Phosphorylation of a Fes-related Protein in Response to Granulocyte-Macrophage Colony Stimulating Factor

Diana Linnekin,Sherry M Mou,Peter Greer,Dan L Longo,Douglas K Ferris

doi:10.1074/jbc.270.9.4950

Abstract

Previous work has suggested that a 97-kDa protein (p97) is involved in the signal transduction pathway of granulocyte-macrophage colony stimulating factor (GM-CSF) as well as interleukin 3, erythropoietin, and interleukin 2. We have examined the relationship of p97 to the protein tyrosine kinase Fes in the GM-CSF signal transduction pathway in erythroid and myeloid cell lines. GM-CSF stimulation of three different cell lines induced tyrosine phosphorylation of p97 as well as a number of other phosphotyrosylproteins. Although each cell line expressed the proto-oncogene product Fes, antisera specific for Fes did not recognize p97 in immunoblotting experiments. Furthermore, immunodepletion of Fes did not reduce the amount of p97 in GM-CSF-treated cells. Two-dimensional gel electrophoresis demonstrated that p97 and Fes have similar charge to mass ratios, and limited proteolytic mapping of p97 and Fes suggested that these proteins may be related but are not identical. Our studies demonstrate that p97 is not Fes but is probably a Fes-related protein.

Highlights

GM-CSF1 stimulates proliferation and differentiation of myeloid progenitor cells as well as activation ofneutrophils [1, 2]
Recombinant GM-CSF was obtained from the Biological Response Modifiers Program (Frederick Cancer Research and Development Center, Frederick, MD) and was produced by Peprotech
Two factor-dependent human cell lines widely used for study of the GM-CSF signal transduction pathway are Mo7e and TF-1 cells

Summary

Introduction

GM-CSF1 stimulates proliferation and differentiation of myeloid progenitor cells as well as activation ofneutrophils [1, 2]. Recent work has suggested that the rapid increases in protein tyrosine phosphorylation stimulated by GM-CSF may be mediated through activation of multiple tyrosine kinases. A number of Src-like tyrosine kinases (Hck, Lyn, and Yes), as well as JAK2, are stimulated in response to GM-CSF [5,6,7,8,9]. The work of Hanazono and co-workers [10, 11] has suggested that Fes is activated in response to GM-CSF as well as IL-3 and erythropoietin. A number of laboratories have reported a phosphotyrosylprotein similar in size to Fes (90-100 kDa) in GM-CSF-, IL-3-, or erythropoietin-stimulated cells [12,13,14,15]. The present study addresses whether p97 is the proto-oncogene product Fes or potentially a Fes-related protein

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Results