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.
Highlights
The class 1 phosphoinositide 3-kinases [PI phosphoinositol 3-kinase (3-K)] are heterodimers consisting of an 85 kDa regulatory/adapter subunit (p85) coupled to a 110 kDa catalytic subunit (p110) with both subunits possessing several isoforms
The class I PI 3-kinases are further subdivided into two subclasses: class Ia and class Ib; the class 1a PI 3-kinases (p110α, p110β and p110δ) signal downstream of tyrosine kinases, while the single class Ib PI 3-K (p110γ ) operates downstream of heterotrimeric GPCRs (G-protein-coupled receptors) [1,2]
A range of oncogenic mutations have been identified in PIK3CA (p110α) and PIK3R1 (p85α) [7,8,9,10,11,12] and these result in elevation of their lipid kinase activity [7,10,13] and protein kinase activity [13,14]
Summary
The class 1 phosphoinositide 3-kinases [PI 3-K (phosphoinositol 3-kinase)] are heterodimers consisting of an 85 kDa regulatory/adapter subunit (p85) coupled to a 110 kDa catalytic subunit (p110) with both subunits possessing several isoforms. Owing to their importance in cell metabolism and cancer, the class 1 PI 3-kinases and oncogenic mutants have become the subjects of intense research efforts focusing on the development of a wide range of small molecule drugs to inhibit the lipid kinase activity of PI 3-K (recently reviewed in [15]). We report that an NT His-tag has no effect on the Km or Vmax, and there is no impact on in-vitro lipid kinase assays, or on IC50 determinations for the reference compounds investigated It did result in a significant increase in the autophosphorylation of the catalytic subunit in oncogenic forms of p110α and elevation of autophosphorylation of all wt (wild-type) isoforms. Inhibitors Wortmannin and LY294002 were from Sigma-Aldrich; TGX-221 was from Symansis; PIK-75, A66 and AS252424 were synthesized in-house as previously described [23,24]
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