Figure 2.

Phosphorylation of Httex1p regulates its ubiquitination, SUMOylation, acetylation, and nuclear localization. (A and B) Phosphorylation of serines 13 and 16 regulates mutant Httex1p ubiquitination and SUMOylation. St14A (A) or HeLa (B) cells were transiently transfected with vector or HIS-ubiquitin (A) or HIS-SUMO-1 (B) and control and mutant 97QP VL* Httex1p. Conjugated proteins were purified under denaturing conditions by Ni-NTA magnetic nickel beads and Htt detected with anti-Htt CAG53b by Western analysis. (C) Mass spectrometry analysis shows that Htt S13 phosphorylation can occur with K9 acetylation. 25QP-HBH was purified from St14A cells cotransfected with IKK-β and CBP and treated for 2 h with histone deacetylase inhibitors 200 mM Trichostatin A/5 mM Nicotinamide. ESI-MS/MS spectra were obtained after chymotryptic digestion and collision-induced dissociation (CID) for a peptide acetylated at K9 and phosphorylated at S13 MAcKAFEpSLKSF, [MH₂]⁺² at m/z 655.30⁺² (M = 1308.60 D). (D) IKK-β overexpression increases phosphorylation of Htt S13 and acetylation of K9. St14A cells were transiently transfected with Httex1p-H4 with 25 or 46Qs, +/− IKK-β or with 46QP QEE-H4. Htt was purified and subjected to Western analysis with anti-K9-Ac, anti-S13-P, and CAG53b. (E) Mimicking phosphorylation significantly increases nuclear localization in primary neurons. Primary cortical neurons were cotransfected with pcDNA3.1-mRFP and 97QP-GFP or 97QP-DD-GFP plasmids. The subcellular distribution of these polypeptides was examined by measuring the fluorescence intensity of GFP, to which they are fused, in regions of the nucleus and cytoplasm for each cell. The extent to which these polypeptides localized preferentially to the nucleus or the cytoplasm was determined by calculating the ratio of nuclear/cytoplasmic GFP fluorescence intensity and comparing the distribution of the two polypeptides by t test. Error bars indicate SEM in arbitrary units.