Interleukin-3 stimulates the tyrosine phosphorylation of the 140-kilodalton interleukin-3 receptor.
Murine interleukin-3 (mIL-3) stimulates the rapid and transient tyrosine phosphorylation of a number of proteins in mIL-3-dependent B6SUtA1 cells. Two of these proteins, p68 and p140, are maximally phosphorylated at tyrosine residues within 2 min of addition of mIL-3. Because 125I-mIL-3 can be cross-linked to both 70- and 140-kDa proteins on intact B6SUtA1 cells, we investigated whether the tyrosine phosphorylated p68 and p140 were these two mIL-3 receptor proteins. Addition of antiphosphotyrosine antibodies (alpha PTyr Abs) to cell lysates from B6SUtA1 cells, to which 125I-mIL-3 had been disuccinimidyl suberate-cross-linked, resulted in the immunoprecipitation of 125I-mIL-3 complexed to both 70- and 140-kDa proteins. To determine if the observed immunoprecipitation pattern was due to the direct interaction of alpha-PTyr Abs with these two mIL-3 receptor proteins or with tyrosine-phosphorylated proteins that were associated with the receptor proteins, cell lysates were treated with 2% sodium dodecyl sulfate, 5% 2-mercaptoethanol, and boiled for 1 min. After removal of sodium dodecyl sulfate and 2-mercaptoethanol, alpha PTyr Abs immunoprecipitated 125I-mIL-3 cross-linked to only the 140-kDa protein. To confirm this finding, 32P-labeled B6SUtA1 cells were treated with biotinylated or fluoresceinated mIL-3. Addition of immobilized streptavidin or antifluorescein antibodies, respectively, to cell lysates from these cells resulted in the enrichment of only a 140-kDa tyrosine phosphorylated protein. Taken together, these results strongly suggest that only the 140-kDa receptor protein is tyrosine phosphorylated upon mIL-3 binding.
Sorensen
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Terry Fox Laboratory, British Columbia Cancer Research Centre, Vancouver, Canada.
Mui
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Krystal
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D000818:Animals; D002460:Cell Line; D007377:Interleukin-3 / Q000494:pharmacology*; D008970:Molecular Weight; D010766:Phosphorylation; D011971:Receptors, Immunologic / Q000378:metabolism*; D016185:Receptors, Interleukin-3; D011994:Recombinant Proteins / Q000494:pharmacology; D014443:Tyrosine
D007377:Interleukin-3; D011971:Receptors, Immunologic; D016185:Receptors, Interleukin-3; D011994:Recombinant Proteins; D014443:Tyrosine