Identifying Substrates of KDAC6

Abstract

Lysine acetylation is a post‐translational modification that occurs on human proteins. The presence of acetylation on these proteins is important for the development of diseases; therefore, deacetylation is also important. KDAC6 is part of a metal‐dependent enzyme family that is responsible for reversing lysine acetylation. To gain insight into how KDAC6 selects substrates to deacetylate, we hypothesized that KDAC6 activity with peptides derived from putative substrate proteins would predict activity with the corresponding full‐length protein. Using an in vitro fluorescence‐based assay, we determined that several such peptides are substrates of KDAC6. However, the peptides derived from the putative substrates were not always predictive of activity of the full‐length protein with KDAC6. To determine whether KDAC6 is responsible for deacetylation of the corresponding full‐length protein in their biological context, we utilized cell‐based studies that detect changes in acetylation status of target proteins dependent on cellular KDAC6 activity. Target proteins were isolated by immunoprecipitation and the acetylation status determined using an acetyl‐specific antibody of the protein. Isolated target proteins were then reacted with a recombinant KDAC in vitro to determine whether the enzyme is capable of deacetylating the full‐length protein. Overall, we observed that only some putative substrates previously attributed to KDAC6 show evidence of a direct deacetylation by KDAC6. Altogether, our data provide robust evidence for biologically‐relevant KDAC6 substrates. Identifying direct targets of KDAC6, as well as other KDACs, is important for understanding the specificity of KDAC6 in a biological context, and will ultimately contribute to our understanding of the role of KDAC6 in disease.