BIOCHEMICAL-CHARACTERIZATION OF A PALMITOYL ACYLTRANSFERASE ACTIVITY THAT PALMITOYLATES MYRISTOYLATED PROTEINS

Dynamic regulation of signal transduction by revers reversible palmito ylation-depalmitoylation cycles has been recently described, However, further understanding of fatty acylation reactions has been hampered b y our lack of knowledge about the specific transferases and thio ester ases involved. Here, we describe an assay for the palmitoyl acyltransf erase (PAT) that palmitoylates ''myrGlyCys'' containing members of the Src family of protein tyrosine kinases (PTKs), Since N-myristoylation of Fyn PTK, a member of the Src family, has been shown to be a prereq uisite for palmitoylation, a new single plasmid vector that allows ove rexpression of myristoylated Fyn substrate in Escherichia coli was dev eloped, Purified myristoylated protein substrates were incubated with [I-125]iodopalmitoyl CoA, a palmitoyl CoA analog, in the presence of b ovine brain lysates, Transfer of radiolabel to the Fyn substrate was d etected by SDS-polyacrylamide gel electrophoresis and autoradiography. This assay was used to partially purify and characterize PAT activity from bovine brain. Here, we demonstrate that PAT is a membrane-bound enzyme, which palmitoylates myristoylated Fyn substrates containing a cysteine residue in position three. The PAT activity attached palmitat e to Fyn proteins via a thioester Linkage and exhibited a fatty acyl C oA preference for long chain fatty acids, It is likely that palmitoyla tion of Fyn and other Src family members by PAT regulates PTK localiza tion and signaling functions.