Limited proteolysis of human histone deacetylase 1.
BACKGROUND
Histone deacetylase (HDAC) proteins are associated with cell proliferation, differentiation, apoptosis, and cancer. Specifically, HDAC1 is linked with cell growth, a hallmark of cancer formation. HDAC1 is a phosphoprotein and phosphorylation at S421 and S423 promotes HDAC1 enzymatic activity and protein association. While single and double point mutants of HDAC1 at S421 and S423 appear functionally similar, the evidence suggests that HDAC1 is phosphorylated simultaneously at both S421 and S423 in vivo. Additional experiments are necessary to probe the role of double phosphorylation of HDAC1 at S421 and S423.


RESULTS
To characterize HDAC1 phosphorylation at S421 and S423, limited proteolysis of HDAC1 was performed for the first time. HDAC1 degraded without production of discrete fragments. By performing concentration-dependent proteolysis, HDAC1 double point mutants with disrupted phosphorylation at S421 and S423 displayed different trypsin sensitivities compared to wild type HDAC1. Unexpectedly, HDAC1 single point mutants with disrupted phosphorylation at either S421 or S423 demonstrated protease sensitivity similar to the wild type HDAC1.


CONCLUSIONS
Concentration-dependent proteolysis experiments provide evidence that phosphorylation of S421 and S423 individually contribute to HDAC1 function. In addition, the limited proteolysis experiments support a model where associated proteins promote HDAC1 enzymatic activity, reinforcing the importance of protein interactions in HDAC1 structure and function. Finally, because HDAC1 does not display distinct regions of protease sensitivity, the proteolysis studies suggest that HDAC1 comprises inter-related structural regions.
Kamath
Nayana
N
Department of Chemistry, Wayne State University, Detroit, MI 48202, USA. nayana@chem.wayne.edu
Karwowska-Desaulniers
Paulina
P
Pflum
Mary Kay H
MK
D000595:Amino Acid Sequence; D015870:Gene Expression; D056284:Histone Deacetylase 1; D006655:Histone Deacetylases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D019169:Jurkat Cells; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010766:Phosphorylation; D011485:Protein Binding; D014357:Trypsin / Q000378:metabolism
D014357:Trypsin; C110550:HDAC1 protein, human; D056284:Histone Deacetylase 1; D006655:Histone Deacetylases