Protein Kinase C Inhibits Type VI Adenylyl Cyclase by Phosphorylating the Regulatory N Domain and Two Catalytic C1 and C2 Domains

Abstract

We previously showed that phosphorylation of Ser10 of the N terminus domain of the type VI adenylyl cyclase (ACVI) partly mediated protein kinase C (PKC)-induced inhibition of ACVI. We now report that phosphorylation of the other two cytosolic domains (C1 and C2), which form the catalytic core complex of ACVI, also contributes to PKC-mediated inhibition. In vitrophosphorylation by PKC of the recombinant C1a and C2 domains, and of the synthetic peptides representing potential PKC phosphorylation sites, suggests that Ser568 and Ser674 of the C1 domain and Thr931 of the C2 domain might act as substrates for PKC. We next created several full-length ACVI mutants in which one or more of the four likely PKC phosphorylation sites (Ser10, Ser568, Ser674, and Thr931) were mutated to alanine. Simultaneous mutation of at least two of the three likely residues located in the N and C1 domains (Ser10, Ser568, and Ser674) was required to render ACVI variants completely insensitive to PKC treatment. In contrast, a single mutation of Thr931 was sufficient to create a functional ACVI mutant that exhibited no detectable PKC-mediated inhibition, demonstrating the essentiality of Thr931 to PKC-mediated regulation. Based on these results, we propose that the three cytosolic domains of ACVI might form a regulatory complex. Phosphorylation of this regulatory complex at different sites might induce a fine-tuning of the catalytic core complex and subsequently lead to alternation in the catalytic activity of ACVI. We previously showed that phosphorylation of Ser10 of the N terminus domain of the type VI adenylyl cyclase (ACVI) partly mediated protein kinase C (PKC)-induced inhibition of ACVI. We now report that phosphorylation of the other two cytosolic domains (C1 and C2), which form the catalytic core complex of ACVI, also contributes to PKC-mediated inhibition. In vitrophosphorylation by PKC of the recombinant C1a and C2 domains, and of the synthetic peptides representing potential PKC phosphorylation sites, suggests that Ser568 and Ser674 of the C1 domain and Thr931 of the C2 domain might act as substrates for PKC. We next created several full-length ACVI mutants in which one or more of the four likely PKC phosphorylation sites (Ser10, Ser568, Ser674, and Thr931) were mutated to alanine. Simultaneous mutation of at least two of the three likely residues located in the N and C1 domains (Ser10, Ser568, and Ser674) was required to render ACVI variants completely insensitive to PKC treatment. In contrast, a single mutation of Thr931 was sufficient to create a functional ACVI mutant that exhibited no detectable PKC-mediated inhibition, demonstrating the essentiality of Thr931 to PKC-mediated regulation. Based on these results, we propose that the three cytosolic domains of ACVI might form a regulatory complex. Phosphorylation of this regulatory complex at different sites might induce a fine-tuning of the catalytic core complex and subsequently lead to alternation in the catalytic activity of ACVI. adenylyl cyclase(s) type VI adenylyl cyclase A2A-R protein kinase A protein kinase C phosphate-buffered saline phenylmethylsulfonyl fluoride guanosine 5′-3-O-(thio)triphosphate wild-type Genes of nine mammalian membrane-bound adenylyl cyclases (ACs)1 have been isolated and characterized (1Taussig R. Gilman A.G. J. Biol. Chem. 1995; 270: 1-4Abstract Full Text Full Text PDF PubMed Scopus (432) Google Scholar, 2Paterson J.M. Smith S.M. Harmar A.J. Antoni F.A. Biochem. Biophys. Res. Commun. 1995; 214: 1000-1008Crossref PubMed Scopus (93) Google Scholar, 3Chern Y. Cell. Signal. 