A fluorescence-based high performance liquid chromatographic method for the characterization of palmitoyl acyl transferase activity

Huntingtin interacting protein 14 is an oncogenic human protein: palmitoyl acyltransferase

“…Furthermore, HIP14 does not modify the Nterminal myristoylated-palmitoylation motif indicating that there are additional PATs expressed in human cells. This is consistent with our previous biochemical characterization of PAT activity in mouse and human tumor cells (Varner et al, 2002(Varner et al, , 2003. It will be interesting to determine if alterations in HIP14 affect subcellular localization of the endogenous RAS protein.…”

“…The in vitro palmitoylation assay developed in this laboratory (Varner et al, 2002) has demonstrated an increase in palmitoylation activity in RAS-transformed NIH 3t3 cells as compared to wild-type cells. This increased PAT activity is specific for a peptide substrate that mimics the C-terminal RAS farnesylated-palmitoylation motif, FarnCNRas(NBD), but not toward one that mimics the N-terminal myristoylated-palmitoylation motif, MyrGCK(NBD) (Varner et al, 2002(Varner et al, , 2003.…”

“…This increased PAT activity is specific for a peptide substrate that mimics the C-terminal RAS farnesylated-palmitoylation motif, FarnCNRas(NBD), but not toward one that mimics the N-terminal myristoylated-palmitoylation motif, MyrGCK(NBD) (Varner et al, 2002(Varner et al, , 2003. These two substrates represent the two palmitoylation motifs identified for cellular proteins (Resh, 1999) that are clear targets for PAT enzymes (Varner et al, 2002(Varner et al, , 2003. RT-PCR analysis of RNA from these same cells has revealed that HIP14 is overexpressed in N-, K-, and H(EJ)-RAS-transformed NIH 3t3 cells, with EJ-RAS cells having the highest level of HIP14 message ( Figure 1).…”

“…The subcellular fractions were prepared as described in Varner et al (2002). Briefly, cells were grown to 70% confluence in 150 mm tissue culture dishes and collected by centrifugation.…”

“…The IVP assay was performed as described in Varner et al (2002Varner et al ( , 2003. Briefly, IVP assays with the MyrGCK(NBD) ((myr)GCK-NBD) substrate consisted of 10 mM deprotected peptide, 2 mM palmitoyl-CoA, 50 mg membrane protein, and acylation buffer (50 mM citrate, 50 mM phosphate, 50 mM Tris, and 50 mM CAPS at pH 7.2) in a total volume of 100 ml.…”