Skip to main content
. Author manuscript; available in PMC: 2007 Jan 12.
Published in final edited form as: J Biol Chem. 2006 Sep 12;281(45):34537–34548. doi: 10.1074/jbc.M606654200

FIGURE 1. Identification of phosphorylation sites in Pah1p.

FIGURE 1

A, schematic representation of the primary structure of Pah1p. The gray and black boxes indicate the conserved N-lipin and HAD-like domains within Pah1p, respectively. Asterisks denote the Ser/Thr-Pro phospho residues identified in this study. Phosphorylated residues in sequences are in bold. B, sequencing of the phospho-Ser-744/Ser-748 peptide of Pah1p by nanoelectrospray MS/MS. Collision-induced dissociation spectrum of a double-charged ion m/z 838.4 is shown. The spectrum containing a y-ion series (y1-y12) and a b-ion series (b2-b6) confirmed the peptide sequence as QIYLELGSPLASPK. The phosphorylation sites were identified by the 167 mass different between y2-y3, and y6-y7, corresponding to phosphoserine 12 and 8. C, mapping of N-terminal Ser/Thr-Pro phosphorylation sites using the MPM-2 antibody. The indicated Pah1p phosphorylation mutants (asterisks indicate the position of the different Ser/Thr to Ala mutations: S168A/S602A/T723A/S744A/S748A, called Pah1p-5P; S168A/S475A/S602A/T723A/S744A/S748A, called Pah1p-6P; S168A/S475A/T522A/S602A/T723A/S744A/S748A, called Pah1p-7P-C; S110A/S114A/S168A/S602A/T723A/S744A/S748A, called Pah1p-7P; S110A/S114A/S168A/S475A/T522A/S602A/T723A/S744A/S748A, called Pah1p-9P) or the wild-type control, tagged with protein A, were affinity-purified using IgG-Sepharose beads and then digested with tobacco etch virus protease to remove the PtA tag. The various pulldowns were resolved by 10% SDS-PAGE and Western-blotted using the anti-MPM2 antibody. Supernatants from each sample were also analyzed using anti-PtA antibodies. D, the effect of alanine substitutions at the phosphorylation sites on the electrophoretic mobility of Pah1p. Protein extracts from pah1Δ (lane 1) or nem1Δ pah1Δ (lanes 2–9) cells transformed with centromeric plasmids expressing wild type (wt) or Pah1p-PtA mutants as indicated were prepared from logarithmically growing cells and resolved by 7% SDS-PAGE followed by Western blot using anti-PtA antibodies.