Synthetic peptides reproducing the EGF-receptor segment homologous to the pp60v-src phosphoacceptor site. Phosphorylation by tyrosine protein kinases.
The octapeptide E-E-K-E-Y-H-A-E, corresponding to the amino acid sequence 841-845 of EGF receptor, whose tyrosine-845 is homologous to the main phosphorylation site of pp60v-src, has been synthesized together with seven shorter peptides encompassing variable segments around the tyrosine residue. The peptides have been employed as model substrates for inspecting the local structural determinants of three tyrosine protein kinases (TPKs), namely; TPK-IIB and TPK-III, isolated from lymphoid cells (Eur. J. Biochem. 172, 451-457 (1988] and the TPK encoded by the oncogene of Abelson murine leukemia virus. The phosphorylation order with the different peptide substrates is variable depending on the TPK used: in particular, the lysine residue at position -2 relative to tyrosine proved especially harmful with TPK-IIB, the peptides K-E-Y-H and K-E-Y-H-A-E being very poor substrates compared with their shorter derivatives devoid of the N-terminal lysine (E-Y-H and E-Y-H-A-E, respectively). Conversely, such a basic residue is well tolerated by the other two TPKs. The negative effect of the N-terminal lysine on TPK-IIB-catalyzed phosphorylation is accounted for by an increase of Km and can be overcome by the presence of additional glutamic acid(s) on that side. On the other hand, the C-terminal acidic doublet Ala-Glu specifically impairs the phosphorylation efficiency of abl-TPK, by lowering the Vmax value, the heptapeptide E-K-E-Y-H-A-E being much less readily phosphorylated than E-K-E-Y-H. Collectively, these results would indicate that the site specificity of tyrosine protein kinases results from the balance of positive and negative determinants whose influence on the catalytic activity of the individual enzymes can differ greatly.
Cola
C
C
Dipartimento di Chimica Biologica, Università di Padova, Italy.
Brunati
A M
AM
Borin
G
G
Ruzza
P
P
Calderan
A
A
De Castiglione
R
R
Pinna
L A
LA
D000595:Amino Acid Sequence; D066246:ErbB Receptors / Q000378:metabolism*; D008969:Molecular Sequence Data; D015688:Oncogene Protein pp60(v-src); D010446:Peptide Fragments / Q000138:chemical synthesis / Q000378:metabolism*; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D012191:Retroviridae Proteins / Q000378:metabolism*; D013379:Substrate Specificity
D010446:Peptide Fragments; D012191:Retroviridae Proteins; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases; D015688:Oncogene Protein pp60(v-src)