Identification of an IGF-1R kinase regulatory phosphatase using the fission yeast Schizosaccharomyces pombe and a GFP tagged IGF-1R in mammalian cells.

AIMS To study the regulation of type 1 insulin like growth factor receptor (IGF-1R) tyrosine kinase activity using the fission yeast Schizosaccharomyces pombe and a green fluorescent protein (GFP) tagged, full length IGF-1R. METHODS The beta chain of the IGF-1R (betawt) was expressed under inducible conditions in the fission yeast S. pombe. Western blot analysis with antiphosphotyrosine antibodies was used to assess the kinase activity of betawt. A GFP tagged IGF-1R (GFP-IGF-1R) was constructed to study the tyrosine kinase activity of the full length IGF-1R. The signalling capabilities of GFP-IGF-1R in response to IGF-1 stimulation were investigated in transiently transfected fibroblasts. Immunofluorescent staining for cellular phosphotyrosine content was used to assess the localisation and tyrosine kinase activity of GFP-IGF-1R. RESULTS The betawt protein displayed functional tyrosine kinase activity in S pombe and phosphorylated endogenous yeast proteins. In response to IGF-1 stimulation, the GFP-IGF-1R became autophosphorylated and also activated the phosphatidylinositol 3-kinase and mitogen activated protein kinase pathways. Tyrosine phosphorylation and kinase activity of the GFP-IGF-1R could be visualised by immunofluorescence with antiphosphotyrosine antibodies. Coexpression of a mammalian tyrosine phosphatase PTP1B with betawt completely inhibited this tyrosine kinase activity in yeast and also reduced the tyrosine phosphorylation in COS cells transfected with the GFP-IGF-1R. CONCLUSIONS Schizosaccharomyces pombe can be used to analyse the tyrosine kinase activity of the IGF-1R beta chain and its regulation by tyrosine phosphatases. In addition, the regulation of IGF-1R tyrosine kinase activity can be studied using a GFP tagged IGF-1R. Using both of these methods, IGF-1R kinase activity was shown to be inhibited by the protein tyrosine phosphatase, PTP1B.