N-acetyl-D-glucosamine kinase interacts with dynein light-chain roadblock type 1 at Golgi outposts in neuronal dendritic branch points.
N-acetylglucosamine kinase (GlcNAc kinase or NAGK) is a ubiquitously expressed enzyme in mammalian cells. Recent studies have shown that NAGK has an essential structural, non-enzymatic role in the upregulation of dendritogenesis. In this study, we conducted yeast two-hybrid screening to search for NAGK-binding proteins and found a specific interaction between NAGK and dynein light-chain roadblock type 1 (DYNLRB1). Immunocytochemistry (ICC) on hippocampal neurons using antibodies against NAGK and DYNLRB1 or dynein heavy chain showed some colocalization, which was increased by treating the live cells with a crosslinker. A proximity ligation assay (PLA) of NAGK-dynein followed by tubulin ICC showed the localization of PLA signals on microtubule fibers at dendritic branch points. NAGK-dynein PLA combined with Golgi ICC showed the colocalization of PLA signals with somal Golgi facing the apical dendrite and with Golgi outposts in dendritic branch points and distensions. NAGK-Golgi PLA followed by tubulin or DYNLRB1 ICC showed that PLA signals colocalize with DYNLRB1 at dendritic branch points and at somal Golgi, indicating a tripartite interaction between NAGK, dynein and Golgi. Finally, the ectopic introduction of a small peptide derived from the C-terminal amino acids 74-96 of DYNLRB1 resulted in the stunting of hippocampal neuron dendrites in culture. Our data indicate that the NAGK-dynein-Golgi tripartite interaction at dendritic branch points functions to regulate dendritic growth and/or branching.
Department of Anatomy, Dongguk Medical Institute, College of Medicine Dongguk University, Gyeongju, Republic of Korea.
Md Ariful
MA
Islam
Department of Anatomy, Dongguk Medical Institute, College of Medicine Dongguk University, Gyeongju, Republic of Korea.
Syeda Ridita
SR
Sharif
Neuroscience Section, Dongguk Medical Institute, College of Medicine Dongguk University, Gyeongju, Republic of Korea.
HyunSook
H
Lee
Departments of Biochemistry, College of Medicine Inje University, Busan, Republic of Korea.
Dae-Hyun
DH
Seog
1] Department of Anatomy, Dongguk Medical Institute, College of Medicine Dongguk University, Gyeongju, Republic of Korea [2] Neuroscience Section, Dongguk Medical Institute, College of Medicine Dongguk University, Gyeongju, Republic of Korea.
Il Soo
IS
Moon
D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D056445:Cytoplasmic Dyneins / Q000737:chemistry / Q000378:metabolism*; D003712:Dendrites / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D057809:HEK293 Cells; D006624:Hippocampus; D006801:Humans; D008969:Molecular Sequence Data; D009474:Neurons / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D060066:Protein Interaction Maps; D017207:Rats, Sprague-Dawley; D014404:Tubulin
D014404:Tubulin; D017853:Phosphotransferases (Alcohol Group Acceptor); C018716:N-acetylglucosamine kinase; D056445:Cytoplasmic Dyneins; C000604283:DYNLRB1 protein, rat