A novel cytosolic dual specificity phosphatase, interacting with glucokinase, increases glucose phosphorylation rate.
A novel protein was cloned from a rat liver cDNA library by interaction with the liver glucokinase. This protein contained 339 residues and possessed a canonical consensus sequence for a dual specificity phosphatase. The recombinant protein was able to dephosphorylate phosphotyrosyl and phosphoseryl/threonyl substrates. We called this protein the glucokinase-associated phosphatase (GKAP). The GKAP partially dephosphorylated the recombinant glucokinase previously phosphorylated, in vitro, by protein kinase A. The GKAP fused with green fluorescent protein was located in the cytosol, where glucokinase phosphorylates glucose, and not in the nucleus where the glucokinase is retained inactive by the glucokinase regulatory protein. More importantly, the GKAP accelerated the glucokinase activity in a dose-dependent manner and with a stoichiometry compatible with a physiological mechanism. This strongly suggested that the interaction between GKAP and glucokinase had a functional significance. The cloning of this novel protein with a dual specificity phosphatase activity allows the description of a possible new regulatory step in controlling the glycolysis flux.
Muñoz-Alonso
M J
MJ
CNRS UPR 1524, 9, rue Jules Hetzel, 92190 Meudon, INSERM U505, 15 rue de l'Ecole de Médecine, 75006 Paris, France.
Guillemain
G
G
Kassis
N
N
Girard
J
J
Burnol
A F
AF
Leturque
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002467:Cell Nucleus / Q000201:enzymology; D003001:Cloning, Molecular; D003600:Cytosol / Q000201:enzymology; D018076:DNA, Complementary / Q000235:genetics; D054637:Dual-Specificity Phosphatases; D005941:Glucokinase / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism*; D005982:Glutathione Transferase / Q000378:metabolism; D049452:Green Fluorescent Proteins; D007700:Kinetics; D008164:Luminescent Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D010749:Phosphoprotein Phosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D018076:DNA, Complementary; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D005982:Glutathione Transferase; D005941:Glucokinase; D010749:Phosphoprotein Phosphatases; C489855:dual specificity phosphatase 12; D054637:Dual-Specificity Phosphatases; D005947:Glucose