2000; 12: 195-204Crossref PubMed Scopus (72) Google Scholar). All of these ACs contain two hydrophobic spans, composed of six transmembrane helices, and three large cytoplasmic domains (N, C1a/b, and C2, see Fig. 1A). The crystal structure of the catalytic domains of AC has been resolved and analyzed in detail (4Yan S.-Z. Hahn D. Huang Z.-H. Tang W.-J. J. Biol. Chem. 1996; 271: 10941-10945Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 5Tesmer J.J.G. Sunahara R.K. Gilman A.G. Sprang S.R. Science. 1997; 278: 1907-1916Crossref PubMed Scopus (680) Google Scholar). It was clearly demonstrated that two cytoplasmic domains (C1a and C2) of ACs form the catalytic core. In addition, Gαs and/or forskolin increase the affinity between C1a and C2 and activate cyclase by changing the relative orientation of the C1 and C2 domains to an active conformation (4Yan S.-Z. Hahn D. Huang Z.-H. Tang W.-J. J. Biol. Chem. 1996; 271: 10941-10945Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 5Tesmer J.J.G. Sunahara R.K. Gilman A.G. Sprang S.R. Science. 1997; 278: 1907-1916Crossref PubMed Scopus (680) Google Scholar). The functional roles of the C1b domain of ACs are relatively more variable. For ACII and ACVII, the C1b domain suppresses the catalytic activity by holding ACs in their basal non-stimulated state (6Yan S.-Z. Beeler J.A. Chen Y. Shelton R.K. Tang W.-J. J. Biol. Chem. 2001; 276: 8500-8506Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Moreover, the C1b domain modulates calmodulin-elicited activation of ACI- and PKA-evoked inhibition of ACVI (7Levin L.R. Reed R.R. J. Biol. Chem. 1995; 270: 7573-7579Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 8Chen Y. Li A. Harry J. Smit M.J. Bai X. Magnusson R. Pieroni J.P. Weng G. Iyengar R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14100-14104Crossref PubMed Scopus (123) Google Scholar). The N terminus domains of ACs are highly variable among ACs and have been demonstrated to play a regulatory role (9Gu C. Cooper D.M.F. J. Biol. Chem. 1999; 274: 8012-8021Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 10Lai H.-L. Lin T.-H. Kao Y.-Y. Lin W.-J. Hwang M.-J. Chern Y. Mol. Pharmacol. 1999; 56: 644-650Crossref PubMed Scopus (39) Google Scholar). The twelve transmembrane segments are responsible for linking together the two catalytic domains (C1a and C2) to achieve a proper functional conformation and are important for membrane targeting of ACs (4Yan S.-Z. Hahn D. Huang Z.-H. Tang W.-J. J. Biol. Chem. 1996; 271: 10941-10945Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 11Gu C. Sorkin A. Cooper D.M.F. Curr. Biol. 2001; 11: 185-190Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). We have previously reported that prolonged activation of the A2A adenosine receptor (A2A-R) in PC12 cells significantly inhibits the activity of type VI adenylyl cyclase (ACVI), which in turn causes a lower response to subsequent stimulation by A2A-R (12Chern Y. Lai H.-L. Fong J.C. Liang Y. Mol. Pharmacol. 1993; 44: 950-958PubMed Google Scholar, 13Chern Y. Chiou J.-Y. Lai H.-L. Tsai M.-H. Mol. Pharmacol. 1995; 48: 1-8PubMed Google Scholar). In addition, stimulation by A2A-R activates the calcium-independent protein kinase C (PKC) that phosphorylates and inhibits ACVI. Suppression of ACVI by protein phosphorylation thus produces a lower response of ACVI to subsequent stimulation by A2A-R in PC12 cells (14Lai H.L. T.-H. Lin Chern Y. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). that the and of ACVI are significantly lower of the PKC the of the the of forskolin or Gαs H.-L. Lin T.-H. Kao Y.-Y. Lin W.-J. Hwang M.-J. Chern Y. Mol. Pharmacol. 1999; 56: 644-650Crossref PubMed Scopus (39) Google Scholar). the state of ACVI the of ACVI to by PKC Lai H.-L. Lin Chern Y. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google that of ACVI important for of the of ACVI J. Chen Iyengar R. