Exploration of the Sequence Specificity of pp60c- Tyrosine Kinase

Abstract

The minimum length required for phosphorylation of a peptide by pp60c-src tyrosine kinase (srcTK) was delineated in this work. Budde (M. D. Anderson University of Texas, personal communication) suggested that the peptide (FGE)3Y(GEF)2GD (peptide I) was a “good” srcTK substrate. Peptide I yielded a 251-fold higher kcat/Km than RRLIEDAEYAARRG, a peptide substrate based upon the autophosphorylation site of srcTK. This was due to a 38-fold lower Km and a 6.6-fold increase in kcat. N-terminal truncation of up to 8 residues in a series of peptides yielded only a 3-fold decrease in activity. Removal of the final N-terminal residue resulted in a 10-fold loss in substrate activity, primarily as a result of an increase in the Km. C-terminal truncations ending in the amide yielded no significant loss in activity until the Y+3 residue was removed, which resulted in a 73-fold decrease in kcat/Km relative to peptide I. The latter was due primarily to an increase in Km. The results from peptides truncated on both termini suggest that subsite recognition N- and C-terminal relative to the site of phosphorylation can be examined independently. In addition, the observation that only 5 residues are required for significant substrate activity suggests that small molecule inhibitors based upon interactions with the phosphoacceptor site may be developed. The minimum length required for phosphorylation of a peptide by pp60c-src tyrosine kinase (srcTK) was delineated in this work. Budde (M. D. Anderson University of Texas, personal communication) suggested that the peptide (FGE)3Y(GEF)2GD (peptide I) was a “good” srcTK substrate. Peptide I yielded a 251-fold higher kcat/Km than RRLIEDAEYAARRG, a peptide substrate based upon the autophosphorylation site of srcTK. This was due to a 38-fold lower Km and a 6.6-fold increase in kcat. N-terminal truncation of up to 8 residues in a series of peptides yielded only a 3-fold decrease in activity. Removal of the final N-terminal residue resulted in a 10-fold loss in substrate activity, primarily as a result of an increase in the Km. C-terminal truncations ending in the amide yielded no significant loss in activity until the Y+3 residue was removed, which resulted in a 73-fold decrease in kcat/Km relative to peptide I. The latter was due primarily to an increase in Km. The results from peptides truncated on both termini suggest that subsite recognition N- and C-terminal relative to the site of phosphorylation can be examined independently. In addition, the observation that only 5 residues are required for significant substrate activity suggests that small molecule inhibitors based upon interactions with the phosphoacceptor site may be developed.