Phosphorylation of the 61-kDa calmodulin-stimulated cyclic nucleotide phosphodiesterase at serine 120 reduces its affinity for calmodulin.
Phosphorylation of the 61-kDa isoform of bovine calmodulin (CaM)-stimulated cyclic nucleotide phosphodiesterase (CaM-PDE) by the catalytic subunit of cyclic AMP-dependent protein kinase A (PKA) results in a decrease in the affinity of the enzyme for calmodulin [Sharma, R. K., & Wang, J. H. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 2603-2607]. In the present study, purified 61-kDa CaM-PDE was phosphorylated in the presence of [gamma-32P]ATP and cleaved with a Lys-C endoproteinase. The resultant phosphopeptides were resolved by reverse-phase HPLC and analyzed by electrospray mass spectrometry and Edman sequencing. Serine residues 120 and 138 were identified as the principal sites of phosphorylation. A cDNA encoding the 61-kDa CaM-PDE [Sonnenburg, W. K., Seger, D., & Beavo, J. A. (1993) J. Biol. Chem. 268, 645-652] was used to generate point mutants in which either or both of these serines were replaced with alanine. The mutants were expressed in COS-7 cells, purified, and phosphorylated. Phosphorylation of the mutant Ser 138-->Ala resulted in a decrease in affinity for CaM that was comparable to that seen with the wild-type enzyme. In contrast, phosphorylation of the mutant Ser 120-->Ala had virtually no effect on CaM affinity. We conclude that phosphorylation of serine 120 by PKA is responsible for the reduction in affinity of the 61-kDa CaM-PDE for CaM.
Florio
V A
VA
Department of Pharmacology, University of Washington, Seattle 98195.
Sonnenburg
W K
WK
Johnson
R
R
Kwak
K S
KS
Jensen
G S
GS
Walsh
K A
KA
Beavo
J A
JA
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002147:Calmodulin / Q000378:metabolism*; D002417:Cattle; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009838:Oligodeoxyribonucleotides; D010766:Phosphorylation; D012694:Serine / Q000378:metabolism*
D002147:Calmodulin; D009838:Oligodeoxyribonucleotides; D012694:Serine; D015105:3',5'-Cyclic-AMP Phosphodiesterases