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google the that are potential PKC phosphorylation sites that the three large cytoplasmic domains (N, C1a/b, and see Fig. 1A). We reported that PKC phosphorylates the highly variable N terminus domain of ACVI at The N terminus domain to at least the inhibition and phosphorylation of ACVI by PKC H.-L. Lin T.-H. Kao Y.-Y. Lin W.-J. Hwang M.-J. Chern Y. Mol. Pharmacol. 1999; 56: 644-650Crossref PubMed Scopus (39) Google Scholar). the ACVI mutant or Ser10 by PKC H.-L. Lin T.-H. Kao Y.-Y. Lin W.-J. Hwang M.-J. Chern Y. Mol. Pharmacol. 1999; 56: 644-650Crossref PubMed Scopus (39) Google that PKC also phosphorylates residues located in the C1 or C2 domain of ACVI. In the we have to the the of PKC on ACVI. suggests that PKC phosphorylates at least four residues (Ser10, Ser568, Ser674, and Thr931) located in the three large cytosolic domains (N, and C2) of ACVI and subsequently an on the catalytic activity of ACVI. Moreover, phosphorylation of ACVI at sites located in different cytosolic domains required to achieve the of that the three large cytosolic domains might other to an active which to by PKC. The of ACVI was by Iyengar of of J. Chen Iyengar R. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). of wild-type and mutant ACVI were in a recombinant the were cells the the ACVI AC activity was as previously Y. Chiou J.-Y. Lai H.-L. Tsai M.-H. Mol. Pharmacol. 1995; 48: 1-8PubMed Google Scholar). cells were in and and a at a of for a of The was at for to the membrane The AC activity was at for in a and of membrane were by of of The was isolated by and as previously Y. Chiou J.-Y. Lai H.-L. Tsai M.-H. Mol. Pharmacol. 1995; 48: 1-8PubMed Google Scholar). The ACVI activity was as the between cyclase in membrane cells the ACVI and the cyclase in cells of the The activity was for to to of membrane PKC phosphorylation sites of ACVI were the of Ser568, Ser674, and Thr931 of ACVI was the the were by were a and in cells as The of Gαs protein was a W.-J. Tang of The Gαs protein was in the as S.-Z. Huang Z.-H. R.K. Tang W.-J. Pharmacol. PubMed Scopus Google Scholar). AC the of Gαs protein was by in a and for at the and domains of ACVI were the was in a of the of a and of of for ACVI J. Chen Iyengar R. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google were as for the C1a domain and and for the domain and The for for the domain were and The for The were to the C terminus of a by and for in the of the recombinant C1a and domains were in the C1a or was for in of these domain were in a recombinant the We protein by a on the Biochem. PubMed Scopus Google the For membrane were and for to the and on to the of PubMed Scopus Google Scholar). the was to a in an (14Lai H.L. T.-H. Lin Chern Y. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google at three of in the were at a for at The were three and the were a The was the recombinant C2 domain of ACVI (14Lai H.L. T.-H. Lin Chern Y. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). the C1a and C2 domains of ACVI are highly the C1a and C2 domains of ACVI. the protein phosphorylation ACVI variants in cells were by In of membrane cells the were in of and at for was by of The was at for were protein A and three Phosphorylation by PKC was in a of of a of phenylmethylsulfonyl fluoride and The phosphorylation was by of PKC for at and was by the of were for analyzed by and the phosphorylation of ACVI by were and were and The and of were by and of the peptides are in Phosphorylation of was in a of of a and of PKC. a at the was by for The peptides were by as A. R. A. R. A. J. J. PubMed Scopus Google Scholar). were two of by two of which were to the at peptides the potential phosphorylation sites of of are The potential PKC phosphorylation sites are in in a The of are The potential PKC phosphorylation sites are in In to phosphorylation of the highly variable N domain at H.-L. Lin T.-H. Kao Y.-Y. Lin W.-J. Hwang M.-J. Chern Y. Mol. Pharmacol. 