Myristic acid analogs as probes for the study of acylation in lymphocytes

Monica J. S Nadler, Purdue University

Abstract

Protein N-myristoylation, catalyzed by myristoyl-CoA:protein N-myristoyltransferase (NMT), results in the cotranslational covalent attachment of myristate to the N-terminal glycine of a target protein. An analog of myristic acid, 2-hydroxymyristic acid (HMA), is metabolically activated to 2-hydroxymyristoyl-CoA which is a potent inhibitor of NMT. N-Myristoylation of the protein tyrosine kinase p56$\sp{lck},$ is necessary for its proper targeting to the plasma membrane. In LSTRA cells, which overexpress p56$\sp{lck},$ incubation with HMA led to the synthesis of nonmyristoylated p56$\sp{lck},$ which was cytosolic, had an altered state of phosphorylation, and was less stable than myristoylated p56$\sp{lck}.$ In cell lines that do not overexpress p56$\sp{lck},$ HMA treatment resulted in a decrease in the amount of p56$\sp{lck}.$ Treatment of CD4$\sp+$ cells with HMA decreased the amount of p56$\sp{lck}$ associated with CD4 and resulted in decreased p56$\sp{lck}$ activation after CD4 crosslinking. HMA treatment of B cell lines also decreased the level of the Src-family tyrosine kinase, p53/56$\sp{lyn},$ without altering the level of the nonmyristoylated tyrosine kinase p72$\sp{syk}.$ Treatment of B cells with 12-methoxydodecanoic acid resulted in a decreased association of p53/56$\sp{lyn}$ with the IgM antigen receptor and in a diminished activation of this kinase after receptor crosslinking. These studies demonstrate the utility of targeting NMT to disrupt N-myristoylated tyrosine kinases involved in signaling. p56$\sp{lck}$ was identified as a dually acylated protein, modified posttranslationally with palmitate and cotranslationally with myristate. The $\lbrack\sp3\rm H\rbrack$palmitoyl-p56$\sp{lck}$ linkage was sensitive to cleavage with neutral hydroxylamine, indicating $\lbrack\sp3\rm H\rbrack$palmitate was attached in an ester bond. Palmitoylation of p56$\sp{lck}$ was readily reversible whereas N-myristoylation was not. p56$\sp{lck}$ is one of the first examples of a protein that is both N-myristoylated and palmitoylated. The substrate specificity of posttranslational acyltransferases is not well understood. 5-Nonanyloxyfuran-2-carboxylic acid, an analog of myristic acid, specifically inhibited the posttranslational incorporation of $\lbrack\sp3\rm H\rbrack$myristate but not $\lbrack\sp3\rm H\rbrack$palmitate or $\lbrack\sp3\rm H\rbrack$stearate into the transferrin receptor. These studies suggest the existence of multiple posttranslational acyltransferases which have distinct substrate specificity toward myristate or palmitate.

Degree

Ph.D.

Advisors

Geahlen, Purdue University.

Subject Area

Biochemistry

COinS