Exploration of the Sequence Specificity of pp60c- Tyrosine Kinase

Abstract

The minimum length required for phosphorylation of a peptide by pp60 c-src tyrosine kinase (srcTK) was delineated in this work. Budde (M. D. Anderson University of Texas, personal communication) suggested that the peptide (FGE) 3 Y(GEF) 2 GD (peptide I) was a "good" srcTK substrate. Peptide I yielded a 251-fold higher k cat /K m than RRLIEDAEYAARRG, a peptide substrate based upon the autophosphorylation site of srcTK. This was due to a 38-fold lower K m and a 6.6-fold increase in k cat . 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 K m . 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 k cat /K m relative to peptide I. The latter was due primarily to an increase in K m . 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.Protein tyrosine kinases (TKs) 1 were initially discovered as either oncogenes or proto-oncogene products, pointing to their therapeutic potential as targets in cancer (for a review of oncogenes, see Pimentel (1989a and1989b)). The phosphorylation equilibrium of protein tyrosine residues is regulated by cytokines and growth factors, pointing to roles for these equilibria in signal transduction pathways. Therefore, control of these equilibria has the potential for therapeutic intervention in diseases including cancer, inflammatory diseases, and diabetes, to name a few.While initially TKs were thought to be nonspecific, more recent work has demonstrated that they do indeed phosphorylate specific substrates in vivo (for example, see Ogawa et al. (1994)). The study of these enzymes has been hampered by the lack of specific peptide substrates. The specificity requirements for amino acid sequences remain to be elucidated. While several groups proposed that the specificity of TKs was not governed by the amino acid sequence surrounding the tyrosine (Tinker et al., 1988;Radziejewski et al., 1989), more recent studies have suggested that there is specific recognition of proximal residues. For example, Garcia et al. (1993) reported differences in the peptide sequences recognized by pp60 v-src 2 and v-abl TK. These workers found that substitution of N 3 for D in the YϪ1 4 position of KKSRGDYMTMQIG, a peptide based upon a phosphorylation site of insulin receptor substrate-1, resulted in complete loss of substrate activity for both TKs. Substitution of I for M in the Yϩ1 position resulted in a 4-fold loss in catalytic efficiency for pp60 v-src but a 10-fold increase in the catalytic...

SH2- and SH3-mediated Interactions between Focal Adhesion Kinase and Src

“…The enzyme was then used to phosphorylate the synthetic substrate RRLIEDAEYAARG. The production of ADP was coupled to the oxidation of NADH using phosphoenolpyruvate, pyruvate kinase, and lactate dehydrogenase and the decrease in absorbance at 340 nm was measured as described (34,38). Initial reaction rates were measured and kinetic parameters were determined by nonlinear regression analysis of the rates using the equation,…”

Protein-tyrosine Kinase and GTPase Signals Cooperate to Phosphorylate and Activate Wiskott-Aldrich Syndrome Protein (WASP)/Neuronal WASP

“…2A and Table 1) (27)(28)(29). Using an autoinhibited construct that consists of the GBD linked to the VCA domain through a short flexible linker (GGS) 2 …”

TLiSA1 (PTA1) Activation Antigen Implicated in T Cell Differentiation and Platelet Activation Is a Member of the Immunoglobulin Superfamily Exhibiting Distinctive Regulation of Expression

“…However, at least a proportion of the 32 P label on PTA1 precipitated from activated platelets resists potassium hydroxide treatment, 4 and although we have not yet carried out phosphoamino acid analysis, this result may indicate a phosphotyrosine residue. Contained within the cytoplasmic tail of TLiSA1/PTA1 around Tyr 304 is the sequence EDIYVNY, which could serve as a substrate for a nonreceptor protein-tyrosine kinase (45). Within this sequence, amino-terminal to the putative phosphotyrosine, are two negatively charged residues that together with the three residues immediately carboxyl-terminal to this tyrosine have the potential to form a recognition site for molecules containing an SH2 domain (46).…”