Identification of an IGF-1R kinase regulatory phosphatase using the fission yeast Schizosaccharomyces pombe and a GFP tagged IGF-1R in mammalian cells.
OBJECTIVE
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.
Buckley
D A
DA
Cell Biology Laboratory, Department of Biochemistry, Biosciences Research Institute, National University of Ireland, Cork, Ireland.
Loughran
G
G
Murphy
G
G
Fennelly
C
C
O'Connor
R
R
D000818:Animals; D019556:COS Cells; D002460:Cell Line; D002478:Cells, Cultured; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D049452:Green Fluorescent Proteins; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D008164:Luminescent Proteins; D051379:Mice; D010766:Phosphorylation; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases / Q000494:pharmacology; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D017526:Receptor, IGF Type 1 / Q000235:genetics / Q000378:metabolism*; D011972:Receptor, Insulin / Q000378:metabolism; D012568:Schizosaccharomyces / Q000201:enzymology*; D014162:Transfection
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D007334:Insulin-Like Growth Factor I; C410467:insulin receptor tyrosine kinase; D011505:Protein-Tyrosine Kinases; D017526:Receptor, IGF Type 1; D011972:Receptor, Insulin; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases; C516609:Ptpn1 protein, mouse