Regulation of N‐myristoyltransferase by Akt/PKB Mediated Phosphorylation

Abstract

Post‐translational modifications of proteins create highly dynamic relay system that regulates the core dynamic signalling pathways in response to the alterations in the cellular microenvironment. N‐myristoyltransferase (NMT) has emerged as a central node upstream of diverse signalling proteins regulating cellular survival. NMT modifies proteins in both co‐and post‐translational manner and modulates functioning of the signalling proteins by addition of the myristoyl moiety to an exposed N‐terminal glycine. The sequence feature of NMT reflects that it harbours an Akt/PKB kinase recognition motif within the aD‐ aE loop region. The in vitro kinase assay on the synthetic peptides corresponding to the aD‐aE loop followed by MALDI‐MS analysis established that the sequence motif is a valid substrate for the Akt/PKB kinase. The MS‐MS analysis of the phosphorylated peptide identified Thr319 within aD‐ aE loop region as the site of phosphorylation. To delineate the effects of phosphorylation, phosphomimetic mutant (Thr319Glu) and a null‐mutant (Thr319Ala) were engineered onto the catalytic domain the NMT. Enzymatic assays of the purified recombinant enzymes reflected a drastic loss in activity by mutations at the site Thr319. The analysis of NMT crystal structures shows that this loop region undergoes a conformational change upon binding of the co‐substrate myristoyl‐CoA (MYA). The binding of peptide substrate follows the MYA binding event and Thr319 in the aD‐ aE loop region lies in proximity to the peptide‐substrate binding site. In summary, we have established the site of phosphorylation and the regulation of NMT by the kinase Akt/PKB. These findings indicate that Akt/PKB kinase restricts NMT activity by modulating the peptide substrate affinity presumably by changing in the interaction network around the substrate‐binding site. Delineating of the functional consequences of this novel phosphorylation event is expected to improve our understanding of NMT regulations in diversified cellular needs.Support or Funding InformationThis work was supported by the Canadian Breast Cancer Foundation‐Prairies/NWT operating grant to RKS (Grant number ‐ 412572).