Erythropoietin-induced Recruitment of Shc via a Receptor Phosphotyrosine-independent, Jak2-associated Pathway

Based on the recently implicated role of Shc as a signaling effector for type I cytokine receptors, factors which mediate the recruitment and phosphorylation of Shc in the erythropoietin receptor (EPOR) system have been studied. FDC-P1 cells stably expressing the wild type murine EPOR supported the EPO-induced association of Shc with Jak2 and its rapid tyrosine phosphorylation. However, this did not depend upon the presence of phosphotyrosine sites within the EPOR and was mediated by a mitogenically deficient receptor form (EPOR329) lacking cytoplasmic tyrosine residues. This was shown both by Western blotting of Shc and Jak2 co-immunoprecipitates and through the development of an in vitro assay for cytokine-induced Shc phosphorylation. The direct association of Shc with Jak2 also was observed and was shown to depend upon EPO-exposure and the SH2 subdomain of Shc. Together, these studies indicate that Jak2, in part, may mediate the EPO-induced phosphorylation of Shc. Based on the recently implicated role of Shc as a signaling effector for type I cytokine receptors, factors which mediate the recruitment and phosphorylation of Shc in the erythropoietin receptor (EPOR) system have been studied. FDC-P1 cells stably expressing the wild type murine EPOR supported the EPO-induced association of Shc with Jak2 and its rapid tyrosine phosphorylation. However, this did not depend upon the presence of phosphotyrosine sites within the EPOR and was mediated by a mitogenically deficient receptor form (EPOR329) lacking cytoplasmic tyrosine residues. This was shown both by Western blotting of Shc and Jak2 co-immunoprecipitates and through the development of an in vitro assay for cytokine-induced Shc phosphorylation. The direct association of Shc with Jak2 also was observed and was shown to depend upon EPO-exposure and the SH2 subdomain of Shc. Together, these studies indicate that Jak2, in part, may mediate the EPO-induced phosphorylation of Shc. The development of hematopoietic cells depends upon exposure to a diverse set of cytokines, many of which activate receptors of the type I superfamily (1Bazan J.F. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6934-6938Crossref PubMed Scopus (1869) Google Scholar, 2Miyajima A. Kitamaura T. Harada N. Yokota T. Arai K.I. Annu. Rev. Immunol. 1992; 10: 295-331Crossref PubMed Scopus (537) Google Scholar). These receptors include those for EPO, 1The abbreviations used are: EPOerythropoietinEPORerythropoietin receptorG-CSFgranulocyte colony-stimulating factorGM-CSFgranulocyte-macrophage colony-stimulating factorILinterleukinLIFleukemia inhibitory factorPAGEpolyacrylamide gel electrophoresisGSTglutathione S-transferase.1The abbreviations used are: EPOerythropoietinEPORerythropoietin receptorG-CSFgranulocyte colony-stimulating factorGM-CSFgranulocyte-macrophage colony-stimulating factorILinterleukinLIFleukemia inhibitory factorPAGEpolyacrylamide gel electrophoresisGSTglutathione S-transferase. IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, GM-CSF, G-CSF, and LIF and are related based on conserved extracellular WSXWS motifs and cysteine residues, homologous cytoplasmic subdomains within certain receptor subunits, and the notable absence of recognizable enzymatic activities (1Bazan J.F. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6934-6938Crossref PubMed Scopus (1869) Google Scholar, 2Miyajima A. Kitamaura T. Harada N. Yokota T. Arai K.I. Annu. Rev. Immunol. 1992; 10: 295-331Crossref PubMed Scopus (537) Google Scholar, 3Murakami M. Narazaki M. Yamata H. Yasukawa K. Hamaguchi M. Taga T. Kishimoto T. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 11349-11353Crossref PubMed Scopus (486) Google Scholar, 4Narazaki M. Witthuhn B.A. Yoshida K. Silvennoinen O. Yasukawa K. Ihle J.N. Kishimoto T. Taga T. