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

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 se-quence(YRRVIS 141 )thatcanbephosphorylatedbycAMP-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

confirming that S. cerevisiae DPMS activity is indeed regulated by the cAMP-dependent protein phosphorylation signal, and the phosphorylation target is serine 141.
Mannosylphosphodolichol (Dol-P-Man), 1 a mannosyl donor in the assembly of the precursor oligosaccharide-lipid Glc 3 Man 9 -GlcNAc 2 -PP-Dol in N-glycosylation of proteins, in the synthesis of glycosylphosphatidylinositol (GPI) anchors, in O-glycosylation of proteins in yeast, and in C-mannosylation of Trp-7 in human ribonuclease 2 (RNase 2) is formed by the transfer of mannose from GDP-mannose to the polyisoprenoid-lipid, dolichylmonophosphate (Dol-P; 1-7). Dol-P-Man is synthesized at the cytoplasmic face of the endoplasmic reticulum (ER) membrane (8 -10), and catalyzed by mannosylphosphodolichol synthase (DPMS; Dol-P-Man synthase, EC 2.4.1.83). Dol-P-Man synthase deficiency has been observed in a Class E Thy-1 lymphoma patient and is unable to elongate Man 5 GlcNAc 2 -PP-Dol to Man 9 GlcNAc 2 -PP-Dol, a pre-requisite for Glc 3 Man 9 GlcNAc 2 -PP-Dol synthesis (11). We have also made a similar observation using in vitro studies with amphomycin, a lipopeptide antibiotic from Streptomyces canas, which forms a complex with Dol-P in a calcium-dependent manner, and inhibiting the Dol-P-Man synthase activity (12,13). Recent reports indicate that partial deficiency of Dol-P-Man synthase causes congenital disorder of glycosylation (14,15). DPMS deficiency in these patients is associated with developmental delay, seizures, hyptonia, and dysmorphic function (16). The DPM1 gene is essential for viability in yeast because disruption of the gene is lethal (17).
Dol-P-Man synthase has been partially purified from mammalian tissue (18,19) and highly purified from the budding yeast, Saccharomyces cerevisiae (20). Cloning of the Dol-P-Man synthase gene DPM1p from S. cerevisiae has shown DPMS to be a structural gene in yeast (17) expressing active protein both in Escherichia coli and in mammalian cells (21). Information on the primary structure of Dol-P-Man synthase obtained from cDNA analysis has indicated that (i) it codes for a protein of 267 amino acids with an apparent mass of 30.36 kDa; (ii) there is a potential membrane spanning domain of 25 amino acids at its carboxyl terminus; (iii) a highly conserved amino acid sequence in the membrane spanning domain originally suggested as a potential dolichol and/or polyisoprene substrate recognition site; and (iv) the predicted sequence contains one positive site for phosphorylation by cAMP-dependent protein kinase (i.e. serine 141 in a sequence RRVIS 141 , a consensus sequence for phosphorylation (17,22,23)). Expression of yeast Dol-P-Man synthase in E. coli and characterization of the purified recombinant enzyme revealed it as a 31-kDa protein. The amino acid composition and sequence of the conserved domain are not critically important for the recognition and binding of Dol-P when the synthase is present in a lipid matrix (24). However, the Dol-P-Man synthase from S. cerevisiae is different from the mammalian Dol-P-Man synthase in its in vitro properties such as sensitivity to nonionic detergents and its ability to interact with phospholipid vesicles (24 -26).
