Myristoylation and membrane binding regulate c-Src stability and kinase activity.
Myristoylation is critical for membrane association of Src kinases, but a role for myristate in regulating other aspects of Src biology has not been explored. In the c-Abl tyrosine kinase, myristate binds within a hydrophobic pocket at the base of the kinase domain and latches the protein into an autoinhibitory conformation. A similar pocket has been predicted to exist in c-Src, raising the possibility that Src might also be regulated by myristoylation. Here we show that in contrast to the case for c-Abl, myristoylation exerts a positive effect on c-Src kinase activity. We also demonstrate that myristoylation and membrane binding regulate c-Src ubiquitination and degradation. Nonmyristoylated c-Src exhibited reduced kinase activity but had enhanced stability compared to myristoylated c-Src. We then mutated critical residues in the predicted myristate binding pocket of c-Src. Mutation of L360 and/or E486 had no effect on c-Src membrane binding or localization. However, constructs containing a T456A mutation were partially released from the membrane, suggesting that mutagenesis could induce c-Src to undergo an artificial myristoyl switch. All of the pocket mutants exhibited decreased kinase activity. We concluded that myristoylation and the pocket residues regulate c-Src, but in a manner very different from that for c-Abl.
Patwardhan
Parag
P
Cell Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA.
Resh
Marilyn D
MD
D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism*; D002645:Chickens; D002522:Chlorocebus aethiops; D005786:Gene Expression Regulation; D019814:Myristic Acid / Q000378:metabolism*; D011485:Protein Binding; D055550:Protein Stability; D050721:Proto-Oncogene Proteins c-cbl / Q000235:genetics / Q000378:metabolism; D054875:Ubiquitination; D019061:src-Family Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D019814:Myristic Acid; D050721:Proto-Oncogene Proteins c-cbl; D019061:src-Family Kinases