Structural elements that regulate pp59c-fyn catalytic activity, transforming potential, and ability to associate with polyomavirus middle-T antigen.
Except for its unique amino-terminal region (residues 1 through 83), which possibly dictates substrate recognition, pp59c-fyn bears a high degree of homology with other members of the src family of tyrosine kinases. Here we show that the carboxy terminus of pp59c-fyn is necessary for stable middle-T-antigen association, that pp59c-fyn is normally phosphorylated on both serine and tyrosine residues, and that Tyr-531 and Tyr-420 are phosphorylation sites in vivo and in vitro, respectively. Analysis of a spontaneously generated mutant encoding a truncated form of pp59c-fyn and of variants specifically mutated at the Tyr-531 and Tyr-420 phosphorylation sites indicates that pp59c-fyn has regulatory elements analogous to those that have already been identified for other src-like tyrosine kinases. However, further examination of the pp59c-fyn variants suggests the likelihood of additional means by which its activities might be regulated. Although alteration of Tyr-531 to phenylalanine (531F) in pp59c-fyn results in a protein which is more active enzymatically that the wild type, the enhancement is much less than that for the analogous variant of pp60c-src. Furthermore, contrary to results of similar experiments on other src-like proto-oncogene products, 531F did not induce transformation of NIH 3T3 cells. Studies involving pp59c-fyn-pp60c-src chimeras in which the unique amino-terminal sequences (residues 1 through 83) of the two kinases were precisely interchanged implied that the inability of 531F to induce transformation is probably not caused by the absence of substrates for pp59c-fyn in NIH 3T3 cells but rather by the insufficient enhancement of pp59c-fyn kinase activity. It is therefore probable that the kinase and transforming activities of pp59c-fyn are repressed by additional regulatory elements possibly located in the amino-terminal half of the molecule.
Cheng
S H
SH
Laboratory of Cellular Regulation, Genzyme Corporation, Framingham, Massachusetts 01701.
Espino
P C
PC
Marshall
J
J
Harvey
R
R
Merrill
J
J
Smith
A E
AE
D000595:Amino Acid Sequence; D000818:Animals; D000952:Antigens, Polyomavirus Transforming; D001483:Base Sequence; D002460:Cell Line; D002678:Chimera; D014644:Genetic Variation; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015345:Oligonucleotide Probes; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000302:isolation & purification; D010766:Phosphorylation; D010957:Plasmids; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000235:genetics*; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000378:metabolism; D051076:Proto-Oncogene Proteins c-fyn; D011519:Proto-Oncogenes; D012689:Sequence Homology, Nucleic Acid
D000952:Antigens, Polyomavirus Transforming; C000715470:MAS1 protein, human; D015345:Oligonucleotide Probes; D010748:Phosphopeptides; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins; D011505:Protein-Tyrosine Kinases; C494957:FYN protein, human; D051076:Proto-Oncogene Proteins c-fyn