Dol-P-Man synthase has now been cloned from a number of species such as Trypanosoma brucei, Ustilago maydis, Schizosaccharomyces pombe, Caenorhabditis briggsiae as well as from humans by complementation of a temperature-sensitive S. cerevisiae DPM1 mutant, or by cDNA cloning (27)(28)(29). Sequence analysis has suggested that the Dol-P-Man synthase can now be divided into two classes: one includes the enzymes from S. cerevisiae, U. maydis, Trypanosoma brucei, and Leishmania mexicana (29,30). They share 50 -60% amino acid identity and have a stretch of hydrophobic amino acid residues near the COOH terminus constituting a transmembrane domain. The other includes enzymes from the human, S. pombe, and Caenorhabditis briggsiae, lack the hydrophobic COOH terminus domain, and thus, the transmembrane domain. In addition, they have also exhibited only Ϫ30% amino acid identity with the other group (21,25,30). Yeast DPM1 DNA complemented both mouse Thy-1 negative lymphoma mutant cells of complementation class E and the Lec15 mutant of Chinese hamster cells (31,32). On the other hand, human and mouse homologs of DPM1, hDPM1, and mDPM1 did not complement the DPMS mutant in Lec15 cells. This led to establish that mammalian DPMS is a multicomponent enzyme represented by the catalytic subunit DPM1 and two accessory proteins DPM2 and DPM3 (33)(34)(35). Most striking, however, is that Dol-P-Man synthase from every known source has a serine residue in the position corresponding to serine 141 in S. cerevisiae DPMS. Serine 141, and the preceding conserved residues meet the criteria for a consensus site for phosphorylation by cAMP-dependent protein kinase in the DPMS (22,23). In addition, a cDNA encoding the Schistosoma mansoni DPMS displaying a high homology with Cricetulus griseus and S. pombe (36) as well as a probable DPMS sequence (241 amino acid residues) in the genome sequence of the fruit fly, Drosophila melanogaster (37), have also been identified.
We have been studying the regulation of Dol-P-Man synthase activity in mammalian cells by a cAMP-dependent protein phosphorylation signal (38). Our results supported enhanced Dol-P-Man synthase activity in endoplasmic reticulum membranes from cells treated with a ␤-agonist isoproterenol, as well as after in vitro phosphorylation of the endoplasmic reticulum membranes by the catalytic subunit of the cAMP-dependent protein kinase (39,40). The increased enzyme activity was because of an increase in the V max and was independent of enhanced transcription (41). This was strongly supported by down-regulation of the synthase activity in a series of cAMPdependent protein kinase (PKA)-deficient Chinese hamster ovary cell mutants (42). To understand the molecular detail of the phosphorylation regulation of Dol-P-Man synthase, we have used here a purified recombinant Dol-P-Man synthase from S. cerevisiae (wild type) as well as a DPMS mutant in which serine 141 has been replaced with alanine (S141A mutant). We report here that both biochemical and kinetic data have supported up-regulation of the wild type recombinant Dol-P-Man synthase activity after in vitro phosphorylation by PKA. SDS-PAGE followed by autoradiography of the 32 P-la-beled Dol-P-Man synthase has detected a 31-kDa phosphoprotein species as well. Identification of the 31-kDa phosphoprotein on the immunoblot probed with anti-phosphoserine antibody further supported that Dol-P-Man synthase is phosphorylated at the Ser-141. To evaluate serine 141 as the phosphorylation target, we have developed a phosphorylation sitedeficient DPMS mutant in which serine 141 has been replaced with alanine by PCR site-directed mutagenesis. The S141A DPMS mutant has been successfully expressed as a functionally active enzyme in E. coli and analyzed after in vitro phosphorylation by PKA.

MATERIALS AND METHODS
E. coli DH5␣ harboring plasmid pDPM6 containing the structural gene DPM1 for Dol-P-Man synthase was originally developed in the laboratory of Dr. P. W. Robbins at MIT and obtained from Dr. J. S. Schutzbach. Dolichyl monophosphate, ATP (Na salt), the catalytic subunit of cAMP-dependent protein kinase (PKA, bovine heart), bovine serum albumin (crystalline), chloramphenicol, ampicillin, IPTG, phenylmethylsulfonyl fluoride, TPCK, soybean trypsin inhibitor, leupeptin, aprotenin, Protein A-Sepharose CL-4B, and biotin-conjugated antiphosphoserine monoclonal antibody were purchased from Sigma. Protein molecular weight markers (high molecular weight, low molecular weight, and kaleidoscope), reagents for protein electrophoresis and immunoblotting, and hydroxyapatite were from Bio-Rad. GDP-[U- 14 C]mannose (307 mCi/mmol; a Ci ϭ 37 Gbq), [␥-32 P]ATP (3.0 Ci/mmol), 14 C-methylated protein mixture, ECL chemiluminescence kit, and Amplify were obtained from GE Healthcare. Mouse monoclonal antibody to yeast Dol-P-Man synthase was obtained from Molecular Probes, Eugene, OR. Oligonucleotides for PCR primers were obtained from Oligos Slc, Ridgefield, CT, as well as from the Molecular Biology Core Facility of the University of Puerto Rico Medical Sciences Campus. Restriction enzymes were acquired from New England BioLabs, Boston, MA.