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2285-2289Crossref PubMed Scopus (251) Google Scholar, 5Quelle Proc. Natl. Acad. Sci. U. S. A. 1991; 88: PubMed Scopus Google Scholar, N. H. 1994; PubMed Google Scholar). This studies of However, recently have for type I tyrosine receptors with the of S. N. Taga T. Kishimoto T. PubMed Scopus Google Scholar, M. M. S. 1994; PubMed Scopus Google Scholar, 1994; PubMed Scopus Google and a of the type I cytokine Jak2, B.A. Silvennoinen O. T. O. Ihle J.N. PubMed Scopus Google Scholar, J.N. Witthuhn B.A. K. Silvennoinen O. Sci. 1994; PubMed Scopus Google Scholar, T. PubMed Scopus Google Scholar). tyrosine receptors A. 1992; PubMed Scopus Google and certain type I receptors also have been shown to to activate and to mediate the tyrosine phosphorylation of and Shc M. K. 1992; PubMed Google Scholar, M. N. 1991; PubMed Google Scholar, 1994; PubMed Scopus Google Scholar, A. 1994; PubMed Google Scholar, 1994; PubMed Scopus Google Scholar, H. N. PubMed Google Scholar, O. N. Ihle J.N. N. 1994; PubMed Google have been tyrosine receptors and the recruitment of a PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, PubMed Scopus Google A. 1992; PubMed Scopus Google Scholar, K. M. A. T. Proc. Natl. Acad. Sci. U. S. A. 1992; PubMed Scopus Google and Shc T. 1992; PubMed Scopus Google Scholar). a SH2 receptor Shc a SH2 and both as as phosphotyrosine sites within certain tyrosine receptors and A. A. PubMed Google Scholar, T. K. M. 1994; PubMed Google Scholar, K. S. K. T. 1994; PubMed Google Scholar). for Shc in and also have been factors Shc phosphorylation and its association with A. A. PubMed Google Scholar, T. K. M. 1994; PubMed Google Scholar, K. S. K. T. 1994; PubMed Google Scholar, S. PubMed Scopus Google in by of Shc A. S. T. 1992; PubMed Scopus Google and by and Shc phosphorylation A. T. Proc. Natl. Acad. Sci. U. S. A. 1992; PubMed Scopus Google Scholar, 1994; PubMed Scopus Google Scholar). that not Shc may to for Shc are and Shc may Shc phosphorylation been shown to by IL-2, IL-3, IL-5, GM-CSF, and 1994; PubMed Google Scholar, PubMed Google Scholar, N. 1994; PubMed Google Scholar, M. H. T. 1994; PubMed Scopus Google Scholar). of Shc by type I cytokine receptors, have the recruitment and phosphorylation of this effector as mediated by receptor indicate that Shc recruitment not depend upon phosphotyrosine receptor sites and sites within Jak2 an also for a role for Jak2 in EPO-induced Shc Proc. Natl. Acad. Sci. U. S. A. 1991; 88: PubMed Scopus Google Scholar, N. H. 1994; PubMed Google in and to and Jak2 to to of in blotting and phosphorylation Shc and Jak2 to and with was with and was by with Western cells for in for with and with and in of Shc and Jak2 in in and to and with and Jak2 by in in vitro Jak2 was with and and with by by and and in of for with of and with of A. 1992; PubMed Scopus Google Scholar, A. S. T. 1992; PubMed Scopus Google of and by and of with a SH2 T. 1992; PubMed Scopus Google was by and and and was in and of to was as for and Jak2 was by in vitro and with of of by and the to Jak2 in cells with and with of by and blotting with to was cells by and was in was by to an and in for with T. PubMed Scopus Google Scholar). by by with studies to that the EPOR as stably in FDC-P1 cells tyrosine phosphorylation of Shc. exposure to EPO, Shc was and was for tyrosine phosphorylation by Western phosphorylation of Shc was by EPO, IL-3, and with GM-CSF, FDC-P1 cells used EPOR in a of Proc. Natl. Acad. Sci. U. S. A. 1991; 88: PubMed Scopus Google EPO-induced tyrosine phosphorylation of Shc with its recruitment to Jak2 to in an in of association of Shc with Jak2 This was observed with to Jak2 and blotting with to Shc with to Shc and blotting with to Jak2 of Jak2 These are to of N. H. 1994; PubMed Google Scholar, H. T. 1994; PubMed Google Scholar, O. N. Witthuhn B.A. Ihle J.N. N. 1994; PubMed Google Scholar). to of receptor Proc. Natl. Acad. Sci. U. S. A. 1991; 88: PubMed Scopus Google Scholar, N. H. 1994; PubMed Google was not to receptors in that EPO-induced association of Jak2 and Shc receptor However, Jak2 recently been shown to to receptors in cells O. N. Witthuhn B.A. Ihle J.N. N. 1994; PubMed Google Scholar). a of the of cytokine-induced Shc tyrosine phosphorylation association and that of these may This that the association Jak2 and Shc may not to