Pcyt2 was phosphorylated within the -specific motif that is spliced out in Pcyt2 and on two PKC consensus serine residues, Ser-215 and Ser-223. activity of endogenous Pcyt2 were dramatically improved with phorbol esters and reduced by specific PKC inhibitors. translated Pcyt2 was phosphorylated by PKC, PKCI, and PKCII. Pcyt2 Ser-215 was also directly phosphorylated with PKC. Mapping of the Pcyt2- and –phosphorylated sites to the solved structure of a human Pcyt2 showed that they clustered within and flanking the central linker region that connects the two catalytic domains and is a novel regulatory segment not present in additional cytidylyltransferases. This study is the 1st to demonstrate variations in phosphorylation between Pcyt2 isoforms and to uncover the part of the PKC-regulated phosphorylation. from ethanolamine and diacylglycerol/alkylacylglycerols through the CDP-ethanolamine-Kennedy pathway and by decarboxylation of phosphatidylserine in the mitochondria. The Kennedy pathway is definitely quantitatively the most important route for the biosynthesis of PE in mammalian cells (6). With this pathway, ethanolamine is definitely 1st phosphorylated by ethanolamine kinase to phosphoethanolamine (P-Etn), which is definitely then converted to CDP-ethanolamine (CDP-Etn) by CTP:phosphoethanolamine cytidylyltransferase (Pcyt2; also known as ET (7, 8) or ECT (9)). In the final step, CDP-ethanolamine:1,2-diacylglycerol ethanolamine phosphotransferase transfers P-Etn from CDP-Etn to DAG/alkylacylglycerol to produce PE/plasmalogens. We have recently examined the part of Pcyt2 in phospholipid homeostasis (10). Recent studies using Pcyt2-deficient mouse models founded a direct link between PE synthesis and triglyceride rate of metabolism (11,C13). Heterozygous Pcyt2 deletion (using the PURExpress protein synthesis kit (New England Biolabs). This system generates a protein product devoid of post-translational modifications. The untagged Pcyt2 cDNA template was generated by PCR using the pcDNA.ET plasmid. We amplified the entire open reading framework and the required flanking sequences, including the ribosomal binding site, but erased the V5 and His tags using Pcyt2-specific primers (ahead primer, 5-taagaaggagatataccaatgatccggaacgggcacgg-3-3; opposite primer, 5-tattcattagtcaatctcccctccaggcggcgcc-3). In the second round of PCR, we added the T7 promoter in the 5-end of Pcyt2 using the common primer (5-gaaattaatacgactcactatagggagaccacaacggtttccctctagaaataattttgtttaactttaagaaggagatatacca-3) and the Pcyt2 reverse primer. Pcyt2 cDNA was purified using a gel extraction kit (Sigma) and 250 ng of the cDNA incubated with combination I (PURExpress) at 37 C for 1 h to generate Pcyt2 protein. The combination was concentrated (60 min at 1,500 at 4 C) using a Microcon YM-100 spin concentrator (Millipore Corp.) and mixed with 0.25 volumes of nickel-nitrilotriacetic acid-agarose beads (Qiagen) (mixture II) and incubated at 4 C for 45 min to remove the translation assay components. Mixture II was then applied to a micro-spin column (Bio-Rad), and the unbound Pcyt2 protein was released by centrifugation (2 min at 1500 at 4 C). Pcyt2 proteins from multiple reactions were combined and pooled samples applied in the (Z)-Capsaicin PKC phosphorylation assays below. In Vitro Pcyt2 Phosphorylation with PKC, PKCI, and BCL2 PKCII Pcyt2 free from tags and post-translational modifications was subjected to phosphorylation with [-32P]ATP and either PKC, PKCI, or PKCII (Enzo Existence Sciences) (28). The reaction was carried out at 30 C for 10 min in 100 l of combination comprising 20 mm HEPES, pH 7.4, 10 mm MgCl2, 0.1 mm CaCl2, 100 m ATP, 100 g/ml phosphatidylserine, 20 g/ml diacylglycerol, 0.03% Triton X-100, 1 l of [-32P]ATP, and different aliquots (20 or 40 l) of Pcyt2 protein. Reactions were initiated by adding 0.4 g of PKC and terminated after 10 min by adding 20 l of 1% BSA and 1 ml of 10% TCA. After 5 min of incubation on ice, the aliquots (40 l) from each reaction were transferred to a nitrocellulose membrane on an aspirator funnel and washed three times with 2 ml of 5% TCA. Membranes were