Analytical Methods-Protein was assayed following precipitation of the protein with trichloroacetic acid using a modification (42) of the procedure of Lowry et al. (43). Bovine serum albumin was used as the standard. Electrophoresis was carried out under reducing conditions in 12% polyacrylamide gels (SDS-PAGE) using the buffer system described by Laemmli (44). The gels were either processed for autoradiography in Amplify TM , according to the procedure described by Bonner and Lasky (45), or they were processed for Western blot analysis.
Dol-P-Man Synthase Assay-Enzymatic transfer of mannose from GDP-mannose was assayed using the procedure described earlier (13) with the following modifications. In vitro phosphorylated or control recombinant Dol-P-Man synthase was incubated at 37°C for 2 min in 30 mM Hepes, pH 7.0, buffer containing 150 M EDTA, 30 M dithiothreitol, 3% glycerol, 90 mM NaCl, 0.25% Triton X-100, 10 mM MnCl 2 , 10 g of Dol-P, 1% Me 2 SO, and 0.125 M GDP-[ 14 C]mannose (specific activity 318 cpm/pmol) in a total volume of 100 l, unless otherwise mentioned. The reaction was stopped with 2 ml of chloroform/methanol (2:1, v/v). Chloroform/methanol extracts containing the Dol-P-Man were washed once with 5 volumes of 0.9% sodium chloride and twice with chloroform/methanol/water (3:47:48, v/v/v) to remove free GDP-[ 14 C]mannose. A lower organic phase containing the Dol-P-Man was then quantified in a liquid scintillation spectrometer.
Recombinant Dol-P-Man synthase was phosphorylated in vitro by incubating with the catalytic subunit from the cAMP-dependent protein kinase (12 units per 30 g of protein) at 30°C for 20 min in the presence of 10 mM NaF, 10 mM MgCl 2 , and 200 M ATP as described earlier (42).
In some experiments, the reactions were started by the addition of 5 Ci of [␥-32 P]ATP (specific activity 3.0 Ci/mmol).
Construction of the Expression Vector BL21(DE3)pDPM1-9 and Preparation of the Enzyme-The expression vector was constructed earlier by Schutzbach et al. (24) to purify the Dol-P-Man synthase (46). In brief, M9ZB broth (47) (4 ϫ 250 ml in 1 liter flasks) containing 100 g/ml ampicillin was inoculated with 10 ml of an overnight culture of BL21(DE3)/pDPM1-9. Cultures were incubated at 37°C on a shaker at 200 rpm. After 3 h (A 600 nm of 0.66) the enzyme was induced by adding 2.5 ml of 100 mM IPTG. The cells were harvested 2 h post-induction by centrifugation at 6,000 rpm for 15 min (Sorvall GSA rotor). All additional procedures were carried out at 0 -4°C or on ice. The cell pellets were washed with 300 ml of water followed by 100 ml of Buffer A, and frozen overnight. The pellets were then thawed, suspended in 10 ml of Buffer A, and the cells were sonically ruptured three times for 30 s with 1-min intervals to allow cooling using a Branson sonifier (Kontes, model W185) at a power setting of 4.5. The suspension was fractionated by centrifugation at 39,000 ϫ g for 20 min (Sorvall SS-34 rotor) at 4°C. The pellet was washed twice with 25 ml of Buffer A and then suspended in 5 ml of the same buffer. The suspension was diluted with 17.5 ml of Buffer B and 2.5 ml of 10% Nonidet P-40 was added. The mixture was centrifuged at 18,000 rpm for 20 min. Both supernatant (which often contained significant amounts of activity), and the pellet were saved. To solubilize additional enzyme, the pellet was suspended in 20 ml of Buffer C. To the dispersed pellets 2.5 ml of 10% Nonidet P-40 was added and homogenized for 10 min in a Tekmer homogenizer. The homogenates were centrifuged as above, and the supernatants were saved. The procedure was repeated once more to yield a third solubilized fraction. Solubilized fractions containing the enzyme activity were pooled and applied to a hydroxyapatite column (2.5 ϫ 11 cm) equilibrated with Buffer B. The column was washed with 30 ml of Buffer B and eluted with a 400-ml linear gradient of 0.0 to 1.0 M NaCl in the same buffer. Fractions containing the enzyme activity were pooled and concentrated by ultrafiltration using YM-10 membranes (Amicon).
