Phosphorylation of G β is augmented by chronic morphine and enhances G βγ stimulation of adenylyl cyclase activity

Abstract

We previously demonstrated (Chakrabarti, et al., 2001) that in vivo phosphorylation of the G β subunit of G proteins, via protein kinase A (PKA) and protein kinase C (PKC), is dramatically increased following chronic morphine. The present study investigates the PKC isoform selectivity of G β phosphorylation and the consequences thereof on the ability of G βγ to stimulate adenylyl cyclase II (ACII). The catalytic subunit of PKC and PKA, as well as the conventional PKC isoform PKCγ, was effective in phosphorylating G β. In contrast, G β was only minimally phosphorylated by another conventional isoform, PKCα or the atypical isoform PKCζ. In the presence of activated recombinant G sα, ACII activity was dose dependently stimulated by G βγ, the magnitude of which was dependent upon its phosphorylation state. The increment in ACII activity produced by G βγ was increased ≈2-fold following in vitro phosphorylation by the catalytic subunit of either PKA or PKC. In contrast, the concomitant or sequential phosphorylation of G βγ by PKA and PKC catalytic subunits did not result in an additive enhancement of its ability to stimulate ACII and, in fact, negated the observed enhancing effect of each kinase, individually. Threonine phosphorylated G β occurs naturally in the spinal cord, the levels of which are augmented (≈60%) by chronic morphine. The natural occurrence of phosphorylated G β in spinal cord, its up-regulation following chronic morphine and the augmented ability of phosphorylated G βγ to stimulate ACII activity, in the aggregate, indicate that phosphorylation of G β could be a regulatory mechanism causally associated with altered cellular signaling.