for the phosphorylation of the and Shc association with Jak2 exposure of cells to exposure of FDC-P1 cells to GM-CSF, to Jak2 and with to Shc to and with to Jak2 with to Jak2 and Shc to in not factors Shc an in vitro phosphorylation assay was this the of cells and the of these with that of and for to a also in and by tyrosine phosphorylation of Shc was as was phosphorylation and residues. This the presence in the of in to the observed in vitro phosphorylation of Shc was this and phosphorylation of and also was observed of an in vitro assay for EPO-induced tyrosine phosphorylation of Shc. exposure of cells to EPO, and for phosphorylation in vitro and to the of and as and for to to in vitro phosphorylation of Shc was tyrosine residues, in vitro was to and in and with and phosphorylation of Shc in exposure of and FDC-P1 cells to in vitro phosphorylation of Shc in was as in the to and and on the observed association of Shc with tyrosine receptors phosphotyrosine sites A. A. PubMed Google Scholar, T. K. M. 1994; PubMed Google Scholar, K. S. K. T. 1994; PubMed Google the that EPO-induced recruitment of Shc to Jak2 depend upon phosphotyrosine sites within the receptor was a which Jak2 deficient in N. H. 1994; PubMed Google and cytoplasmic tyrosine residues. a cytoplasmic residues, and in both Jak2 and for the to mediate Shc supported this to receptor phosphorylation of Shc was for that EPO-induced recruitment of Shc its SH2 these that recruitment not depend upon receptor an this the of to Shc exposure also was of with to Jak2 Shc and direct blotting with to Shc Jak2 with by EPO-induced tyrosine phosphorylation of Shc by this receptor form in cells was observed with Shc and direct blotting with to phosphotyrosine phosphorylation of Shc a receptor form lacking cytoplasmic tyrosine residues. wild type receptor and and the EPO-induced phosphorylation of Shc wild type and vitro phosphorylation as in the to and and phosphorylation of Shc and its association with Jak2 in EPO-induced association of Shc in cells Western blotting of Shc with to Jak2 and of Jak2 with to Shc EPO-induced tyrosine phosphorylation of Shc Western blotting of Shc with to Jak2 upon the SH2 of Shc and the association of these factors the of a to Jak2 was and of Jak2 to was observed by Western blotting of and this was shown to depend upon of cells to this also and as in vitro and association of Shc with of to Jak2 the of with Jak2 was by Western of Jak2 on exposure of cells to as for A. of to and was for to a gel gel certain tyrosine receptors A. A. PubMed Google Scholar, T. K. M. 1994; PubMed Google Scholar, K. S. K. T. 1994; PubMed Google Shc to motifs and to to and A. 1994; PubMed Google Scholar, A. S. T. 1992; PubMed Scopus Google Scholar). However, have been for the receptor in which Shc and are the tyrosine phosphorylation of Shc N. A. K. S. S. T. M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: PubMed Scopus Google Scholar). for the for Shc to a which for N. K. N. Arai K. A. PubMed Scopus Google Scholar). factors that mediate Shc recruitment tyrosine phosphorylation and Shc The in the receptor system these in that a receptor form within a mitogenically subdomain N. H. 1994; PubMed Google the recruitment and phosphorylation of the absence of cytoplasmic tyrosine in this receptor recruitment of Shc through to an an SH2 Based on the SH2 Shc and Jak2 and on in vitro Jak2 to and to Shc motifs for have been T. M. H. H. T. S. 1994; PubMed Scopus Google Scholar). of a for Shc motifs in Jak2 and are in the also that Shc recruitment by factors shown to upon exposure and to with receptors a that co-immunoprecipitates with receptor 1992; PubMed Google Scholar, Proc. Natl. Acad. Sci. U. S. A. 1992; PubMed Scopus Google an tyrosine S. K. H. H. PubMed Google and a shown to with EPOR in cells 1994; PubMed Scopus Google Scholar). However, for and have not been not in EPOR in cells cells O. N. Witthuhn B.A. Ihle J.N. N. 1994; PubMed Google and for these factors in EPO-induced have not been of the receptor in cells also with tyrosine phosphorylation of Shc and its association with a 1994; PubMed Google also an effector of Shc phosphorylation. of the of EPO-induced phosphorylation of by a Jak2 are with this not the of in EPO-induced recently have a form of Jak2 H. T. 1994; PubMed Google Scholar). studies of cells expressing this that EPO-induced phosphorylation of Shc depends upon EPO-induced of and These and studies in the receptor system in both for by and in the for Shc in The development of hematopoietic cells depends upon exposure to a diverse set of cytokines, many of which activate receptors of the type I superfamily (1Bazan J.F. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6934-6938Crossref PubMed Scopus (1869) Google Scholar, 2Miyajima A. Kitamaura T. Harada N. Yokota T. Arai K.I. Annu. Rev. Immunol. 1992; 10: 295-331Crossref PubMed Scopus (537) Google Scholar). These receptors include those for EPO, 1The abbreviations used are: EPOerythropoietinEPORerythropoietin receptorG-CSFgranulocyte colony-stimulating factorGM-CSFgranulocyte-macrophage colony-stimulating factorILinterleukinLIFleukemia inhibitory factorPAGEpolyacrylamide gel electrophoresisGSTglutathione S-transferase.1The abbreviations used are: EPOerythropoietinEPORerythropoietin receptorG-CSFgranulocyte colony-stimulating factorGM-CSFgranulocyte-macrophage colony-stimulating factorILinterleukinLIFleukemia inhibitory factorPAGEpolyacrylamide gel electrophoresisGSTglutathione S-transferase. IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, GM-CSF, G-CSF, and LIF and are related based on conserved extracellular WSXWS motifs and cysteine residues, homologous cytoplasmic subdomains within certain receptor subunits, and the notable absence of recognizable enzymatic activities (1Bazan J.F. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6934-6938Crossref PubMed Scopus (1869) Google Scholar, 2Miyajima A. Kitamaura T. Harada N. Yokota T. Arai K.I. Annu. Rev. Immunol. 1992; 10: 295-331Crossref PubMed Scopus (537) Google Scholar, 3Murakami M. Narazaki M. Yamata H. Yasukawa K. Hamaguchi M. Taga T. Kishimoto T. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 11349-11353Crossref PubMed Scopus (486) Google Scholar, 4Narazaki M. Witthuhn B.A. Yoshida K. Silvennoinen O. Yasukawa K. Ihle J.N. Kishimoto T. Taga T. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2285-2289Crossref PubMed Scopus (251) Google Scholar, 5Quelle Proc. Natl. Acad. Sci. U. S. A. 1991; 88: PubMed Scopus Google Scholar, N. H. 1994; PubMed Google Scholar). This studies of However, recently have for type I tyrosine receptors with the of S. N. Taga T. Kishimoto T. PubMed Scopus Google Scholar, M. M. S. 1994; PubMed Scopus Google Scholar, 1994; PubMed Scopus Google and a of the type I cytokine Jak2, B.A. Silvennoinen O. T. O. Ihle J.N. PubMed Scopus Google Scholar, J.N. Witthuhn B.A. K. Silvennoinen O. Sci. 1994; PubMed Scopus Google Scholar, T. PubMed Scopus Google Scholar). tyrosine receptors A. 1992; PubMed Scopus Google and certain type I receptors also have been shown to to activate and to mediate the tyrosine phosphorylation of and Shc M. K. 1992; PubMed Google Scholar, M. N. 1991; PubMed Google Scholar, 1994; PubMed Scopus Google Scholar, A. 1994; PubMed Google Scholar, 1994; PubMed Scopus Google Scholar, H. N. PubMed Google Scholar, O. N. Ihle J.N. N. 1994; PubMed Google Scholar). erythropoietin erythropoietin receptor colony-stimulating colony-stimulating inhibitory gel S-transferase. erythropoietin erythropoietin receptor colony-stimulating colony-stimulating inhibitory gel S-transferase. have been tyrosine receptors and the recruitment of a PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, PubMed Scopus Google A. 1992; PubMed Scopus Google Scholar, K. M. A. T. Proc. Natl. Acad. Sci. U. S. A. 1992; PubMed Scopus Google and Shc T. 1992; PubMed Scopus Google Scholar). a SH2 receptor Shc a SH2 and both as as phosphotyrosine sites within certain tyrosine receptors and A. A. PubMed Google Scholar, T. K. M. 1994; PubMed Google Scholar, K. S. K. T. 1994; PubMed Google Scholar). for Shc in and also have been factors Shc phosphorylation and its association with A. A. PubMed Google Scholar, T. K. M. 1994; PubMed Google Scholar, K. S. K. T. 