1999; 56: 644-650Crossref PubMed Scopus (39) Google suggests that PKC might other cytosolic domains and C2, the C1 and C2 domains form the catalytic core complex J.J.G. Sunahara R.K. Gilman A.G. Sprang S.R. Science. 1997; 278: 1907-1916Crossref PubMed Scopus (680) Google we the activation state of the catalytic core complex the of ACVI to by PKC. of ACVI was by ACVI Gαs which the activity of ACVI by in Fig. ACVI in the and by PKC to a Based on the structure of J.J.G. Sunahara R.K. Gilman A.G. Sprang S.R. Science. 1997; 278: 1907-1916Crossref PubMed Scopus (680) Google we a of the catalytic core composed of the C1a and C2 domains of ACVI to the two potential PKC phosphorylation sites, are located in the catalytic core of the complex of ACVI. and are among other AC and to the forskolin and sites, these two residues are likely the core catalytic complex at or are likely to to PKC-mediated We next the other PKC phosphorylation residues located in the C1 Ser568, and Ser674) and C2 domains of ACVI by PKC. synthetic peptides have been to the likely phosphorylation sites PubMed Scopus Google Scholar, J.M. J. Biol. Chem. peptides representing the potential PKC phosphorylation sites of the C1 domain were PKC to for the likely PKC phosphorylation sites in the C1 in Fig. peptides Ser568, or Ser674, the were by PKC. In addition, the recombinant C1a domain by PKC might the C1 domain of ACVI, and the likely residues of the C1 domain by PKC are Ser568, and We next to the three likely phosphorylation sites Ser568, or Ser674) of the C1 domain and the ACVI mutants in cells a in Fig. ACVI mutant a single mutation exhibited as that of the wild-type ACVI. In addition, mutation of Ser568 or Ser674 to a in the phosphorylation of ACVI by PKC residues of ACVI might by to that PKC was to a of phosphorylation in and In contrast, mutation of the PKC-mediated phosphorylation In of the we a increase in PKC-mediated phosphorylation of that of the wild-type Fig. the in the PKC-mediated phosphorylation between the and the mutant was at least two residues Ser674) located in the C1 domain of ACVI might by PKC. the functional of Ser568 and Ser674 in PKC-mediated inhibition of ACVI, we next the of PKC on the of and in mutation of Ser568 or Ser674 PKC-mediated inhibition relative of the and were inhibition of ACVI mutants the phosphorylation of and Ser568 and Ser674, might for PKC-mediated inhibition of ACVI It to that of these two residues PKC-mediated inhibition, that residues to the phosphorylation and of ACVI by to PKC-mediated inhibition of ACVI activity by phosphorylation of the and C2 domains of of the mutated of ACVI of the relative activity of between the and mutants were the of the relative activity of between the and mutants were the For PKC-mediated ACVI variants were or PKC for at in the of the of were analyzed by and by for the phosphorylation of these ACVI were to and were as in Fig. Phosphorylation and protein of ACVI variants were relatively was by between the ACVI and the wild-type ACVI were the of the relative activity of between the and mutants were the For PKC-mediated ACVI variants were or PKC for at in the of the of were analyzed by and by for the phosphorylation of these ACVI were to and were as in Fig. Phosphorylation and protein of ACVI variants were relatively was by between the ACVI and the wild-type ACVI were the For PKC-mediated ACVI variants were or PKC for at in the of the of were analyzed by and by for the phosphorylation of these ACVI were to and were as in Fig. Phosphorylation and protein of ACVI variants were relatively was by between the ACVI and the wild-type ACVI were the For PKC-mediated ACVI variants were or PKC for at in the of the of were analyzed by and by for the phosphorylation of these ACVI were to and were as in Fig. Phosphorylation and protein of ACVI variants were relatively was by between the ACVI and the wild-type ACVI were the For PKC-mediated