Upregulation of dendritic arborization by N-acetyl-D-glucosamine kinase is not dependent on its kinase activity.
N-acetylglucosamine kinase (GlcNAc kinase or NAGK; EC 2.7.1.59) is highly expressed and plays a critical role in the development of dendrites in brain neurons. In this study, the authors conducted structure-function analysis to verify the previously proposed 3D model structure of GlcNAc/ ATP-bound NAGK. Three point NAGK mutants with different substrate binding capacities and reaction velocities were produced. Wild-type (WT) NAGK showed strong substrate preference for GlcNAc. Conversion of Cys143, which does not make direct hydrogen bonds with GlcNAc, to Ser (i.e., C143S) had the least affect on the enzymatic activity of NAGK. Conversion of Asn36, which plays a role in domain closure by making a hydrogen bond with GlcNAc, to Ala (i.e., N36A) mildly reduced NAGK enzyme activity. Conversion of Asp107, which makes hydrogen bonds with GlcNAc and would act as a proton acceptor during nucleophilic attack on the γ-phosphate of ATP, to Ala (i.e., D107A), caused a total loss in enzyme activity. The overexpression of EGFP-tagged WT or any of the mutant NAGKs in rat hippocampal neurons (DIV 5-9) increased dendritic architectural complexity. Finally, the overexpression of the small, but not of the large, domain of NAGK resulted in dendrite degeneration. Our data show the effect of structure on the functional aspects of NAGK, and in particular, that the small domain of NAGK, and not its NAGK kinase activity, plays a critical role in the upregulation of dendritogenesis.
Department of Anatomy, Dongguk University College of Medicine, Gyeongju 780-714, Korea.
HyunSook
H
Lee
Departmet of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE, USA.
Samikshan
S
Dutta
Dongguk Medical Institute, Dongguk University College of Medicine, Gyeongju 780-714, Korea.
Il Soo
IS
Moon
D000117:Acetylglucosamine / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D048708:Cell Growth Processes / Q000235:genetics; D002478:Cells, Cultured; D003712:Dendrites / Q000502:physiology*; D006624:Hippocampus / Q000166:cytology*; D051379:Mice; D009154:Mutation / Q000235:genetics; D009410:Nerve Degeneration / Q000235:genetics; D055495:Neurogenesis / Q000235:genetics; D009474:Neurons / Q000502:physiology*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D011487:Protein Conformation; D015202:Protein Engineering; D017434:Protein Structure, Tertiary / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D013329:Structure-Activity Relationship; D013379:Substrate Specificity / Q000235:genetics
D000255:Adenosine Triphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); C018716:N-acetylglucosamine kinase; D000117:Acetylglucosamine