title: Enzyme activity effects of N-terminal His-tag attached to catalytic sub-unit of phosphoinositide-3-kinase.
abstract: NTT (N-terminal tags) on the catalytic (p110) sub-unit of PI 3-K (phosphoinositol 3-kinase) have previously been shown to increase cell signalling and oncogenic transformation. Here we test the impact of an NT (N-terminal) His-tag on in vitro lipid and protein kinase activity of all class-1 PI 3-K isoforms and two representative oncogenic mutant forms (E545K and H1047R), in order to elucidate the mechanisms behind this elevated signalling and transformation observed in vivo. Our results show that an NT His-tag has no impact on lipid kinase activity as measured by enzyme titration, kinetics and inhibitor susceptibility. Conversely, the NT His-tag did result in a differential effect on protein kinase activity, further potentiating the elevated protein kinase activity of both the helical domain and catalytic domain oncogenic mutants with relation to p110 phosphorylation. All other isoforms also showed elevated p110 phosphorylation (although not statistically significant). We conclude that the previously reported increase in cell signalling and oncogenic-like transformation in response to p110 NTT is not mediated via an increase in the lipid kinase activity of PI 3-K, but may be mediated by increased p110 autophosphorylation and/or other, as yet unidentified, intracellular protein/protein interactions. We further observe that tagged recombinant protein is suitable for use in in vitro lipid kinase screens to identify PI 3-K inhibitors; however, we recommend that in vivo (including intracellular) experiments and investigations into the protein kinase activity of PI 3-K should be conducted with untagged constructs.
authors: James M J
authors: JM
authors: Dickson
authors: ‡Department of Molecular Medicine and Pathology, University of Auckland, Auckland, New Zealand.
authors: Woo-Jeong
authors: WJ
authors: Lee
authors: Peter R
authors: PR
authors: Shepherd
authors: Christina M
authors: CM
authors: Buchanan
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry; D019943:Amino Acid Substitution; D000730:Androstadienes / Q000737:chemistry; D000818:Animals; D002867:Chromones / Q000737:chemistry; D058543:Class Ia Phosphatidylinositol 3-Kinase / Q000737:chemistry* / Q000235:genetics; D057075:Enzyme Assays; D006639:Histidine / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D009025:Morpholines / Q000737:chemistry; D009842:Oligopeptides / Q000737:chemistry*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation; D011993:Recombinant Fusion Proteins / Q000737:chemistry*; D061987:Sf9 Cells; D018411:Spodoptera; D000077191:Wortmannin
chemical_list: D000730:Androstadienes; D002867:Chromones; C471213:His-His-His-His-His-His; D009025:Morpholines; D009842:Oligopeptides; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011993:Recombinant Fusion Proteins; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D006639:Histidine; D000255:Adenosine Triphosphate; D058543:Class Ia Phosphatidylinositol 3-Kinase; D000077191:Wortmannin
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