Expression and role in glycolysis of human ADP-dependent glucokinase.
A novel murine enzyme, ADP-dependent glucokinase (ADPGK), has been shown to catalyse glucose phosphorylation using ADP as phosphoryl donor. The ancestral ADPGK gene appears to have been laterally transferred from Archaea early in metazoan evolution, but its biological role has not been established. Here, we undertake an initial investigation of the functional properties of human ADPGK in human tumour cell lines and specifically test the hypothesis that ADPGK might prime glycolysis using ADP under stress conditions such as hypoxia. Recombinant human ADPGK was confirmed to catalyse ADP-dependent glucose phosphorylation in vitro, with an apparent K (M) for glucose of 0.29 mM. Expression databases and western blotting of surgical samples demonstrated high expression in many human tissues, including tumours. Unlike hexokinase-2 (HK2), RNAi studies with exon arrays showed that ADPGK is not a transcriptional target of hypoxia inducible factor-1. Consistent with this, ADPGK protein was not upregulated by hypoxia or anoxia. Surprisingly, stable fivefold overexpression of ADPGK in H460 or HCT116 cells had no apparent effect on proliferation or glycolysis, and did not rescue clonogenicity or glycolysis when HK2 was suppressed by siRNA. Furthermore, suppression of ADPGK by siRNA did not cause detectable inhibition of glycolysis or cell killing by anoxia, although it did induce a statistically significant decrease in plating efficiency of H460 cells under aerobic conditions. Thus, human ADPGK catalyses ADP-dependent phosphorylation of glucose in vitro, but despite its high expression in human tumour cell lines it appears not to make a quantifiable contribution to glycolysis under the conditions evaluated.
Auckland Cancer Society Research Centre, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag, 92019 Auckland, New Zealand.
Susan
S
Richter
Jan P
JP
Richter
Sunali Y
SY
Mehta
Amanda M
AM
Gribble
Andrew J
AJ
Sutherland-Smith
Kathryn M
KM
Stowell
Cristin G
CG
Print
Ron S
RS
Ronimus
William R
WR
Wilson
D000244:Adenosine Diphosphate / Q000378:metabolism; D002384:Catalysis; D015687:Cell Hypoxia / Q000235:genetics; D049109:Cell Proliferation; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects / Q000235:genetics; D005941:Glucokinase / Q000235:genetics* / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism* / Q000494:pharmacology; D006019:Glycolysis; D045325:HCT116 Cells; D019073:HT29 Cells; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism; D019344:Lactic Acid / Q000378:metabolism; D010766:Phosphorylation; D034741:RNA, Small Interfering; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*
C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D019344:Lactic Acid; D000244:Adenosine Diphosphate; C420842:ADP-dependent glucokinase; D005941:Glucokinase; D005947:Glucose