A 31-amino-acid N-terminal extension regulates c-Crk binding to tyrosine-phosphorylated proteins.
Overproduction of v-Crk, but not of c-Crk, in chicken embryo fibroblasts results in cell transformation. The transforming activity of v-Crk mutants correlates with their ability to cause increased tyrosine phosphorylation of specific cellular proteins, a property that depends on the binding of v-Crk to phosphotyrosine residues via its SH2 domain. In this study, proteins translated in rabbit reticulocyte lysates were used to analyze interactions between Crk derivatives and tyrosine-phosphorylated proteins, particularly the epidermal growth factor (EGF) receptor. The results demonstrate that the binding affinity of c-Crk is much lower than that of v-Crk, despite the fact that both proteins contain identical SH2 domains. Moreover, a 31-amino-acid N-terminal extension of c-Crk, resulting from upstream translational initiation at a CUG codon, significantly increases the ability of the resulting protein to bind to phosphotyrosine-containing proteins. Of those 31 amino acids, 24 can be found in the 27-amino-acid region between Gag and Crk sequences in v-Crk, and removal of this region results in a protein with lower affinity toward the EGF receptor. In addition, fusion of Gag to the amino terminus of c-Crk yields a protein with a binding activity that is lower than that of v-Crk but significantly higher than that of c-Crk without the fusion. These data suggest that sequences N terminal to the Crk SH2 regulate binding activity to tyrosine-phosphorylated proteins and that the amino acids encoded immediately 5' to the c-Crk initiator AUG specifically increase binding affinity. In contrast, deletion of one or two SH3 domains of c-Crk proteins did not change their affinity for the EGF receptor. These results were confirmed in vivo by using A431-derived cell lines overproducing either the chicken c-Crk protein or c-Crk with the 31-amino-acid N-terminal extension. Furthermore, the in vivo experiments suggest that binding of Crk proteins to the stimulated EGF receptor results in Crk phosphorylation and subsequent loss of binding affinity.
Fajardo
J E
JE
Rockefeller University, New York, New York 10021.
Birge
R B
RB
Hanafusa
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D002642:Chick Embryo; D004247:DNA / Q000235:genetics; D066246:ErbB Receptors / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D050736:Oncogene Protein v-crk; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D019000:Phosphotyrosine; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000378:metabolism*; D050737:Proto-Oncogene Proteins c-crk; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D015682:Retroviridae Proteins, Oncogenic / Q000235:genetics / Q000378:metabolism; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism
D050736:Oncogene Protein v-crk; D010750:Phosphoproteins; D011518:Proto-Oncogene Proteins; D050737:Proto-Oncogene Proteins c-crk; D012333:RNA, Messenger; D015682:Retroviridae Proteins, Oncogenic; D019000:Phosphotyrosine; D014443:Tyrosine; D004247:DNA; D066246:ErbB Receptors