Immunoprecipitation and Immunoblotting-Recombinant Dol-P-Man synthase after purification was phosphorylated in vitro in the presence of [␥-32 P]ATP as described above. 32 P-Labeled Dol-P-Man synthase was immunoprecipitated with a mouse monoclonal antibody (1: 1,000 dilution) to yeast DPMS for 3 h at 4°C followed by 3 h with 50 g/ml Protein A-Sepharose CL-4B (30 mg/ml) in the presence of aprotinin (1 g/ml), phenylmethylsulfonyl fluoride (1 mM), TPCK (200 M), soybean trypsin inhibitor (1 g/ml), and leupeptin (1 M). The Protein A-Sepharose antigen-antibody complexes were washed twice with NET buffer, twice with washing buffer, and once with phosphate-buffered saline, pH 7.4. The immune complexes were released by boiling at 100°C for 5 min and analyzed by SDS-PAGE (12%) followed by autoradiography (48).
For immunoblotting, the recombinant Dol-P-Man synthase before and after in vitro phosphorylation was separated on 12% SDS-PAGE and transblotted onto 0.2-m nitrocellulose membranes according to the procedure described by Towbin et al. (49). The blots were treated with a blocking solution (5% nonfat dry milk in TTBS) overnight at 15°C and subsequently with (a) anti-Dol-P-Man synthase antibody (diluted 1:2,000 in blocking solution), and (b) biotinylated anti-phosphoserine antibody (diluted 1:2,000 in blocking solution) for 3 h at room temperature. The membranes were rinsed twice with TTBS, washed with one change of TTBS for 15 min and twice more 5 min apart at room temperature. Blot a was treated with horseradish peroxidase-conjugated sheep anti-mouse IgG (1:2,000 dilution) to detect the Dol-P-Man synthase; and blot b was treated with streptavidin-conjugated sheep anti-mouse IgG (1:2,000 dilution) to detect phosphoserine as well as the molecular weight markers. The blots were developed according to the instructions provided with the ECL chemiluminescence kit and exposed to Hyperfilm TM until the desired intensity was achieved.
