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J. Biol. Chem., Vol. 280, Issue 6, 4174-4181, February 11, 2005

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In Vitro Phosphorylation by cAMP-dependent Protein Kinase Up-regulates Recombinant Saccharomyces cerevisiae Mannosylphosphodolichol Synthase^*

Dipak K. Banerjee¶, Elena A. Carrasquillo||, Patsy Hughey**, John S. Schutzbach**, Juan A. Martínez, and Krishna Baksi

From the Department of Biochemistry, School of Medicine, University of Puerto Rico, San Juan, Puerto Rico 00936-5067, the ^**Department of Microbiology, University of Alabama, Birmingham, Alabama 35294-0019, and the Department of Anatomy and Cell Biology, Universidad Central del Caribe, Bayamón, Puerto Rico 00960-6032

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 K_m for GDP-mannose between the in vitro phosphorylated and control recombinant DPMS, but the V_max was increased by 6-fold with the phosphorylated enzyme. In vitro phosphorylated recombinant DPMS also exhibited higher enzyme turnover (k_cat) and enzyme efficiency (k_cat/K_m). 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.

Received for publication, June 22, 2004 , and in revised form, November 15, 2004.

^* This work was supported in part by Grants GM47492 and GM38643 (to J. S. S.) and 2S07 GM50695 (to K. B.) from the National Institutes of Health, and Committee for the Integration of the Development of Scientific Research funds from the University of Puerto Rico Medical Sciences Campus. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Both authors contributed equally to this work.

^|| Supported by Minority Predoctoral Fellowship Program NIGMS National Institutes of Health Grant F31 GM17177.

^¶ To whom correspondence should be addressed: Dept. of Biochemistry, School of Medicine, University of Puerto Rico, San Juan, Puerto Rico 00936-5067. Tel.: 787-758-7090; Fax: 787-274-8724; E-mail: dbanerjee{at}rcm.upr.edu.

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