Cyclic AMP-Dependent Phosphoprotein Components I and II Interact with βγ Subunits of Transducin in Frog Rod Outer Segments

• Published in: Biochemistry (Easton) 1996-01, Vol.35 (1), p.290-298
• Main Authors: Suh, Kyong Hoon, Hamm, Heidi E
• Format: Article
• Language: English
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi9518656

Components I and II (CI&II) in frog rod outer segments (ROS) are prominent cAMP-dependent protein kinase (PK-A) substrates. Their phosphorylation level is high in the dark, and illumination causes dephosphorylation. In order to understand their physiological role in phototransduction, biochemical characterization of CI&II phosphorylation was performed. Fractionation of phosphorylated ROS proteins showed that CI&II in the soluble fraction were highly phosphorylated by endogenous PK-A, whereas those in the membrane-associated protein fractions were not. The latter proteins could be phosphorylated by purified catalytic subunit of PK-A (PK-Acat) while the former proteins were not, suggesting that membrane-bound CI&II are normally much less phosphorylated. Treatments that dissociate the α subunit (αt) of transducin (Gt) from βγ subunits (βγt) and thus produce excess free subunits of Gt in the soluble fraction caused inhibition of CI&II phosphorylation in the soluble fraction and enhancement of CI&II phosphorylation in the peripheral membrane fractions containing less Gt. Unphosphorylated CI&II tightly associated with the washed ROS membranes could be extracted after phosphorylation by PK-Acat. Phosphorylation also caused elution of βγt from the membrane under the same conditions. Cross-linking by the maleimidobenzoyl-N-hydroxysuccinimide ester of the peripheral membrane fraction produced a distinct phosphorylated 50 kDa product with concurrent disappearance of the β subunit of transducin (βt) and phosphorylated CI&II. This phosphorylated cross-linked product was not recognized by a monoclonal anti-αt antibody but was recognized by antiserum against βt, suggesting that the 50 kDa protein is a complex of βγt and CI&II. Amino terminal sequencing of components I and II suggests that they are identical proteins with a unique sequence unrelated to other proteins in protein data bases. Phosphopeptide mapping of phosphorylated CI&II in the soluble fraction yielded two trypsinized phosphopeptides, while that in the peripheral membrane fractions showed only one phosphopeptide. These data suggest that multiple phosphorylation of CI&II alters their cellular localization. We conclude that phosphorylation of CI&II controls their localization in frog ROS and an interaction of CI&II with subunits of Gt regulates their phosphorylation.