Association of the N- and C-terminal Domains of Phospholipase D

Rat brain phospholipase D1 (rPLD1) belongs to a superfamily defined by the highly conserved catalytic motif (H(X)K(X)4D, denoted HKD. rPLD1 contains two HKD domains, located in the N- and C-terminal regions. The integrity of the two HKD domains is essential for enzymatic activity. Our previous studies showed that the N-terminal half of rPLD1 containing one HKD motif can associate with the C-terminal half containing the other HKD domain to reconstruct wild type PLD activity (Xie, Z., Ho, W.-T. and Exton, J. H. (1998)J. Biol. Chem. 273, 34679–34682). In the present study, we have shown by mutagenesis that conserved amino acids in the HKD domains are important for both the catalytic activity and the association between the two halves of rPLD1. Furthermore, we found that rPLD1 could be modified by Ser/Thr phosphorylation. The modification occurred at the N-terminal half of the enzyme, however, the association of the N-terminal domain with the C-terminal domain was required for the modification. The phosphorylation of the enzyme was not required for its catalytic activity or response to PKCα and small G proteinsin vitro, although the phosphorylated form of rPLD1 was localized exclusively in the crude membrane fraction. In addition, we found that the individually expressed N- and C-terminal fragments did not interact when mixed in vitro and were unable to reconstruct PLD activity under these conditions. It is concluded that the association of the N- and C-terminal halves of rPLD1 requires their co-expression in vivo and depends on conserved residues in the HKD domains. The association is also required for Ser/Thr phosphorylation of the enzyme. Rat brain phospholipase D1 (rPLD1) belongs to a superfamily defined by the highly conserved catalytic motif (H(X)K(X)4D, denoted HKD. rPLD1 contains two HKD domains, located in the N- and C-terminal regions. The integrity of the two HKD domains is essential for enzymatic activity. Our previous studies showed that the N-terminal half of rPLD1 containing one HKD motif can associate with the C-terminal half containing the other HKD domain to reconstruct wild type PLD activity (Xie, Z., Ho, W.-T. and Exton, J. H. (1998)J. Biol. Chem. 273, 34679–34682). In the present study, we have shown by mutagenesis that conserved amino acids in the HKD domains are important for both the catalytic activity and the association between the two halves of rPLD1. Furthermore, we found that rPLD1 could be modified by Ser/Thr phosphorylation. The modification occurred at the N-terminal half of the enzyme, however, the association of the N-terminal domain with the C-terminal domain was required for the modification. The phosphorylation of the enzyme was not required for its catalytic activity or response to PKCα and small G proteinsin vitro, although the phosphorylated form of rPLD1 was localized exclusively in the crude membrane fraction. In addition, we found that the individually expressed N- and C-terminal fragments did not interact when mixed in vitro and were unable to reconstruct PLD activity under these conditions. It is concluded that the association of the N- and C-terminal halves of rPLD1 requires their co-expression in vivo and depends on conserved residues in the HKD domains. The association is also required for Ser/Thr phosphorylation of the enzyme. phospholipase D polyacrylamide gel electrophoresis protein kinase C phorbol 12-myristate 13-acetate phosphatidylinositol 4,5-bisphosphate phosphatidylethanolamine phosphatidylcholine immunoprecipitation guanosine 5′-O-(thiotriphosphate) Phospholipase D (PLD)1is a ubiquitous enzyme found in bacteria, fungi, plants, and mammals (1Exton J.H. Biochim. Biophys. Acta. 1998; 1436: 105-115Crossref PubMed Scopus (131) Google Scholar). It hydrolyzes phosphatidylcholine to phosphatic acid and choline. Phosphatic acid is generally recognized as the signaling product of PLD and functions as an effector in different physiological processes. Phosphatic acid can be converted to diacylglycerol by phosphatidate phosphohydrolase or to lysophosphatidic acid by a specific phospholipase A2. Diacylglycerol is a well characterized activator for protein kinase C, while lysophosphatidic acid is a major extracellular signal that acts on specific cell surface receptors. PLD also catalyzes a phosphatidyl transfer reaction using primary alcohols as nucleophilic acceptors to produce phosphatidylalcohols. This reaction is used as a specific measure of PLD activity. The physiological function of PLD is still under investigation. Several studies have shown that PLD plays a role in a variety of signaling pathways (2Exton J.H. Physiol. Rev. 1997; 77: 303-320Crossref PubMed Scopus (388) Google Scholar). Different factors, including protein tyrosine kinases, protein kinase C (PKC), heterotrimeric and small G proteins, and intracellular Ca2+ regulate PLD activity directly or indirectly (2Exton J.H. Physiol. Rev. 1997; 77: 303-320Crossref PubMed Scopus (388) Google Scholar, 3Houle M.G. Bourgoin S. Biochim. Biophys. Acta. 1999; 1439: 135-150Crossref PubMed Scopus (86) Google Scholar, 4Exton J.H. Biochim. Biophys. Acta. 1999; 1439: 121-133Crossref PubMed Scopus (337) Google Scholar). Based on the wide involvement of PLD in signaling pathways and the actions of its products, multiple functions of PLD have been proposed (2Exton J.H. Physiol. Rev. 1997; 77: 303-320Crossref PubMed Scopus (388) Google Scholar), which include signal transduction, membrane trafficking, and cytoskeleton changes. To date, two isoforms of mammalian PLD (PLD1 and PLD2) have been cloned. These isoforms share about 50% amino acid similarity, but exhibit quite different regulatory properties. PLD1 has a low basal activity and responds to PKC and to members of the Rho and ARF families of small G proteins (5Hammond S.M. Altshuller Y.M. Sung T.-C. Rudge S.A. Rose K. Engebrecht J. Morris A.J. Frohman M.A. J. Biol. Chem. 1995; 270: 29640-29643Abstract Full Text Full Text PubMed Scopus Google Scholar, S.M. S. K. S. Frohman M.A. Morris A.J. J. Biol. Chem. 1997; Full Text Full Text PubMed Scopus Google Scholar, W.-T. J.H. J. Biol. Chem. 1997; Full Text Full Text PubMed Scopus Google Scholar, Altshuller Y.M. Frohman M.A. J. 1997; PubMed Scopus Google Scholar). The PKC domain has been to the N-terminal of the J.H. Biophys. 1998; PubMed Scopus Google Scholar, W.-T. J.H. J. Biol. Chem. 1998; Full Text Full Text PubMed Scopus Google Scholar, Morris A.J. Frohman M.A. J. Biol. Chem. 1999; Full Text Full Text PubMed Scopus Google Scholar), while the Rho domain has been localized to the C-terminal of the enzyme H. S. K. H. H. Frohman M.A. J. Biol. Chem. 1999; Full Text Full Text PubMed Scopus Google Scholar). on the other a basal activity and generally response to S. J. Biol. Chem. 1997; Full Text Full Text PubMed Scopus Google Scholar, Sung T.-C. J. S.M. Altshuller Morris A.J. Frohman M.A. Biol. 1997; Full Text Full Text PubMed Scopus Google Scholar, J. Biol. Chem. 1998; Full Text Full Text PubMed Scopus Google Scholar). a has shown that can be tyrosine phosphorylated and its activity in with Frohman M.A. K. J. Biol. Chem. 1998; Full Text Full Text PubMed Scopus Google Scholar). The of the two isoforms is not well have shown that both isoforms are present in a membrane J. Biol. Chem. 1999; Full Text Full Text PubMed Scopus Google Scholar, J.H. S. 1999; PubMed Scopus Google Scholar). has that PLD belongs to a superfamily defined by an denoted A.J. Engebrecht J. Frohman M.A. Full Text PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, Full Text PubMed Scopus Google Scholar). The the exhibit functions and include proteins, and the the of the superfamily the HKD motif to be essential for their enzymatic activity. PLD contains two of the HKD of HKD motif PLD1 and Morris A.J. Frohman M.A. J. Biol. Chem. 1999; Full Text Full Text PubMed Scopus Google Scholar, T.-C. Rudge S.A. S.M. Morris A.J. Engebrecht J. Frohman M.A. J. 1997; PubMed Scopus Google Scholar). and studies of the and that the in the conserved motif is directly in the catalytic reaction by a J. J. Biol. Chem. 1999; Full Text Full Text PubMed Scopus Google Scholar, Biol. 1999; PubMed Scopus Google Scholar, S. 1998; PubMed Scopus (131) Google Scholar). have a PLD1 brain (rPLD1) and have its in vivo and in vitro W.-T. J.H. J. Biol. Chem. 1997; Full Text Full Text PubMed Scopus Google Scholar, J.H. Biophys. 1998; PubMed Scopus Google Scholar, J.H. J. Biol. Chem. 1998; Full Text Full Text PubMed Scopus Google J.H. J. Biol. Chem. 1998; Full Text Full Text PubMed Scopus Google Scholar). Our previous of showed that N-terminal or C-terminal fragments containing one of the HKD domains did not have PLD activity when expressed in W.-T. J.H. J. Biol. Chem. 1998; Full Text Full Text PubMed Scopus Google Scholar). PLD activity was when the two fragments were W.-T. J.H. J. Biol. Chem. 1998; Full Text Full Text PubMed Scopus Google Scholar). Furthermore, the N- and C-terminal fragments were shown to associate W.-T. J.H. J. Biol. Chem. 1998; Full Text Full Text PubMed Scopus Google Scholar). the two HKD domains are to form an catalytic by In study, we mutagenesis to the of conserved amino acids in the HKD domains for the association of the N- and C-terminal halves of rPLD1. In the of we found that rPLD1 can be modified by Ser/Thr phosphorylation and modification requires the association of the N- and C-terminal halves of the enzyme. 12-myristate 13-acetate phosphatidylinositol and were and were and acid were modified and were The and the were were were The and the the and were with were PKCα was Ser/Thr was by The N-terminal rPLD1 or the N- or C-terminal fragments of rPLD1 with to amino acids and were by and at in the of as W.-T. J.H. J. Biol. Chem. 1998; Full Text Full Text PubMed Scopus Google Scholar). The C-terminal rPLD1 containing amino acids to was also by by in the of The was The was The fragments were with the C-terminal The of rPLD1 were as in the the were to the of rPLD1. were in modified with and in were with and were with and with to with in were in in the of the were with and in with for PLD activity was as J.H. J. Biol. Chem. Full Text Full Text PubMed Scopus Google Scholar). were in for were with and with were and the product was by with a were by of were and as The were with of and and The were in and a The cell was at for to The was at for at to the and crude membrane The was with the and a the was The was used for the of protein in the and the crude membrane the of the N- and C-terminal domains was in vitro, the membrane N- or C-terminal or both fragments were in the with the of to a of and to The was mixed as under and at for to and the was to immunoprecipitation as were by on and to The were with and with primary by with were using on were and as The were with and in immunoprecipitation containing and of The cell was a and the cell was at in an for at to the The was by with of and of a of protein for at The was and the was with of and of protein The were with the and in and by or N- and C-terminal halves of rPLD1 were by as and with the to proteins to the protein and an two with and of to the in the The on the protein were in the reaction and with addition, or with the of Ser/Thr M.A. S. J. Biol. Chem. 1999; Full Text Full Text PubMed Scopus Google containing and or with the with a of protein type and S. PubMed Scopus Google Scholar). at the reaction was by the of and by or the were with and used for in vitro PLD The catalytic activity of the or N- and C-terminal halves of rPLD1 was by the of vitro as W.-T. J.H. J. Biol. Chem. 1997; Full Text Full Text PubMed Scopus Google Scholar). containing were used with as The reaction were at for and with The were the and by with a were by Our previous showed that the N-terminal of rPLD1 containing one of the HKD domains can form a with the C-terminal containing the other HKD domain W.-T. J.H. J. Biol. Chem. 1998; Full Text Full Text PubMed Scopus Google Scholar). of PLD1 have shown that of amino acids in the conserved HKD PLD activity T.-C. Rudge S.A. S.M. Morris A.J. Engebrecht J. Frohman M.A. J. 1997; PubMed Scopus Google and of the S. 1998; PubMed Scopus (131) Google and J. J. Biol. Chem. 1999; Full Text Full Text PubMed Scopus Google have that the conserved in the HKD motif directly in the catalytic reaction by a the that the HKD and their are important for the of amino acids containing and found or to the HKD domains are highly conserved mammalian and PLD as in the of these amino acids in the by to the amino acid The in the N-terminal half of rPLD1 were and and the in the C-terminal half of rPLD1 were and In the the wild type N- and C-terminal fragments of rPLD1 are to as and In addition, the used in the was at its C with a and was at its with an These two have been shown to be to associate and reconstruct wild type PLD activity when in W.-T. J.H. J. Biol. Chem. 1998; Full Text Full Text PubMed Scopus Google Scholar). the of the of the amino acids on PLD activity in of wild type with in a PLD activity to wild in the activity to of that of wild while and to of wild type and activity a was with wild type of wild type PLD activity was was at the C-terminal half of activity was when the was with the wild type N-terminal half in when and were PLD activity to was the in the N- or C-terminal halves the PLD although to different The of activity could not be to of the proteins and In to we also to or the the PLD activity to activity was when was to a amino acid of to the PLD activity not or to the PLD activity to not These that these of amino acids are important for PLD activity. To the of these amino acids in the association of the N- and C-terminal fragments of immunoprecipitation were the and fragments were with and The were with the wild type or N- and