Journal: Biology Open

Article Title: Regulation of behavioral circadian rhythms and clock protein PER1 by the deubiquitinating enzyme USP2

doi: 10.1242/bio.20121990

Figure Lengend Snippet: ( A ) PER1 is ubiquitinated in cells. PER1-V5(His) 6 was co-expressed with HA-Ub in HEK293 cells. Cells were treated without or with proteasome inhibitor MG132 for 6 h to accumulate ubiquitin-conjugated proteins and then lysed under denaturing conditions. His-tagged PER1 was affinity-isolated with Ni-NTA agarose beads and blotted with anti-HA antibody to detect ubiquitinated PER1 (lower panel). A broad band of approximately 200 kDa was found to be the major form of PER1. This experiment was done twice, with similar results. ( B ) PER1 is expressed in cells as two major isoforms, of ∼136 kDa and ∼200 kDa. HEK293 cells were transfected with empty plasmid or plasmid expressing V5-tagged PER1. Lysates were then analyzed by immunoblotting with either anti-V5 or anti-PER1 antibody. ( C ) High molecular weight (MW) PER1 contains multiple bands. HEK293 cells were transfected and whole cell lysates were prepared as in (A), PER1 was immunoprecipitated with anti-V5 antibody. The immune complexes were separated by 5% SDS-PAGE to resolve proteins in high molecular weight region, immunoblotted with anti-HA antibody to detect ubiquitinated PER1 (upper panel), and re-blotted with anti-V5 antibody (lower panel). Multiple ubiquitinated PER1 bands were found to migrate in the gel region around 200 kDa. ( D ) High MW PER1 is regulated by the abundance of ubiquitin in cultured cells. PER1-V5(His) 6 was expressed in U2Os-shUb and U2Os-shUb-HAUbWT cells, which were treated with tetracycline to induce expression of the shUb to silence the endogenous ubiquitin genes in both cell lines and simultaneously induce the expression of HA-tagged ubiquitin in the U2Os-shUb-HAUbWT cell line. The ratio of the ubiquitinated to the non-ubiquitinated form of PER1 was determined. This experiment was done twice, with similar results. ( E ) PER1-V5 was expressed in HEK293 cells and the cells were treated with MG132 for 6 h prior to lysis in the absence of phosphatase inhibitors. PER1-V5 was immunoprecipitated with anti-V5 antibody, incubated with alkaline phosphatase, resolved on 5–10% SDS-PAGE, and blotted with anti-V5 antibody. ( F , G ) USP2, but not USP3 or USP19, regulates the ubiquitination of PER1 in HEK293 cells. (F) USP2a wild-type (USP2aWT) or inactive mutant (USP2aCA) was co-expressed with PER1-V5(His) 6 and HA-Ub in HEK293 cells. PER1-V5(His) 6 was isolated and analyzed as in panel (A). The ubiquitinated form of PER1 is decreased in the presence of USP2aWT. This experiment was done twice, with similar results. Note that the ∼200 kDa band is less broad than in other figures due to use of a minigel. (G) PER1-V5(His) 6 was expressed in HEK293 cells alone or with HA-tagged USP2b, Myc-tagged USP19 or USP3. Cells were lysed and cellular proteins were analyzed by immunoblotting with the indicated antibodies. ( H , I ) Levels of ubiquitinated PER1 accumulate in Usp2 KO embryonic fibroblasts. (H) Lysates from either WT or Usp2 KO MEFs (unsynchronized) were analyzed by immunoblotting with anti-PER1 antibodies. The ubiquitinated PER1 protein levels were quantified and normalized to actin. Shown are a representative blot and the mean±SEM from three independent experiments. (I) Lysates were prepared from either WT or Usp2 KO MEFs at the indicated times following synchronization by a serum shock as in Fig. 3 and analyzed by immunoblotting with anti-PER1 antibodies. The ubiquitinated PER1 protein levels were quantified, normalized to γ-tubulin and calibrated to the PER1 levels of WT MEF at time 0. The immunoblots from two series of culture wells processed in parallel gave similar results and one series is shown here. The experiment was done twice using two different sets of MEFs prepared from embryos in different backcross backgrounds and similar results were seen in the two separate experiments.

Article Snippet: The HA-tagged ubiquitin expressing plasmid (HA-Ub/pRK5) was from Addgene.

Techniques: Isolation, Transfection, Plasmid Preparation, Expressing, Western Blot, Molecular Weight, Immunoprecipitation, SDS Page, Cell Culture, Lysis, Incubation, Mutagenesis