Catalytic and non-catalytic domains of the Fujinami sarcoma virus P130gag-fps protein-tyrosine kinase distinguished by the expression of v-fps polypeptides in Escherichia coli.
While protein-tyrosine kinases share a region of sequence identity corresponding to their kinase domains, the specific elements essential for catalysis, substrate binding and substrate specificity are largely undefined. The P130gag-fps transforming protein of Fujinami avian sarcoma virus is a cytoplasmic tyrosine kinase with a complex structure that includes a C-terminal kinase domain. To identify the precise N-terminal border of the v-fps catalytic region and to assess its interactions with non-catalytic domains, C-terminal v-fps polypeptide fragments of decreasing size were expressed in E. coli as trpE-v-fps hybrid proteins. All such polypeptides containing 263 or more residues derived from the C-terminus of P130gag-fps (i.e. residues 920-1182) were enzymatically active as tyrosine kinases. They autophosphorylated at physiological sites in vivo and phosphorylated exogenous substrates such as enolase and poly(glu,tyr) at tyrosine in vitro. Deletion of a further five amino acids from P130gag-fps residues 920-925 abolished all enzymatic activity. This deletion coincides with the predicted N-terminus of the v-fps ATP-binding site at residue 922. These data indicate that the N-terminal border of the ATP-binding site defines the start of the minimal v-fps tyrosine kinase catalytic domain, and show that this minimal domain is competent to bind substrates. More N-terminal non-catalytic sequences appear to functionally interact with the catalytic domain.
D002384:Catalysis; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D004926:Escherichia coli; D008970:Molecular Weight; D010750:Phosphoproteins / Q000378:metabolism; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases / Q000235:genetics*; D009053:Sarcoma Viruses, Murine / Q000201:enzymology / Q000235:genetics*; D013329:Structure-Activity Relationship; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism; D014760:Viral Fusion Proteins / Q000235:genetics*
D010750:Phosphoproteins; D014760:Viral Fusion Proteins; D019000:Phosphotyrosine; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases