Functional effect of phosphorylation of the photoreceptor phosphodiesterase inhibitory subunit by protein kinase C.
In rod outer segments the light activation of cGMP phosphodiesterase (PDE alpha beta gamma 2) is accomplished by removal of the gamma inhibitory subunit (PDE gamma) from the PDE alpha beta catalytic subunits. A light activation of the inositol signaling pathway also occurs, but there is little information linking these two signal transduction pathways. Here we report that protein kinase C (PKC) purified from bovine rod outer segment phosphorylates the bovine PDE gamma with incorporation of 0.9 +/- 0.1 mol of phosphate/mol of PDE gamma. Phosphorylation of PDE gamma increases its ability to inhibit PDE alpha beta catalytic activity (trypsin-activated PDE, tPDE) with an IC50 for phosphorylated PDE gamma of 26 +/- 4 pM and an IC50 of 60 +/- 5 pM for unphosphorylated PDE gamma. Inhibition of tPDE by PDE gamma is characterized by two values of Kd, Kd1 = 34 pM and Kd2 = 760 pM. Phosphorylation of PDE gamma by PKC eliminates the functional heterogeneity of the PDE gamma population resulting in a single value of Kd = 23 pM. Free PDE gamma (without PDE alpha beta catalytic subunits) is a better substrate for PKC than PDE gamma in a complex with PDE alpha beta. Phosphorylation of free PDE gamma by PKC is characterized by a value of Vmax = 1,550 +/- 148 units/mg (Km = 21.0 +/- 1.9 microM). In contrast, phosphorylation of PDE gamma in PDE alpha beta gamma 2 complex has two values of Vmax, Vmax1 = 0.3 +/- 0.1 units/mg of PDE gamma (Km1 = 0.4 +/- 0.2 microM) and Vmax2 = 0.7 +/- 0.2 units/mg of PDE gamma (Km2 = 4.6 +/- 0.9 microM). ROS PKC phosphorylates Thr35 in PDE gamma. We have previously reported (Morrison, D. F., Rider, M. A., and Takemoto, D. J. (1987) FEBS Lett. 222, 266-270; Lipkin, V. M., Udovichenko, I. P., Bodarenko, V. A., Yurovskaya, A. A., Telnykh, E. V., and Skiba, N. P. (1990) Biomed. Sci. (Lond.) 1, 305-308) that the central fragment of PDE gamma (24-45) is responsible for binding to PDE catalytic subunits. The new data suggests that this region of PDE gamma also includes the site for phosphorylation by PKC and that phosphorylation increases the ability of PDE gamma to inhibit PDE catalytic activity. This altered regulation of visual transduction may play a role in desensitization or light adaptation.
Udovichenko
I P
IP
Department of Biochemistry, Kansas State University, Manhattan 66506.
Cunnick
J
J
Gonzalez
K
K
Takemoto
D J
DJ
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D004789:Enzyme Activation; D007700:Kinetics; D046911:Macromolecular Substances; D008954:Models, Biological; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis / Q000378:metabolism; D010449:Peptide Mapping; D010766:Phosphorylation; D011493:Protein Kinase C / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012374:Rod Cell Outer Segment / Q000201:enzymology*; D013379:Substrate Specificity; D014357:Trypsin / Q000494:pharmacology
D046911:Macromolecular Substances; D009842:Oligopeptides; D011994:Recombinant Proteins; D011493:Protein Kinase C; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D014357:Trypsin