1994; PubMed Google Scholar, S. PubMed Scopus Google in by of Shc A. S. T. 1992; PubMed Scopus Google and by and Shc phosphorylation A. T. Proc. Natl. Acad. Sci. U. S. A. 1992; PubMed Scopus Google Scholar, 1994; PubMed Scopus Google Scholar). that not Shc may to for Shc are and Shc may Shc phosphorylation been shown to by IL-2, IL-3, IL-5, GM-CSF, and 1994; PubMed Google Scholar, PubMed Google Scholar, N. 1994; PubMed Google Scholar, M. H. T. 1994; PubMed Scopus Google Scholar). of Shc by type I cytokine receptors, have the recruitment and phosphorylation of this effector as mediated by receptor indicate that Shc recruitment not depend upon phosphotyrosine receptor sites and sites within Jak2 an also for a role for Jak2 in EPO-induced Shc phosphorylation. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: PubMed Scopus Google Scholar, N. H. 1994; PubMed Google in and to and Jak2 to to of in blotting and phosphorylation Shc and Jak2 to and with was with and was by with Western cells for in for with and with and in of Shc and Jak2 in in and to and with and Jak2 by in in vitro Jak2 was with and and with by by and and in of for with of and with of A. 1992; PubMed Scopus Google Scholar, A. S. T. 1992; PubMed Scopus Google of and by and of with a SH2 T. 1992; PubMed Scopus Google was by and and and was in and of to was as for and Jak2 was by in vitro and with of of by and the to Jak2 in cells with and with of by and blotting with to was cells by and was in was by to an and in for with T. PubMed Scopus Google Scholar). by by with Proc. Natl. Acad. Sci. U. S. A. 1991; 88: PubMed Scopus Google Scholar, N. H. 1994; PubMed Google in and Shc to and Jak2 to to of in blotting and phosphorylation Shc and Jak2 to and with was with and was by with Western cells for in for with and with and in of Shc and Jak2 in in and to and with and Jak2 by in in vitro Jak2 was with and and with by by and and in of for with of and with of A. 1992; PubMed Scopus Google Scholar, A. S. T. 1992; PubMed Scopus Google of and by and of with a SH2 T. 1992; PubMed Scopus Google was by and and and was in and of to was as for and Jak2 was by in vitro and with of of by and the to Jak2 in cells with and with of by and blotting with to was cells by and was in was by to an and in for with T. PubMed Scopus Google Scholar). by by with studies to that the EPOR as stably in FDC-P1 cells tyrosine phosphorylation of Shc. exposure to EPO, Shc was and was for tyrosine phosphorylation by Western phosphorylation of Shc was by EPO, IL-3, and with GM-CSF, FDC-P1 cells used EPOR in a of Proc. Natl. Acad. Sci. U. S. A. 1991; 88: PubMed Scopus Google EPO-induced tyrosine phosphorylation of Shc with its recruitment to Jak2 to in an in of association of Shc with Jak2 This was observed with to Jak2 and blotting with to Shc with to Shc and blotting with to Jak2 of Jak2 These are to of N. H. 1994; PubMed Google Scholar, H. T. 1994; PubMed Google Scholar, O. N. Witthuhn B.A. Ihle J.N. N. 1994; PubMed Google Scholar). to of receptor Proc. Natl. Acad. Sci. U. S. A. 1991; 88: PubMed Scopus Google Scholar, N. H. 1994; PubMed Google was not to receptors in that EPO-induced association of Jak2 and Shc receptor However, Jak2 recently been shown to to receptors in cells O. N. Witthuhn B.A. Ihle J.N. N. 1994; PubMed Google Scholar). a of the of cytokine-induced Shc tyrosine phosphorylation association and that of these may This that the association Jak2 and Shc may not to for the phosphorylation of the factors Shc an in vitro phosphorylation assay was this the of cells and the of these with that of and for to a also in and by tyrosine phosphorylation of Shc was as was phosphorylation and residues. This the presence in the of in to the observed in vitro phosphorylation of Shc was this and phosphorylation of and also was observed of an in vitro assay for EPO-induced tyrosine phosphorylation of Shc. exposure of cells to EPO, and for phosphorylation in vitro and to the of and as and for to to in vitro phosphorylation of Shc was tyrosine residues, in vitro was to and in and with and phosphorylation of Shc in exposure of and FDC-P1 cells to