Isolation of Dol-P-Man Synthase S141A Mutant by PCR Site-directed Mutagenesis-The strategy for PCR site-directed mutagenesis consisted on the design of oligonucleotides that contained the desired substitution, and also resulted in a silent mutation with either a new restriction site or elimination of an existing one to aid the analysis. The oligonucleotides were designed with the aid of the DNAsis Program (Hitachi Corp.) and using the DPM1 (S. cerevisiae Dol-P-Man synthase gene sequence GenBank TM J04184) as template. We have used the Silmut computer program (50) to identify suitable restriction site(s) for silent mutagenesis. These oligonucleotides were compatible as PCR primer pairs with the DPM1F and DPM1R. The primer pairs, their corresponding products, and diagnostic restriction enzyme were: DPM-1F, 5Ј-CGGGATCCTATGGCTAGCATCGAATACTCTGTT-3Ј/DPM1R, 5Ј-ATTCTAGCAGTCGACGCAATGACGCGTCTGTACA-3Ј (449 bp); and DPM1F, 5Ј-TGTACAGACGCGTCATTGCGTCGACTGCTAGAAT-3Ј/DPM1R, 5Ј-CTGGATCCTTAAAAGACCAAATGGTATAGCTGGTA-GCA-3Ј (409 bp). The genes were first amplified in halves and then added to a second PCR mixture to fill-in the gaps by the ULTMA DNA polymerase. Hybrids served as templates for DPM1F and DPM1R amplification for the complete mutant gene (824 bp). The PCR reaction conditions were 1ϫ ULTMA reaction buffer (10 mM Tris-HCl, pH 8.8), 10 mM KCl, 0.02% Tween 20 (v/v), 1.25 mM dNTPs, 10 mM each of the corresponding primer pair, and 1.5 units of ULTMA DNA polymerase (PerkinElmer Life Sciences) in a final volume of 45 l. The tubes were incubated at 80°C for 5 min in a DNA Thermal Cycler model 480 (PerkinElmer Life Sciences) and 5 l of 25 mM MgCl 2 was added to a final concentration of 2.5 mM. Mineral oil was added and the mixture was incubated at 97°C for 2 min. Thermal cycling conditions were 95°C for 1 min, 60°C for 1 min, and 72°C for 1 min for 25 cycles. Time delay was at 72°C for 7 min.
To generate the mutant gene, PCR products were separated on a 1.5% agarose gel in 1ϫ TAE (0.04 M Tris acetate, 0.001 M EDTA, pH 8.5) and bands corresponding to PCR halves were excised from the gel slices. An aliquot of the PCR fragment (0.3 l) from each half was used as templates in a second PCR along with DPM1F and DPM1R primers to generate the complete mutant gene. S141A mutant gene acquiring a SalI restriction site was analyzed by SalI digestion (1 ϫ SalI buffer, 0.5 l of 100 ϫ bovine serum albumin, 10 units of SalI, and 5 l of PCR mixture in a total volume of 15 l at 37°C for 1 h) followed by electrophoresis (65 volts for 45 min) on a 1.5% agarose gel in 1ϫ TAE. The DNA fragments (450 bp) were detected under the UV light after staining with ethidium bromide.
Subcloning of the S141A DPMS Mutant into pET3(a)/DPM1-9 and Screening for Colonies-PCR products containing the DPMS mutant gene as well as the wild type gene were excised from pUC19/DPMS constructs with AvrII/NsiI. The fragments were gel purified and ligated with T4 DNA ligase (400,000 units/ml) at 16°C overnight to pDPM1-9 (46). The ligation mixture was diluted 1:20, and 2 l were used to transform DH5␣ cells (Invitrogen; efficiency Ͼ1 ϫ 10 8 colony forming units/g of DNA) following the manufacturer's instructions. The cells were then plated on LB/Amp (50 g/ml) plates and the colonies were directly screened for the complete gene by PCR using DPM1F and DPM1R primers. The wild type as well as the S141A mutant DPMS in pDPM1-9 were sequenced using Sequenase version 2.0 kit (U. S. Biochemicals) according to the manufacturer's instructions. The reaction mixtures were electrophoresed in a 5% Long Ranger, 1ϫ TBE (89 mM Tris base, 89 mM boric acid, 2.5 mM EDTA⅐2H 2 O) gel for 1.5 h at 50 W. The gel was vacuum dried, exposed to Kodak X-Omat x-ray films with intensifying screens (DuPont) at Ϫ70°C for 3 days, and developed.
Expression and Analysis of S141A Mutant DPMS-Transformants containing the mutated DPMS gene were grown in 10 ml of LB containing ampicillin (50 g/ml) and chloramphenicol (34 g/ml) at 37°C in a bacterial shaker incubator with shaking at 200 rpm until the A 600 nm reached between 0.6 and 1.0. The expression of the protein was then achieved by adding IPTG to a final concentration of 0.4 mM, and the cultures were harvested after 3 h.
