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.
Wecker
Lynn
L
Department of Pharmacology and Therapeutics, University of South Florida, College of Medicine, Tampa, Florida 33612-4799, USA. lwecker@hsc.usf.edu
Rogers
Christopher Q
CQ
D000595:Amino Acid Sequence; D001667:Binding, Competitive; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D006801:Humans; D009474:Neurons / Q000378:metabolism*; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D020033:Protein Isoforms / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism*; D011978:Receptors, Nicotinic / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity
D010455:Peptides; D020033:Protein Isoforms; D011978:Receptors, Nicotinic; C403531:nicotinic receptor alpha4beta2; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C