NAMPT-dependent deacetylation of the hematopoietic-specific lyn-substrate 1 (HCLS1)

Recently, we demonstrated an essential role of the hematopoietic cell-specific Lyn substrate 1 (HCLS1 or HS1) protein in myeloid differentiation of human and mouse hematopoietic cells. HCLS1 is an interaction partner of HAX1, which is mutated in patients with severe congenital neutropenia (CN). In these patients HCLS1 expression and functions are severely downregulated, leading to “maturation arrest” of myelopoiesis. We also described the new pathway of myeloid differentiation downstream of G-CSF in healthy individuals and in CN patients: G-CSF induced Nampt and NAD⁺, which activated NAD+-dependent protein deacetylases, sirtuins. A huge amount of proteins are post-translationally modified by acetylation or deacetylation. However, it is unclear whether changes in the acetylation status activates or suppresses functions of proteins.

In the present study we analyzed if HCLS1 protein could be de-/acetylated and if de-/acetylation of HCLS1 affects its functions during myeloid differentiation. We found that HCLS1 could be acetylated on 4 lysines. We generated rabbit polyclonal antibody specific for each acetylated lysine and found that HCLS1 is acetylated on three lysines in the acute myeloid leukemia cell lines NB4 and HL60 as well as in CD34⁺ hematopoietic cells. We also found that SIRT1 and SIRT2 interact with HCLS1 protein in NB4 cells. Moreover, specific inhibition of Nampt (FK866) in NB4 cells led to enhanced acetylation of HCLS1 on Lys 123 and 241. Inhibition of SIRT2 (AC93253) elevated HCLS1 acetylation on Lys 123 and 192. Moreover, treatment of CD34⁺ cells with G-CSF induced interaction between SIRT2 and HCLS1 proteins, migration of SIRT2:HCLS1 complexes and changed acetylation status of HCLS1. Effects of Nampt/SIRT-triggered deacetylation on HCLS1 functions remain to be investigated.