To characterize Ser1029 in STaR at a consensus sequence of phosphorylation site by PKC, two mutants of mS1029A with replacement of Ser1029 to Ala1029 and CΔ1029 lacking 22 amino acids including Ser1029 were prepared. Preincubation of the wild type-STaR (wt-STaR) transfectant with 1 μM PMA caused additional STa-mediated guanylyl cyclase (GC) activation compared to control, whereas the mS1029A- and CΔ1029-transfected cells did not show a similar enhanced GC activation by PMA. After metabolic labeling with [32P]phosphate, transfected cells with wt-STaR and mutants were incubated with 1 μM PMA. Subsequent 32P-radiolabeled proteins were immunoprecipitated using anti-STaR antibody, and analyzed by autoradiography after separation on SDS-PAGE. The immunoprecipitated wt-STaR but not mS1029A and CΔ1029 had a significant radioactivity. These results suggest that the effect of PMA on wt-STaR transfectants may be caused by phosphorylation of Ser1029. The CΔ1012 mutant, with further truncation (Gln1012-Phe1050) of the carboxy terminus, did not show STa-mediated GC activation. Based on these data, these 17 amino acids (Gln1012-Ala1028), essential for signaling of GC activation by STa, have an abundance of basic amino acids which might be functionally influenced by phosphorylation of Ser1029.
