Mutational analysis of a phosphotransfer motif essential for v-fps tyrosine kinase activity.
The catalytic domains of protein-tyrosine kinases such as the P130gag-fps oncoprotein contain the sequence HRDLAARN, followed thirteen residues C-terminal by DFG (P130gag-fps residues 1041-1048 and 1061-1063). These residues define a structural motif conserved among eucaryotic protein kinases (-RD----N, DFG) and shared with several procaryotic 3'aminoglycoside phosphotransferases (H-D----N, D-G). Functional analysis of mutant v-fps proteins employing bacterial and mammalian expression systems indicated that this motif is critical for P130gag-fps kinase activity and oncogenicity. In particular, conservative substitutions of the two invariant aspartates (asp1043, asp1061) with glutamate or asparagine completely eliminated enzymatic activity, suggesting that these residues are essential for catalysis. In contrast, substitution of arg1042 with glutamate decreased but did not eliminate v-fps kinase activity in bacteria. The effects of these and other amino acid substitutions within the phosphotransfer motif and at the nearby autophosphorylation site (tyr1073) of P130gag-fps indicate that these conserved residues are intrinsically essential to the execution or regulation of catalytic activity, and suggest that tight spatial constraints operate within the active centre of the v-fps tyrosine kinase domain.
Moran
M F
MF
Division of Molecular and Developmental Biology, Mount Sinai Hospital Research Institute, Toronto, Ontario, Canada.
Koch
C A
CA
Sadowski
I
I
Pawson
T
T
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D017124:Conserved Sequence; D004252:DNA Mutational Analysis; D015681:Fusion Proteins, gag-onc / Q000096:biosynthesis / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009857:Oncogenes; D011505:Protein-Tyrosine Kinases / Q000096:biosynthesis / Q000378:metabolism*; D051381:Rats; D017386:Sequence Homology, Amino Acid; D014162:Transfection
D015681:Fusion Proteins, gag-onc; D011505:Protein-Tyrosine Kinases; C068415:v-fps oncogene protein, Fujinami sarcoma virus