dried under vacuum and the -32P was incorporated into Pcyt2 protein determined by counting. The assays were performed in triplicate, and the background radioactivity for the reactions without Pcyt2 or PKC served as controls (28). Statistical Analysis All measurements were expressed as means S.D. from at least three impartial experiments. GraphPad Prism 3.0 software (San Diego) was used to perform one- or two-way analysis of variance and Student’s paired test. Differences were considered statistically significant at 0.05. Densitometry analysis was performed by Scion Image software (Scion Inc). RESULTS Pcyt2 and Pcyt2 Catalytic Domains and Phosphorylation Sites Fig. 1 provides a simplified representation of the Pcyt2 and – structures showing the position of the two.Gupta A. by PKC, PKCI, and PKCII. Pcyt2 Ser-215 was also directly phosphorylated with PKC. Mapping of the Pcyt2- and –phosphorylated sites to the solved structure of a human Pcyt2 showed that they clustered within and flanking the central linker region that connects the two catalytic domains and is a novel regulatory segment not present in other cytidylyltransferases. This study is the first to demonstrate differences in phosphorylation between Pcyt2 isoforms and to uncover the role of the PKC-regulated phosphorylation. from ethanolamine and diacylglycerol/alkylacylglycerols through the CDP-ethanolamine-Kennedy pathway and by decarboxylation of phosphatidylserine in the mitochondria. The Kennedy pathway is usually quantitatively the most important route for the biosynthesis of PE in mammalian cells (6). In this pathway, ethanolamine is usually first phosphorylated by ethanolamine kinase to phosphoethanolamine (P-Etn), which is usually then converted to CDP-ethanolamine (CDP-Etn) by CTP:phosphoethanolamine cytidylyltransferase (Pcyt2; also known as ET (7, 8) or ECT (9)). In the final step, CDP-ethanolamine:1,2-diacylglycerol ethanolamine phosphotransferase transfers P-Etn from CDP-Etn to DAG/alkylacylglycerol to produce PE/plasmalogens. We have recently reviewed the role of Pcyt2 in phospholipid homeostasis (10). Recent studies using Pcyt2-deficient mouse models established a direct link between PE synthesis and triglyceride metabolism (11,C13). Heterozygous Pcyt2 deletion (using the PURExpress protein synthesis kit (New England Biolabs). This system produces a protein product devoid of post-translational modifications. The untagged Pcyt2 cDNA template was generated by PCR using the pcDNA.ET plasmid. We amplified the entire open reading frame and the required flanking sequences, including the ribosomal binding site, but deleted the V5 and His tags using Pcyt2-specific primers (forward primer, 5-taagaaggagatataccaatgatccggaacgggcacgg-3-3; reverse primer, 5-tattcattagtcaatctcccctccaggcggcgcc-3). In the second round of PCR, we added the T7 promoter at the 5-end of Pcyt2 using the universal primer (5-gaaattaatacgactcactatagggagaccacaacggtttccctctagaaataattttgtttaactttaagaaggagatatacca-3) and the Pcyt2 reverse primer. Pcyt2 cDNA was purified using a gel extraction kit (Sigma) and 250 ng of the cDNA incubated with mixture I (PURExpress) at 37 C for 1 h to generate Pcyt2 protein. The mixture was concentrated (60 min at 1,500 at 4 C) using a Microcon YM-100 spin concentrator (Millipore Corp.) and mixed with 0.25 volumes of nickel-nitrilotriacetic acid-agarose beads (Qiagen) (mixture II) and incubated at 4 C for 45 min to remove the translation assay components. Mixture II was then applied to a micro-spin column (Bio-Rad), and the unbound Pcyt2 protein was released by centrifugation (2 min at 1500 at 4 C). Pcyt2 proteins from multiple reactions were combined and pooled samples applied in the PKC phosphorylation assays below. In Vitro Pcyt2 Phosphorylation with PKC, PKCI, and PKCII Pcyt2 free from tags and post-translational modifications was subjected to phosphorylation with [-32P]ATP and either PKC, PKCI, or PKCII (Enzo Life Sciences) (28). The reaction was carried out at 30 C for 10 min in 100 l of mixture made up (Z)-Capsaicin of 20 mm HEPES, pH 7.4, 10 mm MgCl2, 0.1 mm CaCl2, 100 m ATP, 100 g/ml phosphatidylserine, 