Phosphorylation of the alpha4 subunit of human alpha4beta2 nicotinic receptors: role of cAMP-dependent protein kinase (PKA) and protein kinase C (PKC).

This study determined whether the alpha4 subunit of human alpha4beta2 neuronal nicotinic receptors is phosphorylated in situ by cyclic AMP-dependent protein kinase (PKA) or protein kinase C (PKC). To accomplish this, human cloned epithelial cells stably transfected with the human alpha4beta2 nicotinic receptor (SH-EP1-halpha4beta2) were incubated with 32P-orthophosphate to label endogenous ATP stores, and the phosphorylation of alpha4 subunits was determined in the absence or presence of PKA or PKC activation. Autoradiographs and immunoblots indicated that alpha4 subunits immunoprecipitated from a membrane preparation of SH-EP1-halpha4beta2 cells exhibited a single 32P-labeled band corresponding to the alpha4 subunit protein; no signals were associated with untransfected SH-EP1 cells. The alpha4 subunits from SH-EP1-halpha4beta2 cells incubated in the absence of the activators exhibited a basal level of phosphorylation that was decreased in the presence of the PKA inhibitor H-89 (5 microM), but unaltered in the presence of the PKC inhibitor Ro-31-8220 (0.1 microM). Activation of PKA by forskolin (10 microM), dibutyryl-cAMP (1 mM), or Sp-8-Br-cAMP (1 mM) enhanced phosphorylation nearly threefold; the inactive isomer, Rp-8-Br-cAMP (1 mM) had no effect. In addition, the forskolin effect was totally blocked by the PKA inhibitor H-89 (5 microM). Activation of PKC by the phorbol esters PDBu (200 nM) or PMA (200 nM) increased alpha4 subunit phosphorylation approximately twofold, and the PDBu effect was blocked by the selective PKC inhibitor Ro-31-8220 (0.1 microM). These findings indicate that the alpha4 subunit of human alpha4beta2 nicotinic receptors is phosphorylated in situ by PKA and PKC.