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

doi:10.1074/jbc.270.12.6886

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Volume 270, Number 12, Issue of March 24, 1995 pp. 6886-6893
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.

A Membrane Proximal Domain of the Human Interleukin-3 Receptor Subunit That Signals DNA Synthesis in NIH 3T3 Cells Specifically Binds a Complex of Src and Janus Family Tyrosine Kinases and Phosphatidylinositol 3-Kinase (*)

(Received for publication, July 19, 1994; and in revised form, January 19, 1995)

Padmini Rao , R. Allan Mufson (§)

From the Holland Laboratory for BioMedical Science, American Red Cross, Rockville, Maryland 20855

ABSTRACT

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.

FOOTNOTES

*: This work was supported by National Institutes of Health Grant Ca 53609 and Grant 3134R2 from the Council for Tobacco Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§: To whom correspondence and reprint requests should be addressed: Holland Laboratory for BioMedical Science, American Red Cross, Immunology Dept., 15601 Crabbs Branch Way, Rockville, MD 20855. Tel.: 301-738-0736; Fax: 301-738-0794.
(): The abbreviations used are: IL-3, interleukin-3; GM-CSF, granulocyte macrophage colony stimulating factor; IL-5, interleukin-5; GST, glutathione S-transferase; PCR, polymerase chain reaction; CAPS, 3-(cyclohexylamino)propanesulfonic acid; PI 3-kinase, phosphatidylinositol 3-kinase; FSBA, 5`-p-fluorosulfonylbenzoyladenosine; GAP, GTPase activating protein.

ACKNOWLEDGEMENTS

We thank Dr. Steven Ullrich for his assistance with the phosphoamino acid analysis and Tammy Krogman for technical assistance.

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