in vitro phosphorylation of Shc in was as in the to and and on the observed association of Shc with tyrosine receptors phosphotyrosine sites A. A. PubMed Google Scholar, T. K. M. 1994; PubMed Google Scholar, K. S. K. T. 1994; PubMed Google the that EPO-induced recruitment of Shc to Jak2 depend upon phosphotyrosine sites within the receptor was a which Jak2 deficient in N. H. 1994; PubMed Google and cytoplasmic tyrosine residues. a cytoplasmic residues, and in both Jak2 and for the to mediate Shc supported this to receptor phosphorylation of Shc was for that EPO-induced recruitment of Shc its SH2 these that recruitment not depend upon receptor an this the of to Shc exposure also was of with to Jak2 Shc and direct blotting with to Shc Jak2 with by EPO-induced tyrosine phosphorylation of Shc by this receptor form in cells was observed with Shc and direct blotting with to phosphotyrosine phosphorylation of Shc a receptor form lacking cytoplasmic tyrosine residues. wild type receptor and and the EPO-induced phosphorylation of Shc wild type and vitro phosphorylation as in the to and and phosphorylation of Shc and its association with Jak2 in EPO-induced association of Shc in cells Western blotting of Shc with to Jak2 and of Jak2 with to Shc EPO-induced tyrosine phosphorylation of Shc Western blotting of Shc with to Jak2 upon the SH2 of Shc and the association of these factors the of a to Jak2 was and of Jak2 to was observed by Western blotting of and this was shown to depend upon of cells to this also and as in vitro and association of Shc with of to Jak2 the of with Jak2 was by Western of Jak2 on exposure of cells to as for A. of to and was for to a gel gel certain tyrosine receptors A. A. PubMed Google Scholar, T. K. M. 1994; PubMed Google Scholar, K. S. K. T. 1994; PubMed Google Shc to motifs and to to and A. 1994; PubMed Google Scholar, A. S. T. 1992; PubMed Scopus Google Scholar). However, have been for the receptor in which Shc and are the tyrosine phosphorylation of Shc N. A. K. S. S. T. M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: PubMed Scopus Google Scholar). for the for Shc to a which for N. K. N. Arai K. A. PubMed Scopus Google Scholar). factors that mediate Shc recruitment tyrosine phosphorylation and Shc The in the receptor system these in that a receptor form within a mitogenically subdomain N. H. 1994; PubMed Google the recruitment and phosphorylation of the absence of cytoplasmic tyrosine in this receptor recruitment of Shc through to an an SH2 Based on the SH2 Shc and Jak2 and on in vitro Jak2 to and to Shc motifs for have been T. M. H. H. T. S. 1994; PubMed Scopus Google Scholar). of a for Shc motifs in Jak2 and are in the also that Shc recruitment by factors shown to upon exposure and to with receptors a that co-immunoprecipitates with receptor 1992; PubMed Google Scholar, Proc. Natl. Acad. Sci. U. S. A. 1992; PubMed Scopus Google an tyrosine S. K. H. H. PubMed Google and a shown to with EPOR in cells 1994; PubMed Scopus Google Scholar). However, for and have not been not in EPOR in cells cells O. N. Witthuhn B.A. Ihle J.N. N. 1994; PubMed Google and for these factors in EPO-induced have not been of the receptor in cells also with tyrosine phosphorylation of Shc and its association with a 1994; PubMed Google also an effector of Shc phosphorylation. of the of EPO-induced phosphorylation of by a Jak2 are with this not the of in EPO-induced recently have a form of Jak2 H. T. 1994; PubMed Google Scholar). studies of cells expressing this that EPO-induced phosphorylation of Shc depends upon EPO-induced of and These and studies in the receptor system in both for by and in the for Shc in studies to that the EPOR as stably in FDC-P1 cells tyrosine phosphorylation of Shc. exposure to EPO, Shc was and was for tyrosine phosphorylation by Western phosphorylation of Shc was by EPO, IL-3, and with GM-CSF, FDC-P1 cells used EPOR in a of Proc. Natl. Acad. Sci. U. S. A. 1991; 88: PubMed Scopus Google Scholar). EPO-induced tyrosine phosphorylation of Shc with its recruitment to Jak2 to in an in of association of Shc with Jak2 This was observed with to Jak2 and