N-Acetyl-d-Glucosamine Kinase Interacts with NudC and Lis1 in Dynein Motor Complex and Promotes Cell Migration

Abstract

Recently, we showed that N-acetylglucosamine kinase (NAGK), an enzyme of amino sugar metabolism, interacts with dynein light chain roadblock type 1 (DYNLRB1) and promotes the functions of dynein motor. Here, we report that NAGK interacts with nuclear distribution protein C (NudC) and lissencephaly 1 (Lis1) in the dynein complex. Yeast two-hybrid assays, pull-down assays, immunocytochemistry, and proximity ligation assays revealed NAGK–NudC–Lis1–dynein complexes around nuclei, at the leading poles of migrating HEK293T cells, and at the tips of migratory processes of cultured rat neuroblast cells. The exogenous expression of red fluorescent protein (RFP)-tagged NAGK accelerated HEK293T cell migration during in vitro wound-healing assays and of neurons during in vitro neurosphere migration and in utero electroporation assays, whereas NAGK knockdown by short hairpin RNA (shRNA) delayed migration. Finally, a small NAGK peptide derived from the NudC interacting domain in in silico molecular docking analysis retarded the migrations of HEK293T and SH-SY5Y cells. These data indicate a functional interaction between NAGK and dynein–NudC–Lis1 complex at the nuclear envelope is required for the regulation of cell migration.

Computational Insights into the Deleterious Impacts of Missense Variants on N-Acetyl-d-glucosamine Kinase Structure and Function

“…Therefore, SNPs in the conserved area are likely to be more pathogenic than those present in the variable region, disrupting structural stability, protein–protein interaction [ 24 ], and catalytic activity [ 25 , 26 ]. Since the NAGK non-canonical function mostly depends on diverse biomolecular interactions [ 1 , 6 , 7 , 12 , 13 , 14 ], we analyze the degree of amino acid conservation of NAGK to investigate further the possible impacts of high-risk SNPs based on evolutionary information. The ConSurf web server was used to predict the evolutionary conservation profile of NAGK, which is shown in Figure S1 ( Supplementary File 2 ).…”

“…Our previous report evidenced the physical interaction of NAGK in the dynein complex, where it interacts with DYNLRB1 by its small domain and promotes dynein-mediated functions [ 11 , 13 , 14 , 34 ]. To analyze the effect of identified NAGK variants in dynein association ( Figure 8 A), we analyze the binding energy between NAGK and DYNLRB1in both wild-type and variants ( Figure 8 B).…”

“…On the other hand, G11R and G120E also showed less binding energy than the wild-type, which is −53.97 kcal/mol and −47.36 kcal/mol, respectively. Figure 8 C represents the deleterious effect of variants in protein–protein interaction, which showed that variants reduced the total binding free energy contributed by hot-spots, especially K 59 AGVDPLVPLR 69 [ 13 , 14 ]. In wild-type, the R69 residue showed total contributing binding energy of −5.58 kcal/mol, which, however, reduced in G11R, G32R, G120E, and A156D to −2.17, 0.34, 0.72, and −1.86 kcal/mol, respectively ( Figure 8 C).…”

“…It has been found that NAGK is involved in different stages of neuronal development, including dendritic arborization and axonal outgrowth, and this effect was unchanged even in the overexpressing kinase-deficient mutant NAGKs [ 11 ]. Mechanistic studies revealed the structural roles of NAGK, where the protein interacts with dynein light chain roadblock 1 (DYNLRB1) in dynein complex and promotes dynein functions in cellular growth [ 1 ], dendrites [ 6 , 7 ] and axon development [ 12 ], neuronal migration [ 13 ], and selective autophagy [ 14 ]. These observations suggest both enzymatic and non-enzymatic importance of NAGK in neuron and brain development.…”

“…Single nucleotide polymorphisms (SNPs) are the most predominant types of genetic variation in humans, linked with various complex genetic and Mendelian disorders [ 12 , 13 ], and commonly occur in both coding and non-coding parts of a genomic region. The nsSNPs, which occurred in the coding region and caused amino acid substitutions, are the most responsible for phenotypic change and are associated with many genetic diseases due to their effect on protein structure, charge, solubility, stability, and function [ 15 , 16 ].…”