Fig. 2.
Identification of the NMNAT-1 phosphorylation site as serine 136. (A) Recombinant NMNAT-1 (25 μg) was 32P-phosphorylated by PKC and then subjected to tryptic digestion. The generated peptides were separated by HPLC. The elution profile is shown. The upper, black trace shows absorbance at 214 nm. The lower, gray trace shows radioactivity of the collected fractions. AUFS, Absorbance units full scale. (B) The fraction containing a radioactive peptide was analyzed by MALDI-TOF mass spectrometry. The relevant part of the mass spectrum is presented. (C) The obtained peptide masses and the corresponding stretches of the NMNAT-1 sequence are shown. (D) WT NMNAT-1 or the S136A mutant was incubated with PKC or HeLa nuclear extracts as indicated in the presence of [γ-32P]ATP. Proteins were separated by SDS/PAGE (PKC Left and Nuclear Extracts Left) and then subjected to autoradiography (PKC Right and Nuclear Extracts Right).