• BMB Reports
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• v.43 no.7
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• pp.485-490
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• 2010

Neurotrophins regulate many aspects of neuronal function through activation of the high affinity Trk receptors. Shc family proteins are implicated in the coupling of RTK to the Ras/mitogen-activated protein kinase signaling cascade. Here we report that the fourth Shc family member, ShcD, associates with TrkB receptor and regulates BDNF-induced MAPK activation. Yeast two-hybrid assay and Co-IP experiments demonstrate ShcD interacts with TrkB in a kinase-activity-dependent manner. Confocal analysis shows ShcD cololizes well with TrkB in transfected 293T cells. Subsequent mapping experiments and mutational analysis indicate that both PTB and SH2 domains are capable of binding to TrkB and PTB domain binds to TrkB NPQY motif. Furthermore, ShcD is involved in BDNF-induced MAPK activation. In summary, we demonstrate that ShcD is a substrate of TrkB and mediates TrkB downstream signaling pathway.

• Proceedings of the Korean Biophysical Society Conference
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• 2003.06a
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• pp.43-43
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• 2003

Large-conductance $Ca^{2+}$-actived $K^{+}$ channels ($BK_{Ca}$ channels) play a key role in setting the pace of contractile activity in muscle and are involved in the regulation of neurotransmitter release in neuron. $BK_{Ca}$ channels are activated by depolarizing membrane potential and the elevated level of intracellular calcium. Using yeast-two hybrid assay, we have identified a novel protein interacting with the cytosolic carboxyl terminus of rSlo, the brain isoform of rat large-conductance $Ca^{2+}$-activated $K^{+}$ channel $\alpha$-subunit. The novel gene encodes 51 kDa protein and is named as SIRK(rSlo-interacting RGS-like protein). SIRK is expressed in various tissues and localized in the cytosolic and the membrane fraction. Biochemical and immunological studies indicated that SIRK physically interacted with the cytosolic region of rSlo. To investigate whether SIRK can modulate the activity of rSlo, GFP-fused SIRK and rSlo were transiently transfected into COS-7 cells and the effects of SIRK was studied using electrophysiological means. We concluded that the overexpression of SIRK alters the surface expression of rSlo channel with only a limited effect on the biophysical characteristics of the channel.the channel.

• Journal of Life Science
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• v.24 no.8
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• pp.820-826
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• 2014

The localization to specific subcellular sites and the regulation of cell surface receptors and channels are crucial for proper functioning. Postsynaptic density-95/Disks large/Zonula occludens-1 (PDZ)-domain is involved in recognition of and interaction between various proteins, by which the localization and the regulation are mediated. Multi-PDZ domain protein 1 (MUPP1) contains 13 PDZ domains. MUPP1 serves a scaffolding function for structure proteins and signaling proteins, but the mechanism how MUPP1 is stabilized and signalized has not yet been elucidated. We used the yeast two-hybrid system to identify proteins that interact with PDZ domains of MUPP1. We found an interaction between MUPP1 and Parkin. Parkin is an E3 ubiquitin ligase. Loss-of-function mutations of Parkin gene are known to cause an autosomal recessive juvenile parkinsonism. Parkin bound to the $12^{th}$ PDZ domain, but not to other PDZ domains of MUPP1. The C-terminal end of Parkin has a type II PDZ-association motif, which was essential for the interaction with MUPP1 in the yeast two-hybrid assay. When co-expressed in HEK-293T cells, Parkin co-localized with MUPP1. When co-expressed with ubiquitin in HEK-293T cells, MUPP1 has been strongly ubiquitinated by Parkin. These findings collectively suggest that MUPP1 is a novel substrate of Parkin and its function or stability could be modulated by Parkin-mediated ubiquitination.

• The Plant Pathology Journal
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• v.36 no.4
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• pp.323-334
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• 2020

Lysin motif (LysM) proteins are reported to be necessary for the virulence and immune response suppression in many herbaceous plant pathogens, while far less is documented in woody plant pathogens. In this study, we preliminarily characterized the molecular function of a LysM protein LtLysM1 in woody plant pathogen Lasiodiplodia theobromae. Transcriptional profiles revealed that LtLysM1 is highly expressed at infectious stages, especially at 36 and 48 hours post inoculation. Amino acid sequence analyses revealed that LtLysM1 was a putative glycoprotein with 10 predicted N-glycosylation sites and one LysM domain. Pathogenicity tests showed that overexpressed transformants of LtLysM1 displayed increased virulence on grapevine shoots in comparison with that of wild type CSS-01s, and RNAi transformants of LtLysM1 exhibited significantly decreased lesion length when compared with that of wild type CSS-01s. Moreover, LtLysM1 was confirmed to be a secreted protein by a yeast signal peptide trap assay. Transient expression in Nicotiana benthamiana together with protein immunoblotting confirmed that LtLysM1 was an N-glycosylated protein. In contrast to previously reported LysM protein Slp1 and OsCEBiP, LtLysM1 molecule did not interact with itself based on yeast two hybrid and co-immunoprecipitation assays. These results indicate that LtLysM1 is a secreted protein and functions as a critical virulence factor during the disease symptom development in woody plants.

• 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.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.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.