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Molecular and Cellular Biology, July 2001, p. 4579-4597, Vol. 21, No. 14
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.14.4579-4597.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
The EphA8 Receptor Regulates Integrin Activity
through p110
Phosphatidylinositol-3 Kinase in a Tyrosine
Kinase Activity-Independent Manner
Changkyu
Gu and
Soochul
Park*
Institute of Environment and Life Science,
Hallym University, Chuncheon 200-702, Korea
Received 19 September 2000/Returned for modification 20 November
2000/Accepted 15 April 2001
Recent genetic studies suggest that ephrins may function in a
kinase-independent Eph receptor pathway. Here we report that expression
of EphA8 in either NIH 3T3 or HEK293 cells enhanced cell adhesion to
fibronectin via
5
1- or
3
integrins. Interestingly, a kinase-inactive EphA8 mutant also markedly
promoted cell attachment to fibronectin in these cell lines. Using a
panel of EphA8 point mutants, we have demonstrated that EphA8 kinase
activity does not correlate with its ability to promote cell attachment
to fibronectin. Analysis using EphA8 extracellular and intracellular
domain mutants has revealed that enhanced cell adhesion is dependent on
ephrin A binding to the extracellular domain and the juxtamembrane
segment of the cytoplasmic domain of the receptor. EphA8-promoted
adhesion was efficiently inhibited by wortmannin, a
phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor. Additionally, we
found that EphA8 had associated PI 3-kinase activity and that the
p110
isoform of PI 3-kinase is associated with EphA8. In vitro
binding experiments revealed that the EphA8 juxtamembrane segment was
sufficient for the formation of a stable complex with p110
. Similar
results were obtained in assay using cells stripped of endogenous
ephrin A ligands by treatment with preclustered ephrin A5-Fc proteins. In addition, a membrane-targeted lipid kinase-inactive p110
mutant was demonstrated to stably associate with EphA8 and suppress
EphA8-promoted cell adhesion to fibronectin. Taken together, these
results suggest the presence of a novel mechanism by which the EphA8
receptor localizes p110
PI 3-kinase to the plasma membrane in a
tyrosine kinase-independent fashion, thereby allowing access to lipid
substrates to enable the signals required for integrin-mediated cell adhesion.
*
Corresponding author. Mailing address: Institute of
Environment and Life Science, Hallym University, 1-Okcheon dong,
Chuncheon 200-702, Korea. Phone: 82-33-240-1793. Fax: 82-33-241-3422. E-mail: scpark{at}sun.hallym.ac.kr.
Molecular and Cellular Biology, July 2001, p. 4579-4597, Vol. 21, No. 14
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.14.4579-4597.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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