Protein kinase A phosphorylates retinal phosducin on serine 73 in situ
Photoreceptors of vertebrate retinas contain a 33,000-dalton phosphoprotein, phosducin, which complexes with the beta, gamma subunits of the photoreceptor G-protein (guanine nucleotide-binding protein), transducin. In situ, the retinal content of phosphorylated phosducin is modulated by light in con...
Gespeichert in:
Veröffentlicht in: | The Journal of biological chemistry 1990-09, Vol.265 (26), p.15860-15866 |
---|---|
Hauptverfasser: | , , |
Format: | Artikel |
Sprache: | eng |
Schlagworte: | |
Online-Zugang: | Volltext |
Tags: |
Tag hinzufügen
Keine Tags, Fügen Sie den ersten Tag hinzu!
|
Zusammenfassung: | Photoreceptors of vertebrate retinas contain a 33,000-dalton phosphoprotein, phosducin, which complexes with the beta, gamma
subunits of the photoreceptor G-protein (guanine nucleotide-binding protein), transducin. In situ, the retinal content of
phosphorylated phosducin is modulated by light in conjunction with light-triggered changes in intracellular cyclic nucleotide
concentration. In vitro, phosducin is phosphorylated by either exogenous or endogenous protein kinase A. 32P-Labeled rat retina
phosducin was isolated by immunoprecipitation either after phosphorylation by protein kinase A in the presence of [gamma-32P]ATP
or after incubation of retinas in darkness with 32Pi. In either case, phosphoamino acid analysis showed that greater than
98% of 32P was linked to serine, with less than 2% to threonine. Two-dimensional peptide mapping showed that [32P]phosphoserine
was associated with the same characteristic set of tryptic peptides. Furthermore, Cleveland peptide analysis using four different
proteases showed that either sample exhibited identical patterns of phosphopeptides which were characteristic of the protease
used. Identical phosphopeptide maps were also obtained from 32P-labeled bovine retina phosducin, indicating that the serine
phosphorylation site for protein kinase A is conserved between rat and bovine. Edman degradation of phosphopeptides derived
from 32P-labeled bovine phosducin showed that radioactive phosphate was incorporated into serine residue 73 which is located
within a consensus phosphorylation sequence for protein kinase A (-R-K-M-S73(P)-). These observations are uniformly in agreement
with protein kinase A being the endogenous kinase that phosphorylates phosducin in vivo. |
---|---|
ISSN: | 0021-9258 1083-351X |
DOI: | 10.1016/S0021-9258(18)55478-8 |