Abstract
We have examined the signal transduction pathways of a number of cytokines that interact with receptors that are members of the hematopoietin receptor superfamily. A 97-kDa protein was phosphorylated on tyrosine in response to stimulation of appropriate target cells with interleukin (IL)-2, IL-3, granulocyte-macrophage colony-stimulating factor (CSF), granulocyte-CSF, or erythropoietin. These data suggest that a 97-kDa phosphotyrosylprotein represents a point of convergence for signal transduction by a number of growth factor receptors that do not have homology with any known protein tyrosine kinase. To address the possibility that p97 may represent a tyrosine kinase involved in multiple signal transduction pathways, we tested the capacity of this protein to bind a tyrosine kinase substrate or ATP. Indeed, a 97-kDa phosphotyrosylprotein purified from IL-2-stimulated lymphoid cells as well as granulocyte-macrophage-CSF-stimulated myeloid cells bound to a polymer of glutamic acid and tyrosine which is a tyrosine kinase substrate. Further, a 97-kDa phosphotyrosylprotein present in both lineages also bound 8-azido-ATP. These data indicate that a 97-kDa phosphotyrosylprotein with properties consistent with those of a protein tyrosine kinase is involved in the signal transduction pathways of certain members of the newly identified hematopoietin receptor superfamily and may represent an early point of convergence in the stimulus-response coupling of multiple cytokine receptors.
Highlights
We have examined thesignal transduction pathways 4, IL- 6, IL-7, GM-CSF, G-CSF, and the p chain of the IL-2 of a number of cytokines that interactwith receptors receptor has demonstrated that these receptors comprise a that are members of the hematopoietin receptor supseurp-erfamily recently termed the hematopoietin receptor SU
To determine if one or more phosphotyrosylproteins represented points of convergencebetween these growth factors, we chose to examine both lymphoid and myeloid cell lines for changes in tyrosine phosphorylation in response to five cytokines whose receptors are within the hematopoietin receptor superfamily
To further characterize the 97-kDa phosphotyrosylprotein observed in cells stimulated with multiple cytokines, we utilized high resolution two-dimensional gel electrophoresis to compare the migration of the 97-kDa phosphotyrosylprotein in GM-CSF-stimulated myeloid cells with that of IL-2-stimulated lymphoidcells after isoelectric focusing.Cellswere radiolabeled with [32P]orthophosphate,stimulated with appropriate growth factor, lysed, and subjected to immunoprecipitation with 1G2, a monoclonal antibody directed against phosphotyrosine
Summary
Bldg. 560, Rm. 21-89A3F,rederick, MD 21702-1201. Tel.: 301- Cell Lines and Growth Factors-The AML-193 cell line war, cul-. The immunoprecipitates were washed six times with 10 mM Hepes, pH 7.4, 30 mM NaC1, 1mM EDTA, 0.05% Triton X-100, and 1%glycerol and eluted with 50 p1 of a 50 mM phenylphosphate solution in 10 mM Hepes, pH 7.4,30 mM NaC1,l mM EDTA, 6 mM 2-mercaptoethanol, 1% glycerol These purified phosphotyrosylproteins were diluted to 1ml in extraction buffer and precleared through a 60-min incubation with 50 pl of a 20% solution (v/v) of poly(G1u)-Sepharose (Sigma) a t 4 "C while rotating. After washing six times with extraction buffer without BSA, protein was removed from the Sepharose with SDS sample buffer, resolved using one-dimensional SDSPAGE and visualized using autoradiography. The immunoprecipitates were incubated for 5 min with ultraviolet irradiation, washed with extraction buffer without BSA, and removed from the Sepharose with SDS sample buffer; proteins were resolved using SDS-PAGE
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