ACVI variants were or PKC for at in the of the of were analyzed by and by for the phosphorylation of these ACVI were to and were as in Fig. Phosphorylation and protein of ACVI variants were relatively was by between the ACVI and the wild-type ACVI were the For PKC-mediated ACVI variants were or PKC for at in the of the of were analyzed by and by for the phosphorylation of these ACVI were to and were as in Fig. Phosphorylation and protein of ACVI variants were relatively was by between the ACVI and the wild-type ACVI were the For PKC-mediated ACVI variants were or PKC for at in the of the of were analyzed by and by for the phosphorylation of these ACVI were to and were as in Fig. Phosphorylation and protein of ACVI variants were relatively was by between the ACVI and the wild-type ACVI were the membrane cells the ACVI were PKC for at and for ACVI AC activity by were of forskolin to in the of and in of ACVI variants was by the of the ACVI as a of the ACVI and was to PKC a on the ACVI the of three to nine between the and of ACVI were the of of the relative activity of between the and mutants were the For PKC-mediated ACVI variants were or PKC for at in the of the of were analyzed by (14Lai H.L. T.-H. Lin Chern Y. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google and by for the phosphorylation of these ACVI were to and were as in Fig. Phosphorylation and protein of ACVI variants were relatively was by between the ACVI and the wild-type ACVI were the in a membrane cells the ACVI were PKC for at and for ACVI AC activity by were of forskolin to in the of and in of ACVI variants was by the of the ACVI as a of the ACVI and was to PKC a on the ACVI the of three to nine between the and of ACVI were the of Scholar). We have previously reported that mutation of Ser10 located in the N terminus domain of ACVI inhibition of ACVI H.-L. Lin T.-H. Kao Y.-Y. Lin W.-J. Hwang M.-J. Chern Y. Mol. Pharmacol. 1999; 56: 644-650Crossref PubMed Scopus (39) Google Scholar). We next mutated two of the three residues (Ser10, Ser568, and Ser674) located in the N and C1 domains to their to PKC-mediated phosphorylation and inhibition. in Fig. phosphorylation of the ACVI mutants of two residues were significantly of these ACVI mutants by PKC Simultaneous phosphorylation of at least two of these three residues of the C1 and N domains to important for PKC-mediated inhibition of ACVI as activity of the ACVI mutant that of three residues was to PKC-mediated inhibition at by PKC likely to located in the C2 thus might also to PKC-mediated phosphorylation and inhibition of ACVI. that of these residues contributes to a of the phosphorylation of ACVI, and are among phosphorylation of single ACVI mutants to of ACVI variants the are of phosphorylation to that PKC-mediated phosphorylation of mutants are lower that of the The C2 domain of ACVI two potential PKC phosphorylation sites and Fig. 1A). located the catalytic core of ACVI and likely to by PKC. this two recombinant C2 domain Fig. of ACVI were and to PKC phosphorylates these two in Fig. PKC the recombinant which Thr931 and the recombinant domain A We Thr931 to the of Thr931 in inhibition of ACVI. phosphorylation of the ACVI mutant were of the wild-type ACVI Moreover, mutation of Thr931 in a functional ACVI mutant that exhibited no PKC-mediated inhibition. Thr931 thus to play a role in PKC-mediated inhibition of ACVI. for and other potential PKC phosphorylation sites are located of the catalytic core of ACVI as by the mutation of the potential PKC phosphorylation sites in the the or protein of ACVI The in the AC activity by forskolin was likely to have been by the and of cells for as previously in cells Sprang S.R. Gilman A.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the of AC of the ACVI were in the or of PKC in the the of The in the different of of the of PKC. In the we that PKC suppresses the activity of ACVI in the and Phosphorylation and that at least four (Ser10, Ser568, Ser674, and Thr931) located in the regulatory N domain and two catalytic domains (C1 and C2) of ACVI might to inhibition and a on several of 10Lai H.-L. Lin T.-H. Kao Y.-Y. Lin W.-J. Hwang M.-J. Chern Y. Mol. Pharmacol. 1999; 56: 644-650Crossref PubMed Scopus (39) Google and this synthetic peptides the potential phosphorylation sites of Ser568, and Ser674 are substrates of PKC. the recombinant N domain C1a domain and domain Thr931) were by PKC. and single and of these four residues the of ACVI to and by PKC. The of the was to residues of ACVI by PKC to the of residues other the four the PKC-mediated inhibition of ACVI to phosphorylation of the three large cytosolic domains and the of domains the catalytic core in the activity of ACVI. phosphorylation of cytosolic domains required to achieve inhibition of ACVI by a fine-tuning by among the three large cytosolic domains play a role in the of ACVI. Phosphorylation one of the important regulatory of the of for ACVI, residues of ACs by reported are located in one cytosolic domain C1b or C2 Y. Li A. Harry J. Smit M.J. Bai X. Magnusson R. Pieroni J.P. Weng G. Iyengar R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14100-14104Crossref PubMed Scopus (123) Google Scholar, J. S. Mol. Cell. Biol. 1996; PubMed Scopus Google Scholar, J. G. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar, C. A. A. Biochem. Biophys. Res. Commun. 1997; PubMed Scopus Google The one or more the of different ACs by are For phosphorylation of the C1b domain of at and by kinase activation of basal catalytic activity J. S. Mol. Cell. Biol. 1996; PubMed Scopus Google phosphorylation of residues of ACVI by PKC catalytic activity 10Lai H.-L. Lin T.-H. Kao Y.-Y. Lin W.-J. Hwang M.-J. Chern Y. Mol. Pharmacol. 1999; 56: 644-650Crossref PubMed Scopus (39) Google and this of ACVI by phosphorylation this AC by at least three different and a kinase Y. Li A. Harry J. Smit M.J. Bai X. Magnusson R. Pieroni J.P. Weng G. Iyengar R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14100-14104Crossref PubMed Scopus (123) Google Scholar, 10Lai H.-L. Lin T.-H. Kao Y.-Y. Lin W.-J. Hwang M.-J. Chern Y. Mol. Pharmacol. 1999; 56: 644-650Crossref PubMed Scopus (39) Google Scholar, 2001; PubMed Scopus Google Scholar). We that PKC phosphorylates the C1b domain of ACVI at Ser674, which the by Y. Li A. Harry J. Smit M.J. Bai X. Magnusson R. Pieroni J.P. Weng G. Iyengar R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14100-14104Crossref PubMed Scopus (123) Google Scholar). Phosphorylation of ACVI by the affinity stimulation by Gαs protein Y. Li A. Harry J. Smit M.J. Bai X. Magnusson R. Pieroni J.P. Weng G. Iyengar R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14100-14104Crossref PubMed Scopus (123) Google phosphorylation of ACVI by PKC causes a in catalytic activity changing forskolin and Gαs H.-L. Lin T.-H. Kao Y.-Y. Lin W.-J. Hwang M.-J. Chern Y. Mol. Pharmacol. 1999; 56: 644-650Crossref PubMed Scopus (39) Google in the of and phosphorylation on of ACVI might to PKC at least three more residues in to Ser674, which the by ACVI in and Y. of ACVI by different might an important to render of stimulation by The of phosphorylation of the highly variable N terminus domain of ACVI at Ser10 has been H.-L. Lin T.-H. Kao Y.-Y. Lin W.-J. Hwang M.-J. Chern Y. Mol. Pharmacol. 1999; 56: 644-650Crossref PubMed Scopus (39) Google Scholar). In the C1 Ser568 and Ser674 are located in relatively on Ser568 in the C1a domain located at the C terminus of the C1a domain located in a and of the C1b which the C1a and C2 domains (6Yan S.-Z. Beeler J.A. Chen Y. Shelton R.K. Tang W.-J. J. Biol. Chem. 2001; 276: 8500-8506Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). We were to single of Ser568, or Ser674 of PKC-mediated inhibition of ACVI, mutation of two or three of these residues to ACVI mutants insensitive to PKC The and H.