Phosphorylation sites within alpha4 subunits of alpha4beta2 neuronal nicotinic receptors: a comparison of substrate specificities for cAMP-dependent protein kinase (PKA) and protein kinase C (PKC).

The present study determined whether putative phosphorylation sites within the M3/M4 cytoplasmic domain of the human alpha4 subunit of alpha4beta2 neuronal nicotinic receptors are substrates for cAMP-dependent protein kinase (PKA) or protein kinase C (PKC). Five peptides corresponding to predicted phosphorylation sequences were synthesized, and phosphorylation was compared with standard peptide substrates for each kinase, that is, Kemptide for PKA and glycogen synthase (GS) 1-8 for PKC. VRCRSRSI had the highest affinity for PKA, with a Km of 44.5 microM; Kemptide had a Km of 7.7 microM. LMKRPSVVK and KARSLSVQH were also phosphorylated by PKA, but had lower affinities of 593 microM and 2896 microM, respectively. LMKRPSVVK had the highest affinity for PKC with a Km of 182 microM; GS 1-8 had a Km of 2.1 microM. VRCRSRSI had a comparative affinity for PKC with a Km of 327 microM. PCKCTCKK was not phosphorylated by PKA, but was a substrate for PKC with a Km of 1392 microM, whereas PGPSCKSP was not phosphorylated by either kinase. Based on these findings, results suggest that Ser-362 and Ser-486 on the human alpha4 subunit may be phosphorylated by either PKA or PKC, Ser-467 is a putative PKA site, and Thr-532 represents a likely PKC substrate; Ser-421 does not appear to be phosphorylated by either kinase.