (KHCs), also called KIF5s, and two light chains (KLCs) that form a heterotetrameric complex. Here, we demonstrate the binding of a neuronal KHC, KIF5A, to the carboxyl (C)-terminal tail region of Fig4 (also known as Sac3), a phosphatase that removes the 5-phosphate from phosphatidylinositol-3,5-bisphosphate ($PtdIns(3,5)P_2$). Fig4 bound to the C-terminal region of KIF5A but not to other KHCs (KIF5B and KIF5C) and KLC1 in yeast two-hybrid assays. The interaction was further confirmed in a glutathione S-transferase pull-down assay and by co-immunoprecipitation. Anti-KIF5A antibody co-immunoprecipitated Fig4 with KIF5A from mouse brain extracts. These results suggest that kinesin-1 could transport the Fig4-associated protein complex or cargo in cells.