Banerjee Dipak K, Carrasquillo Elena A, Hughey Patsy, Schutzbach John S, Martínez Juan A, Baksi Krishna
Department of Biochemistry, School of Medicine, University of Puerto Rico, San Juan, Puerto Rico 00936-5067.
J Biol Chem. 2005 Feb 11;280(6):4174-81. doi: 10.1074/jbc.M406962200. Epub 2004 Nov 17.
DPM1 is the structural gene for mannosylphosphodolichol synthase (i.e. Dol-P-Man synthase, DPMS) in Saccharomyces cerevisiae. Earlier studies with cDNA cloning and sequence analysis have established that 31-kDa DPMS of S. cerevisiae contains a consensus sequence (YRRVIS141) that can be phosphorylated by cAMP-dependent protein kinase (PKA). We have been studying the up-regulation of DPMS activity by protein kinase A-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 severalfold 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 enzyme efficiency (kcat/Km). SDS-PAGE followed by autoradiography of the 32P-labeled DPMS detected a 31-kDa phosphoprotein, and immunoblotting with anti-phosphoserine 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. The S141A DPMS mutant exhibited more than half-a-fold reduction in catalytic activity compared with 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 signal, and the phosphorylation target is serine 141.
DPM1是酿酒酵母中甘露糖基磷酸多萜醇合酶（即Dol-P-Man合酶，DPMS）的结构基因。早期利用cDNA克隆和序列分析的研究表明，酿酒酵母的31-kDa DPMS含有一个共有序列（YRRVIS141），该序列可被环磷酸腺苷依赖性蛋白激酶（PKA）磷酸化。我们一直在研究高等真核生物中蛋白激酶A介导的磷酸化对DPMS活性的上调作用，并在本研究中使用来自酿酒酵母的重组DPMS来进一步增进我们的认识。当重组蛋白在体外被磷酸化时，DPMS的催化活性确实提高了几倍。磷酸化酶的催化速率和催化幅度都更高。当将体外磷酸化的重组DPMS作为外源磷酸多萜醇（Dol-P）浓度增加的函数进行测定时，也观察到催化活性有类似的增加。动力学研究表明，体外磷酸化的重组DPMS与对照重组DPMS相比，GDP-甘露糖的Km没有变化，但磷酸化酶的Vmax增加了6倍。体外磷酸化的重组DPMS还表现出更高的酶周转率（kcat）和酶效率（kcat/Km）。对32P标记的DPMS进行SDS-PAGE后放射自显影检测到一种31-kDa的磷蛋白，用抗磷酸丝氨酸抗体进行免疫印迹确定了体外磷酸化的重组DPMS中存在磷酸丝氨酸残基。为了证实重组DPMS的磷酸化激活作用，通过PCR定点诱变将共有序列中的丝氨酸141替换为丙氨酸。与野生型相比，体外磷酸化后分析时，S141A DPMS突变体的催化活性降低了一半以上。因此，证实酿酒酵母DPMS活性确实受环磷酸腺苷依赖性蛋白磷酸化信号调节，磷酸化靶点是丝氨酸141。
