In Vitro Phosphorylation By cAMP-dependent Protein Kinase Upregulates Recombinant S. cerevisiae Mannosylphospho Dolichol Synthase

Dpm1 is the structural gene for mannosylphospho dolichol synthase (i.e., Dol-P-Man synthase, DPMS) in S. cerevisiae. Earlier studies with cDNA cloning and sequence analysis have established that Mr 31 kDa DPMS of S. cerevisiae contains a consensus sequence (YRRVIS) that can be phosphorylated by cAMP-dependent protein kinase (PKA). We have been studying the upregulation of DPMS activity by PKA-mediated phosphorylation in higher eukaryotes, and used the recombinant DPMS from S. cerevisiae in this study to advance our knowledge further. DPMS catalytic activity was indeed enhanced several folds when the recombinant protein was phosphorylated in vitro. The rate as well as the magnitude of catalysis was higher with the phosphorylated enzyme. A similar increase in the catalytic activity was also observed when the in vitro phosphorylated recombinant DPMS was assayed as a function of increasing concentrations of exogenous dolichylmonophosphate (Dol-P). Kinetic studies indicated that there was no change in the Km for GDP-mannose between the in vitro phosphorylated and control recombinant DPMS, but the Vmax was increased by 6-fold with the phosphorylated enzyme. In vitro phosphorylated recombinant DPMS also exhibited higher enzyme turnover (kcat) and the enzyme efficiency (kcat/Km). SDS-PAGE followed by autoradiography of the Plabeled DPMS detected a Mr 31-kDa phosphoprotein, and immunoblotting with antiphosphoserine antibody established the presence of a phosphoserine residue in in vitro phosphorylated recombinant DPMS. To confirm the phosphorylation activation of recombinant DPMS, serine-141 in the consensus sequence was replaced with alanine by PCR-site directed mutagenesis. S141A DPMS mutant exhibited 50% reduction in catalytic activity compared to the wild type when both were analyzed after in vitro phosphorylation. Thus, confirming that S. cerevisiae DPMS activity is indeed regulated by the cAMP-dependent protein phosphorylation by gest on O cber 5, 2017 hp://w w w .jb.org/ D ow nladed from -5signal, and the phosphorylation target is serine-141.