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Mol Path 2002;55:46-54 doi:10.1136/mp.55.1.46
  • Original Article

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

  1. D A Buckley,
  2. G Loughran,
  3. G Murphy,
  4. C Fennelly,
  5. R O'Connor
  1. Cell Biology Laboratory, Department of Biochemistry, Biosciences Research Institute, National University of Ireland, Cork, Ireland
  1. Correspondence to:
 Dr R O'Connor, Cell Biology Laboratory, Department of Biochemistry, Biosciences Research Institute, National University of Ireland, Cork, Ireland;
 r.oconnor{at}ucc.ie
  • Accepted 7 June 2001

Abstract

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.

Footnotes