Biochemical Characterization of a Palmitoyl Acyltransferase Activity That Palmitoylates Myristoylated Proteins

doi:10.1074/jbc.270.38.22399

Biochemical Characterization of a Palmitoyl Acyltransferase Activity That Palmitoylates Myristoylated Proteins (*)

From the Department of Cell Biology and Genetics, Memorial Sloan Kettering Cancer Center, New York, New York 10021

^¶ A Rita Allen Foundation Scholar and an Established Scientist of the American Heart Association. To whom correspondence should be addressed:
Dept. of Cell Biology and Genetics, Memorial Sloan Kettering Cancer Ctr., 1275 York Ave., Box 143, New York, NY 10021
. Tel.: 212-639-2514; Fax: 212-717-3317.

Abstract

Dynamic regulation of signal transduction by reversible palmitoylation-depalmitoylation cycles has been recently described. However, further understanding of fatty acylation reactions has been hampered by our lack of knowledge about the specific transferases and thioesterases involved. Here, we describe an assay for the palmitoyl acyltransferase (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 prerequisite for palmitoylation, a new single plasmid vector that allows overexpression of myristoylated Fyn substrate in Escherichia coli was developed. Purified myristoylated protein substrates were incubated with iodopalmitoyl CoA, a palmitoyl CoA analog, in the presence of bovine brain lysates. Transfer of radiolabel to the Fyn substrate was detected 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 palmitate to Fyn proteins via a thio-ester linkage and exhibited a fatty acyl CoA preference for long chain fatty acids. It is likely that palmitoylation of Fyn and other Src family members by PAT regulates PTK localization and signaling functions.

Footnotes

↵^§ Supported by a Postdoctoral fellowship from the Fonds de la Recherche en Santé du Québec. Present address: Lipid and Lipoprotein Research Group, Dept. of Anatomy and Cell Biology and Biochemistry, University of Alberta, 328 Heritage Medical Research Ctr., Edmonton T6G 2S2, Canada.
↵* This research was supported by National Institutes of Health Grant CA52405 and American Cancer Society Grant BE-235. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

NMT

N-myristoyl transferase

FynSH432His

Fyn protein-tyrosine kinase truncated after the SH2 domain to which a hexahistidine tag was appended

IC16

iodohexadecanoic acid (a palmitate analog)

myr

myristate

pal

palmitate

PAT

palmitoyl acyltransferase

PTK

protein-tyrosine kinase

SH4

SH3, and SH2, Src homology domains 4, 3, and 2, respectively

PAGE

polyacrylamide gel electrophoresis

PCR

polymerase chain reaction

WT

wild type.
↵²L. Berthiaume and M. D. Resh, unpublished results.
↵³L. Berthiaume and M. D. Resh, unpublished observations.
↵⁴L. Berthiaume and M. D. Resh, unpublished data.
- Received June 22, 1995.
- Revision received July 24, 1995.