Role of N-myristoyltransferase inhibitor protein in differentiation of myeloid progenitors (U937 cells)

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The human cSrc proto-oncogene encodes a non-receptor tyrosine kinase, pp60^c-src, which plays a significant role in the growth mediated signaling pathway leading to cellular proliferation and transformation. Elevated in vitro protein-tyrosine kinase activity of pp60^src in colon carcinomas compared to the normal mucosa adjacent to the tumor established its role in carcinogenesis. Myristoylation of pp60^c-src leads to its membrane association and consequently its activation. Myristoylation of proteins is catalyzed by N-myristoyltransferase (NMT). We have shown earlier the elevated activity and expression of NMT in colon and gallbladder carcinomas. In this report we investigated the role of pp60^c-src, NMT and its inhibitor protein (NIP) during phorbol 12-myristate 13-acetate (PMA) induced differentiation of U937 cells. NMT assay mixture contained 40 mM Tris-HCl, pH7.4, 0.5 mM EGTA, 0.45 mM 2-mercaptoethanol, 1 % Triton X-100, 500 μM peptide substrate and NMT in a total volume of 25 μL. The transferase reaction was initiated by the addition of freshly generated [³H]myristoyl-CoA and was incubated at 30 °C for 30 minutes. The reaction was terminated by spotting 15 μL aliquots of incubation mixture onto P81 phosphocellulose paper discs and radioactivity was quantified. One unit of NMT activity was expressed as 1 pmol of myristoyl peptide formed per min. Western and Northern blot analysis, kinase assay, Immunofluorescence analysis and Immunoprecipitation were carried out using standard procedures. Src kinase activity and protein expression increased during the differentiation process and reached a maximum at 72 h. NMT expression also reached maximum at 72 h and remained elevated. However, NMT activity followed protein expression profile of NMT up to 48 h and then decreased considerably and remained so till further differentiation process. In addition, NIP was observed to be expressed after 48 h. These results suggest that induction of NIP after 48 h regulates NMT activity. Furthermore, during the differentiation process src and NIP translocates to the Golgi apparatus and so does NIP. Regulation of NMT activity by NIP during the differentiation process is novel and provides insight into the myristoylation of proteins involved in signal transduction and cellular differentiation. The concerted translocation of c-src and NIP from cytosol to Golgi apparatus is reported for the first time and may play a significant role in the differentiation process. *This work is supported by the Canadian Institutes of Health Research, Canada.