A membrane proximal domain of the human interleukin-3 receptor beta c subunit that signals DNA synthesis in NIH 3T3 cells specifically binds a complex of Src and Janus family tyrosine kinases and phosphatidylinositol 3-kinase.
The high affinity human interleukin-3 receptor is a heterodimeric protein consisting of an alpha and beta c subunit. The beta c subunit is responsible for receptor signal transduction. We have shown that a membrane proximal domain of the cytoplasmic tail of the human beta c subunit (amino acids 451-517) is minimally required for human IL-3 to signal DNA synthesis in quiescent transfected NIH 3T3 cells. Glutathione S-transferase (GST) fusion proteins of this 451-517 region and another region 451-562 that includes an acidic domain previously shown in other receptors to bind Src family kinases were constructed. Purified Lyn and Lck kinase, but not Fes, could phosphorylate tyrosines in both domains. Adsorption with lysates from the human IL-3-dependent hematopoietic cell line (TF-1) or 3T3 cells and in vitro phosphorylation showed that both these domains were intensely phosphorylated. Phosphoamino acid analysis, however, revealed that the majority of phosphorylation was on serine and threonine rather than tyrosine. Adsorption of these domains with 3T3 or TF-1 cell lysates, followed by immunoblotting, showed that cytoplasmic tyrosine kinases Lyn, Fes, and JAK-2 could also stably associate with both domains; however, Src family kinases are more strongly recognized by both regions than JAK-2 kinase. In addition, phosphatidylinositol 3-kinase from cell lysates was also found stably associated with these domains, but GTPase activating protein, Vav, Sos1, or Grb2 were not.
Rao
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Holland Laboratory for BioMedical Science, American Red Cross, Rockville, Maryland 20855.
Mufson
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RA
D016475:3T3 Cells; D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000096:biosynthesis*; D051380:GRB2 Adaptor Protein; D020690:GTPase-Activating Proteins; D053614:Janus Kinase 2; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D051379:Mice; D008969:Molecular Sequence Data; D019869:Phosphatidylinositol 3-Kinases; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism; D011518:Proto-Oncogene Proteins; D016185:Receptors, Interleukin-3 / Q000235:genetics / Q000502:physiology*; D019061:src-Family Kinases
D048868:Adaptor Proteins, Signal Transducing; D051380:GRB2 Adaptor Protein; D020690:GTPase-Activating Proteins; C495949:Grb2 protein, mouse; D011506:Proteins; D011518:Proto-Oncogene Proteins; D016185:Receptors, Interleukin-3; D004247:DNA; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D011505:Protein-Tyrosine Kinases; C507924:JAK2 protein, human; C507926:Jak2 protein, mouse; D053614:Janus Kinase 2; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); C060902:lyn protein-tyrosine kinase; D019061:src-Family Kinases