C-terminal fragments of rPLD1 and the cell and were with and by with and a was with a protein was by in in a was with and a protein to the of was by in and was also and the of is in a N- or C-terminal fragments were with their wild type or N-terminal we found that showed a as wild type to associate with or to associate with its wild type or N-terminal half in and which is with their to reconstruct a wild type PLD activity for the and of or association with their N-terminal halves was with its on PLD its to associate with the C-terminal the did not the association between and In addition, of or to the association to the or not of the of the N- or C-terminal fragments in showed that the were expressed at with that of the wild type and and were localized in the crude membrane not Based on the we that these amino acids are important for the association between the N- and C-terminal halves of rPLD1. The PLD activity by the with their on the that the of catalytic activity is to the association by the of these amino Several in the HKD as and have been shown to the enzymatic activity of PLD1 T.-C. Rudge S.A. S.M. Morris A.J. Engebrecht J. Frohman M.A. J. 1997; PubMed Scopus Google Scholar). the and in the N-terminal half and in the C-terminal half of rPLD1. with the with these rPLD1 the to reconstruct PLD activity when with their wild type halves not Furthermore, we found that these also the between the N- and of rPLD1 although these were expressed in of PLD1 in the conserved HKD domains has that were by the T.-C. Rudge S.A. S.M. Morris A.J. Engebrecht J. Frohman M.A. J. 1997; PubMed Scopus Google Scholar). In addition, of HKD in by and of J. J. Biol. Chem. 1999; Full Text Full Text PubMed Scopus Google Scholar). the of the in the HKD on the association and catalytic activity or rPLD1 were to be by of the In the shown that conserved amino acids in or to the HKD are important for the association between the N- and C-terminal domains. amino as the in the conserved in both the catalytic reaction and the association between the N- and C-terminal Several were in the of the in the HKD when the N-terminal half of rPLD1 was with its C-terminal the N-terminal half of rPLD1 was modified as by the of with when the cell in and or the in were modification of the N-terminal was when was not or with a the in In addition, the C-terminal as a showed of modification. the in the HKD were was found that in the N- or C-terminal domains that or the or also the modification of the N-terminal and the other the that did not or the association and still or modification with wild type and In the of in which association was and a low of modification was to These that rPLD1 can be modified in the N-terminal and modification requires not the of the C-terminal half association with the C-terminal In to the of the the N- and C-terminal halves were and the using as under the modification in the N-terminal half of rPLD1 was to phosphorylation. The were at for with a Ser/Thr M.A. S. J. Biol. Chem. 1999; Full Text Full Text PubMed Scopus Google or with the by a of type and S. PubMed Scopus Google Scholar). a the were also with for on or at The reaction were by with to the N-terminal of rPLD1. shown in the of modification was not when the were with when the containing the N- and C-terminal halves were with the Ser/Thr the with and was of the protein with which to the of the N-terminal the Ser/Thr was mixed with and to the the of the was in To the modification was also with rPLD1 and to the of Ser/Thr rPLD1 with was and by of the enzyme was also as by the of a protein with that of rPLD1 and in The with a low was to by the Ser/Thr which converted to a which the form This of the was also by The modification in the N-terminal half or rPLD1 was also to by not but not by with the catalytic domain of the protein tyrosine J. J. 1999; PubMed Scopus Google not These that the modification in rPLD1 in is Ser/Thr phosphorylation and that on the N-terminal half of the enzyme. The modification on multiple Ser/Thr in an of the N-terminal of rPLD1 to The association between the N- and C-terminal halves is for the modification of rPLD1. the modification was a of N-terminal was with the C-terminal half in the the C-terminal domain plays an important role in the Ser/Thr modification of the N-terminal the is not is that the C-terminal the kinase that the Ser/Thr phosphorylation. is that a by the association of N- and C-terminal domains of rPLD1 the phosphorylation to the Our previous W.