Aliquots from uninduced and induced cultures were pelleted at 14,000 rpm for 30 s. The cell pellets were boiled in 1ϫ SDS-PAGE sample buffer for 5 min, and a 10-l aliquot from each sample (representing 200 l of culture was loaded into 12% polyacrylamide gels and separated at 180 volts for 45 min according to Laemmli's procedure (43). One gel was stained with 0.1% Coomassie Blue (R-250) for protein detection, and the other gel was used for immunoblotting with yeast anti-DPMS monoclonal antibody as described above.
The cultures were scaled up, and the DPMS activity was solubilized in 1% Nonidet P-40 as mentioned above. The soluble enzyme was recovered after centrifuging at 100,000 ϫ g for 60 min at 4°C (Beckman 50Ti rotor). DPMS content in each extract was quantified by densitometry (Bio-Rad model GS470).

Effect of in Vitro Phosphorylation on the Dol-P-Man Synthase
Activity-Mannosylphosphodolichol synthesis was monitored with in vitro phosphorylated and control recombinant DPMS from S. cerevisiae. The time course of the synthase activity, when examined for a period of 0 -5 min under the conditions described above, indicated that the specific activity of Dol-P-Man synthase was severalfold higher in the in vitro phosphorylated enzyme compared with control (Fig. 1). In addition, Dol-P-Man synthesis remained high at all time points with the in vitro phosphorylated enzyme.
Dependence of GDP-Mannose and Dol-P Concentrations on Synthase Activity-Based on a single point assay, the Dol-P-Man synthase activity was found to be nearly 3-fold higher (p Ͻ 0.05) in the in vitro phosphorylated enzyme compared with control (Table I). However, the kinetic analysis at the initial rate (Figs. 2, a and b) of the recombinant DPMS indicated that the K m for GDP-mannose for in vitro phosphorylated and control enzymes were 1.1 ϫ 10 Ϫ6 M and 1.2 ϫ 10 Ϫ6 M, respectively, whereas the corresponding V max values were 135.8 and 23.6 nmol/min/mg of protein, respectively. The in vitro phosphorylated enzyme also exhibited almost 6-fold higher turnover (k cat ϭ 70.9) and enzyme efficiency (k cat /K m ϭ 6.4 ϫ 10 7 ) as compared with the control enzyme (k cat ϭ 12.1 and k cat /K m ϭ 1 ϫ 10 7 ) ( Table II). The apparent K m for Dol-P was approximately a magnitude higher than that of GDP-mannose but the values did not differ between the in vitro phosphorylated and control enzymes (data not shown).
Characterization of Dol-P-Man Synthase as a Phosphoprotein-Recombinant Dol-P-Man synthase was phosphorylated in vitro in the presence of [␥-32 P]ATP according to the conditions described under "Materials and Methods." The phosphorylated enzyme was immunoprecipitated with a mouse monoclonal antibody to yeast Dol-P-Man synthase, and subjected to a 12% SDS-PAGE followed by autoradiography on X-AR films. It detected Dol-P-Man synthase as a 31-kDa phosphoprotein (Fig. 3). To identify that serine 141 is the target for PKAmediated phosphorylation, Dol-P-Man synthase was separated on a 12% SDS-PAGE after in vitro phosphorylation and trans-blotted onto a nitrocellulose membrane. One-half of the membrane was processed with anti-DPMS antibody, and the other half with anti-phosphoserine antibody. Both anti-DPMS antibody and the anti-phosphoserine antibody detected a 31-kDa protein on the blots (Fig. 4, a and b) supporting that Dol-P-Man synthase was indeed phosphorylated at serine 141 by the PKA. As a control, cell extracts from IPTG-uninduced and -induced E. coli carrying the wild type Dpm1 gene or its S141A mutant were examined by Western blot analysis against anti-phosphoserine antibody. These results (Fig. 4c) were negative, suggesting the presence of no other phosphorylated protein equivalent to the 31-kDa DPMS E. coli cell extracts.
