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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 and  subunit. The  subunit is responsible for
receptor signal transduction. We have shown that a membrane proximal
domain of the cytoplasmic tail of the human  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.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.

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Copyright © 1995 by the American Society for Biochemistry and Molecular Biology.
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