Protein kinase C inhibits type VI adenylyl cyclase by phosphorylating the regulatory N domain and two catalytic C1 and C2 domains.
We previously showed that phosphorylation of Ser(10) 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 vitro phosphorylation by PKC of the recombinant C1a and C2 domains, and of the synthetic peptides representing potential PKC phosphorylation sites, suggests that Ser(568) and Ser(674) of the C1 domain and Thr(931) 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 (Ser(10), Ser(568), Ser(674), and Thr(931)) were mutated to alanine. Simultaneous mutation of at least two of the three likely residues located in the N and C1 domains (Ser(10), Ser(568), and Ser(674)) was required to render ACVI variants completely insensitive to PKC treatment. In contrast, a single mutation of Thr(931) was sufficient to create a functional ACVI mutant that exhibited no detectable PKC-mediated inhibition, demonstrating the essentiality of Thr(931) 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.
Lin
Ting-Hui
TH
Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan, Republic of China.
Lai
Hsing-Lin
HL
Kao
Yu-Ya
YY
Sun
Chung-Nan
CN
Hwang
Ming-Jing
MJ
Chern
Yijuang
Y
D000067956:Adenylyl Cyclase Inhibitors; D000262:Adenylyl Cyclases / Q000737:chemistry; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D020134:Catalytic Domain; D003198:Computer Simulation; D017931:DNA Primers; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011493:Protein Kinase C / Q000378:metabolism*; D017433:Protein Structure, Secondary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D000067956:Adenylyl Cyclase Inhibitors; D017931:DNA Primers; D010446:Peptide Fragments; D011994:Recombinant Proteins; D010768:Phosphoserine; D011493:Protein Kinase C; D000262:Adenylyl Cyclases; C121100:adenylyl cyclase 6