Two N-myristoyltransferase isozymes play unique roles in protein myristoylation, proliferation, and apoptosis.
N-myristoyltransferases (NMT) add myristate to the NH(2) termini of certain proteins, thereby regulating their localization and/or biological function. Using RNA interference, this study functionally characterizes the two NMT isozymes in human cells. Unique small interfering RNAs (siRNA) for each isozyme were designed and shown to decrease NMT1 or NMT2 protein levels by at least 90%. Ablation of NMT1 inhibited cell replication associated with a loss of activation of c-Src and its target FAK as well as reduction of signaling through the c-Raf/mitogen-activated protein kinase/extracellular signal-regulated kinase kinase/extracellular signal-regulated kinase pathway. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assays showed that depletion of either NMT isozyme induced apoptosis, with NMT2 having a 2.5-fold greater effect than NMT1. Western blot analyses revealed that loss of NMT2 shifted the expression of the BCL family of proteins toward apoptosis. Finally, intratumoral injection of siRNA for NMT1 or for both NMT1 and NMT2 inhibited tumor growth in vivo, whereas the same treatment with siRNA for NMT2 or negative control siRNA did not. Overall, the data indicate that NMT1 and NMT2 have only partially overlapping functions and that NMT1 is critical for tumor cell proliferation.
Ducker
Charles E
CE
Apogee Biotechnology Corporation, Hershey, PA, USA.
Upson
John J
JJ
French
Kevin J
KJ
Smith
Charles D
CD
D000217:Acyltransferases / Q000737:chemistry*; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D004305:Dose-Response Relationship, Drug; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005260:Female; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D020287:In Situ Nick-End Labeling; D051379:Mice; D008807:Mice, Inbred BALB C; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009227:Myristic Acids / Q000737:chemistry; D020033:Protein Isoforms; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection; D019061:src-Family Kinases / Q000378:metabolism
D000970:Antineoplastic Agents; D009227:Myristic Acids; D020033:Protein Isoforms; D034741:RNA, Small Interfering; D049452:Green Fluorescent Proteins; D000217:Acyltransferases; C050480:glycylpeptide N-tetradecanoyltransferase; D019061:src-Family Kinases; D019908:Proto-Oncogene Proteins c-raf; D048049:Extracellular Signal-Regulated MAP Kinases