Ubiquitination of HDAC4, HDAC5, and HDAC6. (A and B) 293 cells were transfected with flag-tagged HDAC4, HDAC5, HDAC6, or HDAC6Δzf (comprising amino acids 1–1111) with or without HA-Ub. Before harvesting, cells were treated with 80 μM proteasome inhibitor, MG-132, for 2 h. Cell lysates were immunoprecipitated with flag antibody and blotted for HA. Brackets denote ubiquitin conjugated HDACs (Upper). The corresponding flag Western blots of whole cell lysates (5% immunoprecipitation input). Asterisk denotes a nonspecific band that cross reacts with the flag antibody (Lower). A and B are run on SDS/8% and 10% PAGE, respectively. (C) In vitro ubiquitination of HDAC5 and HDAC6. [³⁵S]methionine-labeled HDACs were incubated with HeLa nuclear extract in the absence and presence of the proteasome inhibitor, MG-132, and the DUB inhibitor, Ub-al. Reactions were electrophoresed on SDS/6.5% PAGE and analyzed by PhosphoImager.

The C terminus of HDAC6 interacts with polyubiquitin in yeast two-hybrid and GST pull-down assays. (A) Schematic diagram of the HDAC6 baits and polyubiquitin library clone 6.3. (B) Interaction of polyubiquitin clone 6.3 (as indicated by blue color) with different regions of HDAC6, HDAC4, or HDAC 5. (C) Yeast strains expressing mutations in residue K48 within the 6.3 library clone were mated to yeast expressing HDAC6 or HDAC6Δzf (in duplicate) and tested for β-galactosidase activity. (D) Interaction of HDAC6 and the isolated zinc finger domain (amino acids 1045–1216) with polyubiquitin clone 6.3 and monoubiquitin (in duplicate). β-galactosidase assay with amino acids 1045–1216 were terminated after 20 min, whereas the assay with full-length HDAC6 was carried out for 80 min. (E) HDAC6 interacts with GST-polyubiquitin in vitro. GST, GST-monoubiquitin, and GST-polyubiquitin were purified on glutathione Sepharose beads and incubated with 293 lysates from cells mock transfected, transfected with flag-HDAC6, or flag-HDAC6Δzf. Proteins binding to the GST fusion proteins were run on SDS/8% PAGE and blotted with flag antibodies. Asterisks denote HDAC 6 or HDAC6Δzf in whole cell lysates. Several bands in the mock input lanes nonspecifically react with the flag antibodies.

Deubiquitinating activity copurifies with full-length HDAC6 and HDAC6Δzf. Flag HDAC6 and HDAC6Δzf were immunopurified from 293 cells and were assayed for DUB activity in vitro. Reaction mixtures were resolved on SDS/15% PAGE and Western blotted with anti-ubiquitin antibodies.

Pulse–chase analysis of HDAC5 and HDAC6. Cos-1 cells expressing flag-HDAC5 or HDAC6 were pulse labeled with [³⁵S]methionine for 30 min and chased for the indicated times with media containing an excess of cold methionine. HDACs were immunoprecipitated with flag antibody and analyzed on SDS/8% PAGE by autoradiography.

Amino acid alignment of the zinc finger of HDAC6 with human DUBs and BRCA1 associated proteins. Asterisks denote conserved C and H residues. USPs are members of the UBP family.