Enhanced phosphorylation of Src family kinase substrates containing SH2 domain binding sites.
Src family protein-tyrosine kinases possess several modular domains important for regulation of catalytic activity and interaction with potential substrates. Here, we explore interactions between the SH2 domain of Hck, a Src family kinase, and substrates containing SH2 domain-binding sites. We have synthesized a series of peptide substrates containing a high affinity SH2 domain binding site, (phospho)Tyr-Glu-Glu-Ile. We show that the presence of this sequence in a peptide results in a dramatic increase in the phosphorylation rate of a second tyrosine located at the N terminus. Enhanced phosphorylation is not a consequence of stimulation of enzymatic activity by C-terminal tail displacement but is imparted instead by a 10-fold reduction in the Km of the phosphotyrosine-containing peptide when compared with a control. The isolated catalytic domain of the non-receptor tyrosine kinase Abl does not show a preference for the pYEEI motif-containing peptide; however, the preference is restored when the SH2 domain of Src is introduced into Abl. Furthermore, enhanced phosphorylation is dependent on the distance between SH2 domain-binding site and phosphorylatable tyrosine, with the minimum distance requirement being seven amino acids. Reversing the orientation of the pYEEI motif with respect to the substrate sequence decreases phosphorylation by down-regulated Hck, but both orientations are utilized equally well by activated Hck. We discuss the possible implications of these results for processive phosphorylation of substrates in vivo by Src family kinases.
Pellicena
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Department of Physiology and Biophysics, School of Medicine, State University of New York at Stony Brook, Stony Brook, New York 11794-8661, USA.
Stowell
K R
KR
Miller
W T
WT
D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051572:Proto-Oncogene Proteins c-hck; D018909:src Homology Domains; D019061:src-Family Kinases / Q000378:metabolism*
D010455:Peptides; D011518:Proto-Oncogene Proteins; D011505:Protein-Tyrosine Kinases; D051572:Proto-Oncogene Proteins c-hck; D019061:src-Family Kinases