FIGURE 4.

In vivo and in vitro regulation of Pcyt2 with PKC. Phosphorylation (A) and catalytic activity (B) of endogenous Pcyt2 were analyzed after a short term treatment (0.5, 1, and 2 h) of serum-starved MCF-7 cells with the PKC activator PMA. PMA (25 nm)-induced Pcyt2 phosphorylation (C) and catalytic activity (D) were measured after 0.5 h of treatment with specific PKC inhibitors Gö6976 (50 nm) and EGF-R (50 nm); Pcyt2 immunoprecipitation and activity assays were performed as described under “Experimental Procedures.” Endogenous Pcyt2 was immunoprecipitated with Pcyt2_total antibody, and phosphorylated Pcyt2 was identified by Ser(P) antibody. Pcyt2 enzyme activity was determined under the same conditions using [¹⁴C]phosphoethanolamine as a substrate. Pcyt2 activity is in picomoles/mg/protein ± S.D. at p < 0.05 (*), p <0.01 (**), and p <0.001 (***). Results are from three independent experiments. E, in vitro transcribed-translated Pcyt2α (produced untagged and devoid of modifications by phosphorylation) was phosphorylated with [³²P]ATP and PKCα, PKC βI, or PKCβII. The incorporation of [³²P]ATP was measured in triplicate (dpm ± S.D.) at two Pcyt2α concentrations (0.1 μg (hatched bars) and 0.2 μg (open bars). The ³²P activity incorporated into Pcyt2α was compared with assays without Pcyt2 or PKC added. F and G, overexpressed V5/His-tagged Pcyt2α was purified from serum-treated MCF-7 cells (conditions established to produce minimal phosphorylation) and was subjected to the PKCα assays as above, however, using unlabeled ATP. The phosphorylated peptides were separated and characterized by MALDI-Qq-TOF tandem mass spectroscopy. Shown are the two m/z fragmentations for PKCα-treated Pcyt2α. Both patterns identify Pcyt2α phosphorylation at Ser-215.