Dol-P-Man Synthase Activity of the Phosphorylation Sitedeficient (S141A) Mutant after in Vitro Phosphorylation by cAMP-dependent Protein Kinase-To establish further that serine 141 of the DPMS is indeed the target for PKA-mediated phosphorylation, serine 141 was replaced by alanine by sitedirected mutagenesis. Alanine substitution at serine 141 of the Dol-P-Man synthase was confirmed by (a) restriction analysis of the DNA constructs; and (b) DNA sequencing. The restriction enzyme analysis demonstrated that the wild type Dol-P-Man synthase gene contained only the BstU1 site but not SalI site. On the other hand, the S141A Dol-P-Man synthase mutant gene contained both a BstU1 and a SalI site. DNA sequencing of the wild type and mutant DPMS genes demonstrated the presence of the TCC sequence (a codon for serine) at amino acid residue 141 of the wild type gene but the corresponding sequence in the mutant was GCC (a codon for alanine; data not shown). It should be noted here that there were no other sequence differences among the wild type, S141A mutant, and published S. cerevisiae DPMS sequence.
Analysis of the expressed protein by 12% SDS-PAGE as well as by immunoblotting with a yeast anti-DPMS monoclonal antibody indicated that IPTG-induced cultures expressed high levels of S141A mutant DPMS as a 31-kDa protein (Fig. 5, a and b). DPMS activity of the mutant enzyme was determined and compared with that of the wild type before and after in vitro phosphorylation. Basal DPMS activity in S141A mutant extracts was ϳ1.5-fold higher than in control extracts. Wild type DPMS was activated by 1.5-fold as opposed to 1-fold in the S141A mutant. This means if the wild type DPMS was activated by 2-fold after phosphorylation, the S141A mutant DPMS was activated less  Enzymatic transfer of mannose from GDP-mannose to Dol-P was measured by incubating the control or in vitro phosphorylated enzyme at 37°C for 0-5 min in 30 mM Hepes, pH 7.0, buffer containing 150 M EDTA, 30 M dithiothreitol, 3% glycerol, 90 mM NaCl, 0.25% Triton X-100, 10 mM MnCl 2 , 10 g of Dol-P, 1% Me 2 SO, and 0.125 M GDP-[ 14 C]mannose (specific activity 318 cpm/pmol) in a total volume of 100 l. Dol-P-Man was extracted and quantified in a liquid scintillation spectrometer. The results are the average from two determinations. E, control; •, in vitro phosphorylated. than one-half of 2-fold (Fig. 6, a and b). E. coli cells transfected with vector alone had no DPMS activity. DISCUSSION Using biochemical parameters we have proposed earlier that mammalian Dol-P-Man synthase is up-regulated by a cAMPdependent protein kinase-mediated phosphorylation event (39). In addition, we have also shown that the mammalian Dol-P-Man synthase activity is associated with a 32-kDa phosphoprotein (40). We have now shown using somatic cell genetics that Chinese hamster ovary cells deficient in cAMP-depend-ent protein kinase exhibited reduced Dol-P-Man synthase activity as compared with the wild type (41). Increased enzymatic activity of in vitro phosphorylated recombinant DPMS from S. cerevisiae has thus supported the observations made earlier with mammalian cells. It has also substantiated the fact that the consensus sequence for PKA-dependent phosphorylation in S. cerevisiae DPMS is indeed functionally active. Enhancing the initial rate of the transferase activity with in vitro phosphorylated recombinant enzyme was not because of a change in the K m for GDP-mannose but because of an increase in the V max . This increase in enzyme activity is corroborated with the enzyme turnover (k cat ) as well as its catalytic efficiency (k cat /K m ). DPMS also exhibited and elevated the level of activity when in vitro phosphorylated enzyme was analyzed in the presence of increasing concentrations of Dol-P (data not shown). These results have strongly supported that increased synthase activity in phosphorylated DPMS is not because of a mere change in the affinity for the substrate, but most likely because of a conformational change of the enzyme. Future analysis of the DPMS protein structure by x-ray crystallography is expected to clarify this hypothesis.
