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Identification and characterization of an interleukin-3 receptor-associated 110-kDa serine/threonine kinase
1Terry Fox Laboratory, British Columbia Cancer Research Centre, Vancouver, Canada.
We recently reported that interleukin-3 (IL-3) stimulation of the murine IL-3-responsive cell line, B6SUtA1, results in the rapid phosphorylation of the beta subunit of the IL-3 receptor (IL-3R), not only on tyrosine residues but on serine/threonine (Ser/Thr) residues as well. Since this occurred even at 4 degrees C, it suggested that a Ser/Thr-specific kinase might be closely associated with the IL-3R. To test this possibility, IL-3R complexes were isolated with anti-IL-3R (alpha IL-3R) antibodies, and in vitro phosphorylation studies were undertaken. These revealed the presence of a 110-kDa protein that was heavily phosphorylated in vitro on serine and threonine residues and that bound selectively to gamma-ATP-Sepharose beads. Moreover, this protein, which was not the 110-kDa subunit of phosphatidylinositol 3-kinase, was tyrosine phosphorylated in response to IL-3 and was specifically labeled in vitro with azido-[32P]ATP. These data, together with in vitro kinase inhibitor studies, suggest that an as yet uncharacterized H7- and staurosporine-sensitive 110-kDa Ser/Thr kinase may be constitutively associated with the IL-3R and activated following IL-3 stimulation. A comparison of IL-3R and erythropoietin receptor complexes suggests that this 110-kDa protein may be preferentially associated with the IL-3R.
We recently reported that interleukin-3 (IL-3) stimulation of the murine IL-3-responsive cell line, B6SUtA1, results in the rapid phosphorylation of the beta subunit of the IL-3 receptor (IL-3R), not only on tyrosine residues but on serine/threonine (Ser/Thr) residues as well. Since this occurred even at 4 degrees C, it suggested that a Ser/Thr-specific kinase might be closely associated with the IL-3R. To test this possibility, IL-3R complexes were isolated with anti-IL-3R (alpha IL-3R) antibodies, and in vitro phosphorylation studies were undertaken. These revealed the presence of a 110-kDa protein that was heavily phosphorylated in vitro on serine and threonine residues and that bound selectively to gamma-ATP-Sepharose beads. Moreover, this protein, which was not the 110-kDa subunit of phosphatidylinositol 3-kinase, was tyrosine phosphorylated in response to IL-3 and was specifically labeled in vitro with azido-[32P]ATP. These data, together with in vitro kinase inhibitor studies, suggest that an as yet uncharacterized H7- and staurosporine-sensitive 110-kDa Ser/Thr kinase may be constitutively associated with the IL-3R and activated following IL-3 stimulation. A comparison of IL-3R and erythropoietin receptor complexes suggests that this 110-kDa protein may be preferentially associated with the IL-3R.