Fig. 7.

Effects of PS and DAG on the Pkc1 phosphorylations of Nem1-ΔTM, Spo7-ΔTM, and the Nem1 and Spo7 peptides. Nem1-ΔTM (80 μg/ml) (A), Spo7-ΔTM (30 μg/ml) (B), Nem1 peptide (197-LRAQSVKSRPR-207, 100 μM) (C), or Spo7 peptide (17-SASIVSGPRRR-27, 100 μM) (D) was incubated for 20 min with 70 ng Pkc1, 50 μM [γ-³²P]ATP, and 10 mM MgCl₂ in the presence of 0.5 mM PS, 0.15 mM DAG, or both. A, B: CaCl₂ (1.7 mM) was also included in the phosphorylation of Nem1-ΔTM and Spo7-ΔTM. The reaction mixtures were resolved by SDS-PAGE (12% gel) and the gel was dried and subjected to phosphorimaging analysis. The levels of Nem1-ΔTM and Spo7-ΔTM phosphorylations were normalized to their maximum phosphorylations in the presence of DAG and PS. C, D: CaCl₂ was not included in the phosphorylation of the Nem1 and Spo7 peptides. The reaction mixtures were spotted onto the P81 phosphocellulose paper, which was washed with 75 mM phosphoric acid and subjected to scintillation counting. The levels of the peptide phosphorylation were normalized to their maximum phosphorylations (Nem1 peptide, 82 nmol/min/mg; Spo7 peptide, 22 nmol/min/mg) in the presence of DAG and PS. The data shown are the averages of three experiments ± SD (error bars).