Hematopoietic-specific lyn-substrate 1 (HCLS) is deacetylated by NAMPT/NAD+/SIRT1 pathway

Recently we identified HCLS1 protein to be an essential player in the G-CSFR signalling pathway in myeloid differentiation (Skokowa et al., 2012). We also described a new G-CSFR signalling through activation of Nicotinamide phosphoribosyltransferase (NAMPT)/NAD⁺/Sirtuins in healthy individuals and in CN patients. We found that G-CSF-triggered deacetylation of myeloid specific factors is essential for myeloid differentiation (Skokowa et al., 2009).

In the present study we aimed to investigate whether HCLS1 is regulated by NAMPT/NAD⁺/Sirtuin pathway via deacetylation. We identified four acetyl-lysine sites in HCLS1 protein and generated rabbit polyclonal antibodies specifically recognized each of acetylated lysines. We also generated lentiviral constructs contained HCLS1 cDNA in which one of acetylated lysines of HCLS1 protein is replaced by a single amino acid. Using immunoprecipitation with anti-acetyl-lysine antibody we found that HCLS1 is acetylated in HL60 myeloid cells upon pre-treatment of cells with histone deacetylase inhibitors. We also showed that treatment of HL60 and NB4 cells with high doses of Nicotinamide but not Trichostatin A enhanced HCLS1 acetylation on lysine 123 and 241, suggesting that class III histone deacetylases (Sirtuin family) could deacetylate HCLS1 protein.

We further found that NAD⁺ and NAMPT treatment of the acute myeloid leukemia cell line HL60 decreased HCLS1 acetylation on both lysines 123 and 241. Similar effects were observed in HL60 cells transduced with lentiviral construct expressing NAMPT cDNA. Inhibition of NAMPT using specific inhibitor FK866 increased HCLS1 acetylation on acetyl-lysines 123 and 241 in NB4 and on acetyl-lysine 241 in HL60 cells. To evaluate the mechanism of NAMPT-dependent deacetylation of HCLS1, we performed immunoprecipitation experiments in cell lysates of HL60 cells using anti-SIRT1 antibody and found interaction between endogenous HCLS1 and SIRT1 proteins. Moreover, interaction between HCLS1 and SIRT1 proteins was detected by immunoprecipitation in 293T cells over-expressing HCLS1 and SIRT1. Taken together, we concluded that HCLS1 is deacetylated through NAMPT/NAD⁺/SIRT1 pathway and that in patients with severe congenital neutropenia not only diminished expression and phosphorylation but also elevated NAMPT-triggered deacetylation may contribute to the neutropenic phenotype.