• 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.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.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.