blotting with to Shc with to Shc and blotting with to Jak2 of Jak2 These are to of N. H. 1994; PubMed Google Scholar, H. T. 1994; PubMed Google Scholar, O. N. Witthuhn B.A. Ihle J.N. N. 1994; PubMed Google Scholar). to of receptor Proc. Natl. Acad. Sci. U. S. A. 1991; 88: PubMed Scopus Google Scholar, N. H. 1994; PubMed Google was not to receptors in that EPO-induced association of Jak2 and Shc receptor However, Jak2 recently been shown to to receptors in cells O. N. Witthuhn B.A. Ihle J.N. N. 1994; PubMed Google Scholar). a of the of cytokine-induced Shc tyrosine phosphorylation association and that of these may This that the association Jak2 and Shc may not to for the phosphorylation of the factors Shc an in vitro phosphorylation assay was this the of cells and the of these with that of and for to a also in and by tyrosine phosphorylation of Shc was as was phosphorylation and residues. This the presence in the of in to the observed in vitro phosphorylation of Shc was this and phosphorylation of and also was observed Based on the observed association of Shc with tyrosine receptors phosphotyrosine sites A. A. PubMed Google Scholar, T. K. M. 1994; PubMed Google Scholar, K. S. K. T. 1994; PubMed Google the that EPO-induced recruitment of Shc to Jak2 depend upon phosphotyrosine sites within the receptor was a which Jak2 deficient in N. H. 1994; PubMed Google and cytoplasmic tyrosine residues. a cytoplasmic residues, and in both Jak2 and for the to mediate Shc supported this to receptor phosphorylation of Shc was for that EPO-induced recruitment of Shc its SH2 these that recruitment not depend upon receptor an this the of to Shc exposure also was of with to Jak2 Shc and direct blotting with to Shc Jak2 with by EPO-induced tyrosine phosphorylation of Shc by this receptor form in cells was observed with Shc and direct blotting with to phosphotyrosine to Jak2 upon the SH2 of Shc and the association of these factors the of a to Jak2 was and of Jak2 to was observed by Western blotting of and this was shown to depend upon of cells to this also and as in vitro and certain tyrosine receptors A. A. PubMed Google Scholar, T. K. M. 1994; PubMed Google Scholar, K. S. K. T. 1994; PubMed Google Shc to motifs and to to and A. 1994; PubMed Google Scholar, A. S. T. 1992; PubMed Scopus Google Scholar). However, have been for the receptor in which Shc and are the tyrosine phosphorylation of Shc N. A. K. S. S. T. M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: PubMed Scopus Google Scholar). for the for Shc to a which for N. K. N. Arai K. A. PubMed Scopus Google Scholar). factors that mediate Shc recruitment tyrosine phosphorylation and Shc The in the receptor system these in that a receptor form within a mitogenically subdomain N. H. 1994; PubMed Google the recruitment and phosphorylation of the absence of cytoplasmic tyrosine in this receptor recruitment of Shc through to an an SH2 Based on the SH2 Shc and Jak2 and on in vitro Jak2 to and to Shc motifs for have been T. M. H. H. T. S. 1994; PubMed Scopus Google Scholar). of a for Shc motifs in Jak2 and are in the also that Shc recruitment by factors shown to upon exposure and to with receptors a that co-immunoprecipitates with receptor 1992; PubMed Google Scholar, Proc. Natl. Acad. Sci. U. S. A. 1992; PubMed Scopus Google an tyrosine S. K. H. H. PubMed Google and a shown to with EPOR in cells 1994; PubMed Scopus Google Scholar). However, for and have not been not in EPOR in cells cells O. N. Witthuhn B.A. Ihle J.N. N. 1994; PubMed Google and for these factors in EPO-induced have not been of the receptor in cells also with tyrosine phosphorylation of Shc and its association with a 1994; PubMed Google Scholar). Jak2 also an effector of Shc phosphorylation. of the of EPO-induced phosphorylation of by a Jak2 are with this not the of in EPO-induced recently have a form of Jak2 H. T. 1994; PubMed Google Scholar). studies of cells expressing this that EPO-induced phosphorylation of Shc depends upon EPO-induced of and These and studies in the receptor system in both for by and in the for Shc in and of for the of EPO, GM-CSF, and Shc used in these