Structural requirement of CRK SH2 region for binding to phosphotyrosine-containing proteins. Evidence from reactivity to monoclonal antibodies.
The SH2 region of signal-transducing proteins mediates binding to phosphotyrosine-containing proteins. We analyzed the structure-function relationship of the SH2 region of the human CRK protein by using a series of monoclonal antibodies (mAbs). Seventeen mAbs against the CRK SH2 region were classified into 5 groups according to the reactivity with mutant CRK proteins expressed in COS7 cells and in Escherichia coli and by epitope scanning with synthetic nonapeptides. Two groups of mAbs (groups A and B) were reactive only with intact SH2. Mutation(s) in either the amino-terminal B box or the carboxyl-terminal C box, which are the two subdomains of SH2, abolished the reactivity of the CRK mutants to the mAbs of groups A and B. Group A mAbs competed the binding of the CRK SH2 region to the phosphotyrosine-containing proteins. Moreover, the spectrum of the CRK mutants which were recognized by group A mAbs coincided with that of the CRK mutants which could bind phosphotyrosine-containing proteins, suggesting that group A mAbs were directed against the site of binding to phosphotyrosine-containing proteins. Group C mAbs, directed against the region between the B and C boxes, were reactive with both wild-type and mutant CRK proteins and did not affect the capacity of SH2 to bind phosphotyrosine-containing proteins. Contrary to group A and B mAbs, mAbs belonging to groups D and E, which were mapped onto the C box, did not bind well to the native CRK proteins, but bound to CRK mutants with mutation(s) in either the B or the C box. These results suggest that the B and C boxes, which are separated by a hinge region, coordinately form the functional SH2 domain that binds to the phosphotyrosine-containing proteins and to the group A mAbs.
Matsuda
M
M
Department of Pathology, National Institute of Health, Tokyo, Japan.
Nagata
S
S
Tanaka
S
S
Nagashima
K
K
Kurata
T
T
D000818:Animals; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D015153:Blotting, Western; D016203:CDC2 Protein Kinase / Q000378:metabolism; D002460:Cell Line; D002522:Chlorocebus aethiops; D000939:Epitopes; D006801:Humans; D006825:Hybridomas; D009154:Mutation; D010750:Phosphoproteins / Q000378:metabolism*; D019000:Phosphotyrosine; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D050737:Proto-Oncogene Proteins c-crk; D015800:Protozoan Proteins; D013329:Structure-Activity Relationship; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000939:Epitopes; D010750:Phosphoproteins; D011518:Proto-Oncogene Proteins; D050737:Proto-Oncogene Proteins c-crk; D015800:Protozoan Proteins; D019000:Phosphotyrosine; D014443:Tyrosine; D016203:CDC2 Protein Kinase