A peptide model system for processive phosphorylation by Src family kinases.
The Src homology 2 (SH2) and Src homology 3 (SH3) domains of Src family kinases are involved in substrate recognition in vivo. Many cellular substrates of Src kinases contain a large number of potential phosphorylation sites, and the SH2 and SH3 domains of Src are known to be required for phosphorylation of these substrates. In principle, Src could phosphorylate these substrates by either a processive mechanism, in which the enzyme remains bound to the peptide substrate during multiple phosphorylation events, or a nonprocessive (distributive) mechanism, where each phosphorylation requires a separate binding interaction between enzyme and substrate. Here we use a synthetic peptide system to demonstrate that Hck, a Src family kinase, can phosphorylate substrates containing an SH2 domain ligand by a processive mechanism. Hck catalyzes the phosphorylation of these sites in a defined order. Furthermore, we show that addition of an SH3 domain to a peptide can enhance its phosphorylation both by activating Hck and by increasing the affinity of the substrate. On the basis of our observations on the role of the SH2 and SH3 domains in substrate recognition, we present a model for substrate targeting in vivo.
Scott
M P
MP
Department of Physiology and Biophysics, School of Medicine, State University of New York at Stony Brook, Stony Brook, New York 11794-8661, USA.
Miller
W T
WT
D000595:Amino Acid Sequence; D007700:Kinetics; D008024:Ligands; D008956:Models, Chemical; D008969:Molecular Sequence Data; D015688:Oncogene Protein pp60(v-src) / Q000378:metabolism; D050736:Oncogene Protein v-crk; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D011506:Proteins; D050717:Retinoblastoma-Like Protein p130; D015682:Retroviridae Proteins, Oncogenic / Q000378:metabolism; D013379:Substrate Specificity; D018909:src Homology Domains / Q000502:physiology; D019061:src-Family Kinases / Q000378:metabolism*
D008024:Ligands; D050736:Oncogene Protein v-crk; D010455:Peptides; D010750:Phosphoproteins; D011506:Proteins; D050717:Retinoblastoma-Like Protein p130; D015682:Retroviridae Proteins, Oncogenic; D015688:Oncogene Protein pp60(v-src); D019061:src-Family Kinases