N-acetyl-D-glucosamine kinase interacts with dynein light-chain roadblock type 1 at Golgi outposts in neuronal dendritic branch points

Abstract

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.

N-Acetyl-D-Glucosamine Kinase Promotes the Axonal Growth of Developing Neurons

“…We previously showed that NAGK interacts with DYNLRB1 and NAGK-dynein-Golgi tripartite interactions occur in somata and dendrites, especially in dendritic branch points in stage 4 neurons, and that the NAGK-DYNLRB1 interaction is critical during dendritogenesis ( Islam et al, 2015 ). To investigate NAGK-dynein-Golgi colocalization in axons, we conducted a proximity ligation assay (PLA) using anti-NAGK and anti-TGN38 antibodies followed by ICC with anti-tubulin antibody in stage 3 hippocampal neurons ( Fig.…”

“…An 18-amino acid peptide termed ‘DYNLRB1 (59–76)’ and a 23-amino acid peptide termed ‘DYNLRB1 (74–96)’, which were both derived from the NAGK-binding region of DYNLRB1, were custom made by Anygen (Korea) at a purity of 98%. Peptide transfection was performed in neurons after 24 h of plating with a Chariot protein transfection kit (Active Motif, California) using a modified version of the manufacturer’s instructions as previously described ( Islam et al, 2015 ). After incubation for 48 h, neurons were fixed and stained using a β-galactosidase staining kit (Active Motif).…”

“…Generic in situ PLA was conducted using a Duolink In Situ kit (Olink Bioscience, Sweden) using a modified version of the manufacturer’s instructions and as described by Islam et al (2015) . Briefly, after incubating fixed cells with primary antibodies overnight at 4°C, secondary antibodies conjugated with oligonucleotides, PLA probe anti-mouse MINUS and PLA probe anti-rabbit PLUS, were added and incubated for 2 h at 37°C in a humidity chamber.…”

“… Sharif et al (2015) described the expression of NAGK in different subnuclear compartments, such as, speckles and paraspeckles, and on the outer nuclear membrane. Our lab also reported that NAGK interacts with several intracellular proteins, including, dynein light chain roadblock type-1 (DYNLRB1), small nuclear ribonucleoprotein-associated protein N (snRNPN), p54NRB, and a general transcription factor IIH polypeptide 5 (GTF2H5) ( Islam et al, 2015 ; Sharif et al, 2015 ). We also reported the functional role played by the NAGK-DYNLRB1 interaction during dendrite development, which is noteworthy because it is associated with somatic Golgi stacks and dendritic Golgi outposts ( Islam et al, 2015 ).…”

DYNLRB1 is Essential for Dynein Mediated Transport and Neuronal Survival

“…DYNLRB protein sequence is highly conserved among different species; with two isoforms, DYNLRB1 and DYNLRB2, sharing 98% sequence similarity in mammals [13]. Mammalian DYNLRB1 has been studied mainly as an adaptor linking specific modules to the dynein complex, including SMAD2 complexes activated by TGFβ receptors [11,[14][15][16][17][18] and Rab6 or N-acetyl-Dglucosamine kinase (NAGK) for interactions with the Golgi compartment [19][20][21].…”