Journal: Molecular and Cellular Biology
Article Title: N-Terminal Phosphorylation of HP1? Promotes Its Chromatin Binding ▿
Figure Lengend Snippet: Identification of phosphorylation sites of mouse HP1α. (A) The phosphorylation patterns of mouse HP1α analyzed by phos-tag Western blotting (phos-tag WB). Whole-cell lysates (WCLs) of NIH 3T3 cells were resolved on an SDS-PAGE gel containing phos-tag acrylamide (phos-tag) or on a standard SDS-PAGE gel (mock). As an unphosphorylated control, SAP-treated WCL was loaded on the same gel. HP1α was detected using an anti-HP1α antibody. (B) Phosphorylation patterns of mouse HP1α in cells arrested at different cell cycle stages. WCLs were prepared from NIH 3T3 cells grown under the indicated conditions and analyzed by phos-tag WB. The arrowhead indicates an HP1α with basal band shift. The asterisk represents an HP1α species with metaphase-associated phosphorylation. The same membrane was immunoblotted with anti-α-tubulin and anti-TIF1β antibodies for loading controls. (C) Amino acid sequence of mouse HP1α. Light- and dark-shaded boxes indicate the CD and CSD, respectively. The sites of serine (S)-to-alanine (A) substitutions introduced in the following experiments are shown in boldface. (D) Phos-tag and standard WB analysis of wild-type and mutant HP1αs. WCLs were prepared from NIH 3T3 cells transiently expressing FLAG-tagged wild-type HP1α or HP1α with N-terminal (N-term: S11-14A) mutations, hinge (Hinge: S92, 93, 95, 97A) mutations, or both (N-term + hinge). Each FLAG-HP1α with an SAP-treated control was analyzed by phos-tag WB using an anti-FLAG antibody. The phosphorylation state of HP1α represented by each protein band was deduced from the distance of migration level of each band (Rf) and is indicated to the right of the phos-tag WB panel. (E) Phosphorylation state of FLAG-HP1α with each single amino acid substitution in the N-terminal region. The phosphorylation patterns of the indicated FLAG-HP1α species were analyzed by phos-tag WB using a higher concentration (80 μM) of phos-tag acrylamide. (F) Phosphorylation state of FLAG-HP1α with each single amino acid substitution in the hinge region. (G) The numbers of phosphorylated peptides identified by liquid chromatography (LC)-MS/MS analysis of affinity-purified HP1α. (H) Summary of the phosphorylation sites of mouse HP1α deduced from the experiments described above.
Article Snippet: For dephosphorylation, an aliquot of whole-cell lysate (WCL) was incubated with 0.08 U/ml shrimp alkaline phosphatase (SAP) (TaKaRa) for 3 to 6 h at 37°C.
Techniques: Western Blot, SDS Page, Electrophoretic Mobility Shift Assay, Sequencing, Mutagenesis, Expressing, Migration, Concentration Assay, Liquid Chromatography, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Affinity Purification