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.