-T. J.H. J. Biol. Chem. 1998; Full Text Full Text PubMed Scopus Google showed that the N- or C-terminal half of rPLD1 containing one HKD domain did not exhibit PLD activity when The enzymatic activity of rPLD1 required the association of the N- and C-terminal domains W.-T. J.H. J. Biol. Chem. 1998; Full Text Full Text PubMed Scopus Google and The shown that association is also required for Ser/Thr phosphorylation of the enzyme. The the phosphorylation is required for catalytic activity of rPLD1. To we expressed and N-terminal rPLD1 in The rPLD1 was and with or was by with Ser/Thr or not with at for the the were with and for in activity. The C-terminal half of which has catalytic was also expressed and and used as a in the in vitro PLD shown in activity was for the rPLD1 that was with at for This PLD activity was by of PKCα and the were with the or with activity was also which was also to with PKCα and the Ser/Thr phosphorylation of rPLD1 was not essential for the basal catalytic activity of the enzyme or its response to PKCα In the the activity of the enzyme in the or of PKCα The of the to with an of the response to the small G proteins was we found that the Ser/Thr phosphorylation was not required for the response of rPLD1 to or in vitro with the or enzyme, the rPLD1 a response to the of or in the of The of two enzyme fragments to catalytic activity of a enzyme has been not for but also for well characterized as and J. Biol. Chem. Full Text Google J. J. Biol. PubMed Scopus Google Scholar). studies have also that the catalytic activity of and phospholipase can be by the of two protein fragments that catalytic domains S. PubMed Scopus Google Scholar, K. S. PubMed Scopus Google Scholar). In the of the two catalytic domains and can be expressed in and mixed in the enzymatic activity S. PubMed Scopus Google Scholar). To the N- and C-terminal domains of rPLD1 could catalytic activity in vitro, we the N- and C-terminal halves of rPLD1 or in in the N-terminal was with and the C-terminal was with an of the were and membrane and were The N- and C-terminal fragments were localized in the crude membrane fraction. the membrane was in containing and to membrane proteins, and used for the of the N- and C-terminal domains in a the membrane the N-terminal was mixed with the and N-terminal fragments were by in the by protein to the of the N-terminal was by in the the membrane of with the N- and C-terminal fragments when the membrane the N- or C-terminal were was by in the although the expressed N- and C-terminal fragments were mixed at to in It is that the present in the immunoprecipitation the between the individually expressed N- and C-terminal fragments in the in vitro association between the two halves using the which did not the N-terminal was by in the when the N- and C-terminal fragments were in the C, In the was shown that the N- or C-terminal fragments were also mixed at to in with the to associate in vitro, PLD activity was when the individually expressed N- and C-terminal fragments the membrane or were mixed and by in vitro or not The are to be to of the fragments the N- and C-terminal fragments were to the association and PLD activity. these that the association of the N- and C-terminal fragments of rPLD1 requires their in studies of the of the N- and C-terminal halves of rPLD1 that the phosphorylated form which with was in the membrane This form was also in the membrane In the N-terminal that was with the C-terminal was as a in the the rPLD1 was the modified form that was also in the membrane but not in the a small of the N- and C-terminal fragments of rPLD1 in the protein was or the was to that the form with a could be the fraction. these did not the modified form of rPLD1 that is located exclusively in the membrane fraction. studies of rPLD1 in vitro have shown that has PLD activity and responds to as small G proteins W.-T. J.H. J. Biol. Chem. 1997; Full Text Full Text PubMed Scopus Google Scholar). activity that was by PKCα could be in the of with rPLD1 or with the N- and C-terminal halves of rPLD1 the rPLD1 and N-terminal half of rPLD1 present in the are not modified these are with the immunoprecipitation of in that that the phosphorylation modification of rPLD1 is not essential for catalytic activity of the enzyme in Our previous studies of rPLD1 showed that the N-terminal containing one HKD motif can associate with the C-terminal containing the other HKD motif to form a Morris A.J. Frohman M.A. J. Biol. Chem. 1999; Full Text Full Text PubMed Scopus Google Scholar). the domains in the association and the of the were not we found that to of conserved amino acids or to the conserved HKD domains the association between the N- and C-terminal domains although to The of these on the association with their on the of the domains to reconstruct PLD activity in with previous studies W.