-L. Lin T.-H. Kao Y.-Y. Lin W.-J. Hwang M.-J. Chern Y. Mol. Pharmacol. 1999; 56: 644-650Crossref PubMed Scopus (39) Google and suggests that between the N domain and the catalytic core complex are important for the activity of ACVI. mediated by phosphorylation of a has other Y. A. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). significantly to PKC-mediated inhibition of ACVI Thr931 located in a relatively at the of the C2 domain of ACVI in an to the conformation of the catalytic core complex. In to was for other single mutants of ACVI and of Thr931 in a functional ACVI insensitive to PKC-mediated inhibition. Phosphorylation of the C2 domain at Thr931 thus for of ACVI by PKC. in the that the catalytic of ACs by the C2 S. J.M. C. 1999; PubMed Scopus Google Scholar). In addition, the of Ser674 and Thr931 the that linking transmembrane domains and cytosolic are important for the of ACs J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In the of four other AC ACVI, and also a potential PKC phosphorylation that and by PKC J. G. S. Y. Y. J. Biol. Chem. Full Text PDF PubMed Google Scholar). the of by PKC It of to and by PKC. the catalytic core of and ACVI are highly the that PKC has an on on ACVI are potential PKC phosphorylation sites in the three cytosolic domains of least two different PKC at and catalytic activity J. G. S. Y. Y. J. Biol. Chem. Full Text PDF PubMed Google Scholar). these potential PKC phosphorylation sites, are in the as of ACVI Ser568, and least two PKC phosphorylation sites and Ser674) of ACVI are in The of Ser674 by PKC and in ACVI 8Chen Y. Li A. Harry J. Smit M.J. Bai X. Magnusson R. Pieroni J.P. Weng G. Iyengar R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14100-14104Crossref PubMed Scopus (123) Google and this and as the potential phosphorylation in which by phosphorylation G. J. Y. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). by phosphorylation at Ser674 of ACVI and of might the catalytic core The of PKC-mediated phosphorylation at the of might to the of a of PKC on the catalytic the N terminus domains of and ACVI For ACVI, we have reported that PKC phosphorylates N domain at a PKC in and that the N domain might catalytic core to an active conformation to PKC-mediated phosphorylation 10Lai H.-L. Lin T.-H. Kao Y.-Y. Lin W.-J. Hwang M.-J. Chern Y. Mol. Pharmacol. 1999; 56: 644-650Crossref PubMed Scopus (39) Google and this the regulatory roles of the N domain that have been for two AC (9Gu C. Cooper D.M.F. J. Biol. Chem. 1999; 274: 8012-8021Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 10Lai H.-L. Lin T.-H. Kao Y.-Y. Lin W.-J. Hwang M.-J. Chern Y. Mol. Pharmacol. 1999; 56: 644-650Crossref PubMed Scopus (39) Google to that the N domain of which six potential PKC phosphorylation sites, might by PKC and that a role in the on the catalytic activity of on the N terminus H.-L. Lin T.-H. Kao Y.-Y. Lin W.-J. Hwang M.-J. Chern Y. Mol. Pharmacol. 1999; 56: 644-650Crossref PubMed Scopus (39) Google we have demonstrated that PKC phosphorylates at least four residues in the three large cytosolic domains, and phosphorylation of these contributes to PKC-mediated inhibition. The of the to that the three cytosolic domains of ACVI might form a regulatory complex that phosphorylation at sites, the relative of the C1 and C2 domains to the catalytic Based on the by the of the catalytic core and the likely of Ser568 and Ser674, also to that PKC phosphorylates the C1 domain at a to the Gαs and by the Sprang S.R. Gilman A.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In this of to that of ACVI a to different on the inhibition of the mediated by a receptor or PKC Lai H.-L. Lin Chern Y. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). We for mutant and Lin for in the We are also to and for the and Fig.