20 g/ml diacylglycerol, 0.03% Triton X-100, 1 l (Z)-Capsaicin of [-32P]ATP, and different aliquots (20 or 40 l) of Pcyt2 protein. Reactions were initiated by adding 0.4 g of PKC and terminated after 10 min by adding 20 l of 1% BSA and 1 ml of 10% TCA. After 5 min of incubation on ice, the aliquots (40 l) from each reaction were transferred to a nitrocellulose membrane on an aspirator funnel and washed three times with 2 ml of 5% TCA. Membranes were dried under vacuum and the -32P was incorporated into Pcyt2 protein determined by counting. The assays were performed in triplicate, and the background radioactivity for the reactions without Pcyt2 or PKC served as controls (28). Statistical Analysis All measurements were expressed as means S.D. from at least three impartial experiments. GraphPad Prism 3.0 software (San Diego) was used to perform one- or two-way analysis of variance and Student’s paired test. Differences were considered statistically significant at 0.05. Densitometry analysis was performed by Scion Image software (Scion Inc). RESULTS Pcyt2 and Pcyt2 Catalytic Domains and Phosphorylation Sites Fig. 1 provides a simplified representation of the Pcyt2 and – structures showing.M., Martinez A. on two PKC consensus serine residues, Ser-215 and Ser-223. Single and double mutations of PKC consensus sites reduced Pcyt2 phosphorylation, activity, and phosphatidylethanolamine synthesis by 50C90%. The phosphorylation and activity of endogenous Pcyt2 were dramatically increased with phorbol esters and reduced by specific PKC inhibitors. translated Pcyt2 was phosphorylated by PKC, PKCI, and PKCII. Pcyt2 Ser-215 was also directly phosphorylated with PKC. Mapping of the Pcyt2- and –phosphorylated sites towards the resolved structure of the human Pcyt2 demonstrated that they clustered within and flanking the central linker area that connects both catalytic domains and it is a book regulatory segment not really present in additional cytidylyltransferases. This research is the 1st to demonstrate variations in phosphorylation between Pcyt2 isoforms also to uncover the part from the PKC-regulated phosphorylation. from ethanolamine and diacylglycerol/alkylacylglycerols through the CDP-ethanolamine-Kennedy pathway and by decarboxylation of phosphatidylserine in the mitochondria. The Kennedy pathway can be quantitatively the main path for the biosynthesis of PE in mammalian cells (6). With this pathway, ethanolamine can be 1st phosphorylated by ethanolamine kinase to phosphoethanolamine (P-Etn), which can be then changed into CDP-ethanolamine (CDP-Etn) by CTP:phosphoethanolamine cytidylyltransferase (Pcyt2; also called ET (7, 8) or ECT (9)). In the ultimate stage, CDP-ethanolamine:1,2-diacylglycerol ethanolamine phosphotransferase exchanges P-Etn from CDP-Etn to DAG/alkylacylglycerol to create PE/plasmalogens. We’ve recently evaluated the part of Pcyt2 in phospholipid homeostasis (10). Latest research using Pcyt2-lacking mouse models founded a direct hyperlink between PE synthesis and triglyceride rate of metabolism (11,C13). Heterozygous Pcyt2 deletion (using the PURExpress proteins synthesis package (New Britain Biolabs). This technique produces a proteins product without post-translational adjustments. The untagged Pcyt2 cDNA template was generated by PCR using the pcDNA.ET plasmid. We amplified the complete open reading framework and the mandatory flanking sequences, like the ribosomal binding site, but erased the V5 and His tags using Pcyt2-particular primers (ahead primer, 5-taagaaggagatataccaatgatccggaacgggcacgg-3-3; opposite primer, 5-tattcattagtcaatctcccctccaggcggcgcc-3). In the next circular of PCR, we added the T7 promoter in the 5-end of Pcyt2 using the common primer (5-gaaattaatacgactcactatagggagaccacaacggtttccctctagaaataattttgtttaactttaagaaggagatatacca-3) as well as the Pcyt2 change primer. Pcyt2 cDNA was purified utilizing a gel removal package (Sigma) and 250 ng from the cDNA incubated with blend I (PURExpress) at 37 C for 1 h to create Pcyt2 proteins. The blend was focused (60 min at 1,500 at 4 C) utilizing a Microcon YM-100 spin concentrator (Millipore Corp.) and blended with 0.25 volumes of nickel-nitrilotriacetic acid-agarose beads (Qiagen) (mixture II) and incubated at 4 C for 45 min to eliminate the translation assay components. Mixture II was after that put on a micro-spin column (Bio-Rad), as well as the unbound Pcyt2 proteins premiered by centrifugation (2 min at 1500 at 4 C). Pcyt2 protein from multiple reactions had been mixed and pooled examples used in the PKC phosphorylation assays below. In Vitro Pcyt2 Phosphorylation with PKC, PKCI, and PKCII Pcyt2 clear of tags and post-translational adjustments was put through phosphorylation with [-32P]ATP and either PKC, PKCI, or PKCII (Enzo Existence Sciences) (28). The response was completed at 30 C for 10 min in 100 l of blend including 20 mm HEPES, pH 7.4, 10 mm MgCl2, 0.1 mm CaCl2, 100 m ATP, 100 g/ml phosphatidylserine, 20 g/ml diacylglycerol, 0.03% Triton X-100, 1 l of [-32P]ATP, and various aliquots (20 or 40 l) of Pcyt2 proteins. Reactions had been initiated with the addition of 0.4 g of PKC and terminated after 10 min with the addition of 20 l of 1% BSA and 1 ml of 10% TCA. After 5 min of incubation on snow, the aliquots (40 l) from each response had been used in a nitrocellulose membrane with an aspirator funnel and cleaned 3 x with 2 ml of 5% TCA. Membranes had been dried out under vacuum as well as the -32P was integrated into Pcyt2 proteins determined by keeping track of. The assays had been performed in triplicate, and the backdrop radioactivity for the reactions without Pcyt2 or PKC offered as settings (28). Statistical Evaluation All measurements had been indicated as means S.D. from at least three 3rd party tests. GraphPad Prism 3.0 software program (NORTH PARK) was used to execute one- or two-way evaluation of variance and Student’s paired check. Differences had been regarded as statistically significant at 0.05. Densitometry evaluation was performed by Scion Picture software program (Scion Inc). Outcomes Pcyt2 and Pcyt2 Catalytic Domains and Phosphorylation Sites Fig. 1 offers a simplified representation from the Pcyt2 and – constructions showing the positioning of both catalytic (CT) domains. Both splice isoforms differ just in the space from the linker linking the.Folger O., Jerby L., Frezza C., Gottlieb E., Ruppin E., Shlomi T. and Ser-223. Solitary and dual mutations of PKC consensus sites decreased Pcyt2 phosphorylation, activity, and phosphatidylethanolamine synthesis by 50C90%. The phosphorylation and activity of endogenous Pcyt2 had been improved with phorbol esters and reduced by particular PKC inhibitors dramatically. translated Pcyt2 was phosphorylated by PKC, PKCI, and PKCII. Pcyt2 Ser-215 was also straight phosphorylated with PKC. Mapping from the Pcyt2- and –phosphorylated sites towards the resolved structure of the human Pcyt2 demonstrated that they clustered within and flanking the central linker area that connects both catalytic domains and is a novel regulatory segment not present in additional cytidylyltransferases. This study is the 1st to demonstrate variations in phosphorylation between Pcyt2 isoforms and to uncover the part of the PKC-regulated phosphorylation. from ethanolamine and diacylglycerol/alkylacylglycerols through the CDP-ethanolamine-Kennedy pathway and by decarboxylation of phosphatidylserine in the mitochondria. The Kennedy pathway is definitely quantitatively the most important route for the biosynthesis of PE in mammalian cells (6). With this pathway, ethanolamine is definitely 1st phosphorylated by ethanolamine kinase to phosphoethanolamine (P-Etn), which is definitely then converted to CDP-ethanolamine (CDP-Etn) by CTP:phosphoethanolamine cytidylyltransferase (Pcyt2; also known as ET (7, 8) or ECT (9)). In the final step, CDP-ethanolamine:1,2-diacylglycerol ethanolamine phosphotransferase transfers P-Etn from CDP-Etn to DAG/alkylacylglycerol to produce PE/plasmalogens. We have recently examined the part of Pcyt2 in phospholipid homeostasis (10). Recent studies using Pcyt2-deficient mouse models founded a direct link between PE synthesis and triglyceride rate of metabolism (11,C13). Heterozygous