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Title
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Peptide inhibitors of src SH3-SH2-phosphoprotein interactions.
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Authors
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T.Gilmer,
M.Rodriguez,
S.Jordan,
R.Crosby,
K.Alligood,
M.Green,
M.Kimery,
C.Wagner,
D.Kinder,
P.Charifson.
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Ref.
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J Biol Chem, 1994,
269,
31711-31719.
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PubMed id
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Abstract
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Activated pp60c-src has been implicated in a number of human malignancies
including colon carcinoma and breast adenocarcinoma. Association of the src SH2
domain with tyrosine-phosphorylated proteins plays a role in src-mediated signal
transduction. Inhibitors of src SH2 domain-phosphoprotein interactions are,
thus, of great interest in defining the role(s) of src in signal transduction
pathways. To facilitate such studies, an enzyme-linked immunosorbent assay
(ELISA) was developed to detect inhibitors of src SH2-phosphoprotein
interactions. This assay measures inhibition of binding of a fusion construct
(glutathione S-transferase src SH3-SH2) with autophosphorylated epidermal growth
factor receptor tyrosine kinase domain. Activities of phosphopeptide segments
derived from potential src SH2 cognate phosphoprotein partners were determined,
with the focal adhesion kinase-derived segment VSETDDY*AEIIDE yielding the
highest inhibitory activity. Structure activity studies starting from acetyl
(Ac)-Y*EEIE have identified Ac-Y*Y*Y*IE as the most active compound screened in
the ELISA. This compound is at least 20-fold more active than the parent peptide
Ac-Y*EEIE. A high resolution (2 A) crystal structure of human src SH2 complexed
with Ac-Y*EEIE was obtained and provided a useful framework for understanding
the structure-activity relationships. Additionally, Ac-Y*EEIE was able to block
interactions between src and its cellular phosphoprotein partners in
vanadate-treated cell lysates from MDA-MB-468 breast carcinoma cells. However,
it is unable to abrogate proliferation of MDA-MB-468 cells in culture,
presumably because of poor cell penetration and/or lability of the phosphate
group on tyrosine.
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