Sequencing of yeast genome has detected 113 conventional protein kinases in budding yeast. This corresponds to ϳ2% of the total genes and more than 60% of these protein kinases have either known or suspected function (46). Therefore, the presence of a consensus sequence (YRRVIS 141 ST) in Dol-P-Man synthase from S. cerevisiae where serine 141 is a target for PKA phosphorylation (17) is reasonable. In vitro phosphorylation of the Dol-P-Man synthase with [␥-32 P]ATP followed by SDS-PAGE and autoradiographic analysis as well as by immunoblotting with anti-phosphoserine antibody have supported the phosphoprotein nature of the yeast DPMS. To confirm that serine 141 is the target for PKA phosphorylation, and is responsible for enhanced activity of the phospho-

TABLE II Kinetic constants of recombinant Dol-P-Man synthase before and after phosphorylation by cAMP-dependent protein kinase
Recombinant Dol-P-Man synthase was assayed as described in Table I  rylated DPMS, we have substituted the serine residue with alanine by PCR site-directed mutagenesis. Restriction enzyme analysis as well as DNA sequencing have confirmed the S141A substitution in the DPM1 gene. It is important to note that the S141A mutant exhibits a small increase in basal DPMS activity compared with that of the wild type. This may be because of the in vitro assay condition, or because of a change in the protein conformation in this mutant, or it may be a combination of the two and can only be explained by x-ray crystallographic studies in the future. In vitro phospho-  5. Expression of wild type and S141A Dol-P-Man synthase mutant. a, analysis by SDS-PAGE: 1-ml aliquots from uninduced and induced E. coli cultures were pelleted by centrifugation (14,000 rpm for 30 s in a microcentrifuge) and boiled for 5 min in 1ϫ sample buffer. 10-l aliquots representing 200 l of culture were applied to a 12% SDS-PAGE and electrophoresed at 180 volts for 45 min. The gel was stained with 0.1% Coomassie Blue (R-250) to visualize the protein bands. Lane M, molecular weight markers; lane U (WT), wild type DPMS before induction; lane I (WT), wild type DPMS after induction; lane U (S141A), S141A mutant DPMS before induction; lane I (S141A), S141A mutant DPMS after induction. b, analysis by immunoblotting: the wild type and S141A mutant DPMS proteins were separated on a 12% SDS-PAGE as mentioned above and transblotted onto a 0.2-m nitrocellulose membrane. The blot was treated with the blocking solution overnight at 15°C and subsequently with yeast anti-Dol-P-Man synthase monoclonal antibody (1:2,000 dilution in blocking solution) for 3 h at room temperature. The membrane was rinsed twice with TTBS, washed with one change of TTBS for 15 min, and twice for 5 min each at room temperature. The blot was then treated with horseradish peroxidase-conjugated sheep anti-mouse IgG (1:2,000 dilution) to detect the Dol-P-Man synthase and streptavidinconjugated sheep anti-mouse IgG (1:2,000 dilution) to detect the biotinylated molecular weight markers. The blot was developed with the ECL kit and exposed to a Hyperfilm until the desired intensity was achieved. Lane M, biotinylated molecular mass markers; lane WT, wild type DPMS; lane S141A, phosphorylation site-deficient DPMS mutant. rylated S141A mutant DPMS is slightly more active (less than one-half of a fold) than its wild type counterpart. If serine 141 were the only target for PKA-mediated phosphorylation, then one would expect no increase in enzyme activity in the S141A DPMS mutant following phosphorylation. The only convincing argument at this point could be the presence of serine 142 in the DPMS sequence. If there is a change in the protein conformation because of a S141A mutation then it may be overriding the significance of the serine 141 mutation, and making serine 142 accessible to PKA with a lower affinity. On the other hand, serine 142 could play as an alternative phosphorylation site in the absence of serine 141 to respond to cAMP-related stimuli. Thus, we conclude that serine 141 is the primary target for PKA-mediated phos-phorylation of DPMS while responding to cAMP signaling. This valuable information is a step toward understanding the fundamentals of the closely related DPMS family of proteins, and the development of congenital disorder of glycosylation.