Peptide inhibitors of src SH3-SH2-phosphoprotein interactions.
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.
Gilmer
T
T
Glaxo Research Institute, Research Triangle Park, North Carolina 27709.
Rodriguez
M
M
Jordan
S
S
Crosby
R
R
Alligood
K
K
Green
M
M
Kimery
M
M
Wagner
C
C
Kinder
D
D
Charifson
P
P
D000595:Amino Acid Sequence; D015815:Cell Adhesion Molecules / Q000378:metabolism; D002455:Cell Division; D018360:Crystallography, X-Ray; D066246:ErbB Receptors / Q000378:metabolism*; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D006801:Humans; D066298:In Vitro Techniques; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000737:chemistry / Q000378:metabolism*; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism*; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
D015815:Cell Adhesion Molecules; D010748:Phosphopeptides; D011993:Recombinant Fusion Proteins; D019000:Phosphotyrosine; D014443:Tyrosine; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases; D020794:Receptor Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human; D016392:Proto-Oncogene Proteins pp60(c-src)