Pcyt2 deletion (using the PURExpress protein synthesis kit (New England Biolabs). This system produces a protein product devoid of post-translational modifications. The untagged Pcyt2 cDNA template was generated by PCR using the pcDNA.ET plasmid. We amplified the entire open reading framework and the required flanking sequences, including the ribosomal binding site, but erased the V5 and His tags using Pcyt2-specific primers (ahead primer, 5-taagaaggagatataccaatgatccggaacgggcacgg-3-3; opposite primer, 5-tattcattagtcaatctcccctccaggcggcgcc-3). In the second round of PCR, we added the T7 promoter in the 5-end of Pcyt2 using the common primer (5-gaaattaatacgactcactatagggagaccacaacggtttccctctagaaataattttgtttaactttaagaaggagatatacca-3) and the Pcyt2 reverse primer. Pcyt2 cDNA was purified using a gel extraction kit (Sigma) and 250 ng of the cDNA incubated with combination I (PURExpress) at 37 C for 1 h to generate Pcyt2 protein. The combination was concentrated (60 min at 1,500 at 4 C) using a Microcon YM-100 spin concentrator (Millipore Corp.) and mixed with 0.25 volumes of nickel-nitrilotriacetic acid-agarose beads (Qiagen) (mixture II) and incubated at 4 C for 45 min to remove the translation assay components. Mixture II was then applied to a micro-spin column (Bio-Rad), and the unbound Pcyt2 protein was released by centrifugation (2 min at 1500 at 4 C). Pcyt2 proteins from multiple reactions were combined and pooled samples applied in the PKC phosphorylation assays below. In Vitro Pcyt2 Phosphorylation with PKC, PKCI, and PKCII Pcyt2 free from tags and post-translational modifications was subjected to phosphorylation with [-32P]ATP and either PKC, PKCI, or PKCII (Enzo Existence Sciences) (28). The reaction was carried out at 30 C for 10 min in 100 l of combination comprising 20 mm HEPES, pH 7.4, 10 mm MgCl2, 0.1 mm CaCl2, 100 m ATP, 100 g/ml phosphatidylserine, 20 g/ml diacylglycerol, 0.03% Triton X-100, 1 l of [-32P]ATP, and different aliquots (20 or 40 l) of Pcyt2 protein. Reactions were initiated by adding 0.4 g of PKC and terminated after 10 min by adding 20 l of 1% BSA and 1 ml of 10% TCA. After 5 min of incubation on snow, the aliquots (40 l) from each reaction were transferred to a nitrocellulose membrane on an aspirator funnel and washed three times with 2 ml of 5% TCA. Membranes were dried under vacuum and the -32P was integrated into Pcyt2 protein determined by counting. The assays were performed in triplicate, and the background radioactivity for the reactions without Pcyt2 or PKC served as settings (28). Statistical Analysis All measurements were indicated as means S.D. from at least three self-employed experiments. GraphPad Prism 3.0 software (San Diego) was used to perform one- or two-way analysis of variance and Student’s paired test. Differences were regarded as statistically significant at 0.05. Densitometry analysis was performed by Scion Image software (Scion Inc). RESULTS Pcyt2 and Pcyt2 Catalytic Domains and Phosphorylation Sites Fig. 1 provides a simplified representation of the.J. were dramatically improved with phorbol esters and reduced by specific PKC inhibitors. translated Pcyt2 was phosphorylated by PKC, PKCI, and PKCII. Pcyt2 Ser-215 was also directly phosphorylated with PKC. Mapping of the Pcyt2- and –phosphorylated sites to the solved structure of a human Pcyt2 showed that they clustered within and flanking the central linker region that connects the two catalytic domains and is a novel regulatory segment not present in additional cytidylyltransferases. This study is the 1st to demonstrate variations in phosphorylation between Pcyt2 isoforms and to uncover the part of the PKC-regulated phosphorylation. from ethanolamine and diacylglycerol/alkylacylglycerols through the CDP-ethanolamine-Kennedy pathway and by decarboxylation of phosphatidylserine in the mitochondria. The Kennedy pathway is definitely quantitatively the most important route for the biosynthesis of PE in mammalian cells (6). With this pathway, ethanolamine is definitely 1st phosphorylated by ethanolamine kinase to phosphoethanolamine (P-Etn), which is definitely