RT Journal Article
SR Electronic
T1 Identification of an IGF-1R kinase regulatory phosphatase using the fission yeast <em>Schizosaccharomyces pombe</em> and a GFP tagged IGF-1R in mammalian cells
JF Molecular Pathology
JO Mol Pathol
FD BMJ Publishing Group Ltd and Association of Clinical Pathologists
SP 46
OP 54
DO 10.1136/mp.55.1.46
VO 55
IS 1
A1 D A Buckley
A1 G Loughran
A1 G Murphy
A1 C Fennelly
A1 R O'Connor
YR 2002
UL http://mp.bmj.com/content/55/1/46.abstract
AB 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 β chain of the IGF-1R (βwt) 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 βwt. 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 βwt 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 βwt 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 β 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.