-T. J.H. J. Biol. Chem. 1998; Full Text Full Text PubMed Scopus Google these that the association of the N- and C-terminal domains is important of the catalytic activity of Furthermore, that the HKD to This is with the of the between the two the amino acids in the HKD Biol. 1999; PubMed Scopus Google Scholar). This association the two HKD domains to form a catalytic of and have that the conserved in the HKD directly in the catalytic reaction by as a in the J. J. Biol. Chem. 1999; Full Text Full Text PubMed Scopus Google Scholar, Biol. 1999; PubMed Scopus Google Scholar, S. 1998; PubMed Scopus (131) Google Scholar). This to PLD as a of the conserved in the HKD motif also the activity of the was in the we found that not the catalytic activity of but also the association between the N- and C-terminal halves the conserved amino acids in the HKD have in both the catalytic reaction and the It be that other amino acid or the two HKD domains also be in the studies of PLD be required to we found that the association of the N- and C-terminal domains was also required for Ser/Thr phosphorylation modification of rPLD1 at the N-terminal half PLD1 and have been found to be in different cell when with Frohman M.A. K. J. Biol. Chem. 1998; Full Text Full Text PubMed Scopus Google Scholar, J. Bourgoin S. J. Biol. Chem. 1997; Full Text Full Text PubMed Scopus Google Scholar, J.H. J. Biol. Chem. 1998; Full Text Full Text PubMed Scopus Google Scholar). The Ser/Thr phosphorylation of rPLD1 in in the of and the function of the modification investigation. The of that Ser/Thr phosphorylation is not essential for the association of the N- and C-terminal halves a major of the N-terminal fragments found in the were The of and also that the phosphorylation is not for the catalytic activity of rPLD1 or its response to PKCα in In rPLD1 was found to have a basal activity and not Ser/Thr phosphorylation of has also been when the were with a Ser/Thr protein H. Biochim. Biophys. Acta. PubMed Scopus Google Scholar). The phosphorylation the catalytic activity of while the H. Biochim. Biophys. Acta. PubMed Scopus Google Scholar). Ser/Thr phosphorylation of PLD1 and a role in the catalytic activity of these of rPLD1 that was by the modification was the of the enzyme. shown in rPLD1 was located exclusively in the membrane fraction. phosphorylation modification has been in a PLD S.A. Morris A.J. Engebrecht J. J. Biol. 1998; PubMed Scopus Google Scholar). This modification did not its catalytic vitro, however, the of and was found to be essential for the of the membrane PLD1 has been found not in intracellular but also in J.H. S. 1999; PubMed Scopus Google Scholar), which are specific domains on that are in signaling is that the phosphorylation of rPLD1 be essential for the physiological function of the enzyme by its in rPLD1 that the Ser/Thr phosphorylation could be used in studies to the phosphorylation the catalytic activity and function of the enzyme in of the association of the N- and C-terminal domains in vitro showed that the individually expressed N- and C-terminal halves did not interact in vitro when the association was when the N- and the C-terminal domains were in the the association of the N- and C-terminal domains their and specific of the two This has been for the conserved catalytic domains of the enzyme, and can interact and reconstruct the catalytic activity when in K. S. PubMed Scopus Google Scholar). of catalytic activity by two fragments has also been for although the two catalytic domains, and can be individually expressed and mixed in vitro to enzymatic activity S. PubMed Scopus Google Scholar). In addition, the enzymatic activity of can be in the of the association of the two domains by of the enzyme S. PubMed Scopus Google Scholar). It is that the activity of PLD also be by the association between N- and C-terminal fragments of the amino acids required for and association at the HKD a in the association catalytic activity. of PLD could regulate its enzymatic activity by the studies have shown that conserved amino acids in the HKD domains are important for both the catalytic activity and association of studies be to the domains associate and a catalytic The that the enzyme is Ser/Thr phosphorylated on its N-terminal half and that modification is in the form also requires to the of the phosphorylation and to the and protein and also to the role of the C-terminal half in the phosphorylation. J. for the Ser/Thr and J. for a also for we for the