then converted to CDP-ethanolamine (CDP-Etn) by CTP:phosphoethanolamine cytidylyltransferase (Pcyt2; also known as ET (7, 8) or ECT (9)). In the final step, CDP-ethanolamine:1,2-diacylglycerol ethanolamine phosphotransferase transfers P-Etn from CDP-Etn to DAG/alkylacylglycerol to produce PE/plasmalogens. We have recently examined the part of Pcyt2 in phospholipid homeostasis (10). Recent studies using Pcyt2-deficient mouse models founded a direct link between PE synthesis and triglyceride rate of metabolism (11,C13). Heterozygous Pcyt2 deletion (using the PURExpress protein synthesis kit (New England Biolabs). This system produces a proteins product without post-translational adjustments. The untagged Pcyt2 cDNA template was generated by PCR using the pcDNA.ET plasmid. We amplified the complete open reading body and the mandatory flanking sequences, like the ribosomal binding site, but removed the V5 and His tags using Pcyt2-particular primers (forwards primer, 5-taagaaggagatataccaatgatccggaacgggcacgg-3-3; slow primer, 5-tattcattagtcaatctcccctccaggcggcgcc-3). In the next circular of PCR, we added the T7 promoter on the 5-end of Pcyt2 using the general primer (5-gaaattaatacgactcactatagggagaccacaacggtttccctctagaaataattttgtttaactttaagaaggagatatacca-3) as well as the Pcyt2 change primer. Pcyt2 cDNA was purified utilizing a gel removal package (Sigma) and 250 ng from the cDNA incubated with mix I (PURExpress) at 37 C for 1 h to create Pcyt2 proteins. The mix was focused (60 min at 1,500 at 4 C) utilizing a Microcon YM-100 spin concentrator (Millipore Corp.) and blended with 0.25 volumes of nickel-nitrilotriacetic acid-agarose beads (Qiagen) (mixture II) and incubated at 4 C for 45 min to eliminate the translation assay components. Mixture II was after that put on a micro-spin column (Bio-Rad), as well as the unbound Pcyt2 proteins premiered by centrifugation (2 min at 1500 at 4 C). Pcyt2 protein from multiple reactions had been mixed and pooled examples used in the PKC phosphorylation assays below. In Vitro Pcyt2 Phosphorylation with PKC, PKCI, and PKCII Pcyt2 clear of tags and post-translational adjustments was put through phosphorylation with [-32P]ATP and either PKC, PKCI, or PKCII (Enzo Lifestyle Sciences) (28). The response was completed at 30 C for 10 min in 100 l of mix formulated with 20 mm HEPES, pH 7.4, 10 mm MgCl2, 0.1 mm CaCl2, 100 m ATP, 100 g/ml phosphatidylserine, 20 g/ml diacylglycerol, 0.03% Triton X-100, 1 l of [-32P]ATP, and various aliquots (20 or 40 l) of Pcyt2 proteins. Reactions had been initiated with the addition of 0.4 g of PKC and terminated after 10 min with the addition of 20 l of 1% BSA and 1 ml of 10% TCA. After 5 min of incubation on glaciers, the aliquots (40 l) from each response had been used in a nitrocellulose membrane with an aspirator funnel and cleaned 3 x with 2 ml of 5% TCA. Membranes had been dried out under vacuum as well as the -32P was included into Pcyt2 proteins determined by keeping track of. The assays had been performed in triplicate, and the backdrop radioactivity for the reactions without (Z)-Capsaicin Pcyt2 or PKC offered as handles (28). Statistical Evaluation All measurements had been portrayed as means S.D. from at least three indie tests. GraphPad Prism 3.0 software program (NORTH PARK) was used to execute one- or two-way evaluation of variance and Student’s paired check. Differences had been regarded statistically significant at 0.05. Densitometry evaluation was performed by Scion Picture software program (Scion Inc). Outcomes Pcyt2 and Pcyt2 Catalytic Domains and Phosphorylation Sites Fig. 1 offers a simplified representation from the Pcyt2 and – buildings showing the positioning of both catalytic (CT) domains. Both splice isoforms differ just in the distance from the linker hooking up both CT domains the following: 74 residues for the isoform and 56 residues for Pcyt2. Putative phosphorylation sites had been deduced with the Scan-Prosite proteomic server (ExPASy-Swiss Institute of Bioinformatics). Information on how the particular phosphorylations proven in Fig. 1 had been characterized are defined below..