Cyclic AMPdependent protein kinase (PKA) - Wiley Online Library

Journal of Neurochemistry, 2007, 103, 456–466

doi:10.1111/j.1471-4159.2007.04853.x

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*Department of Molecular Pharmacology and Physiology, University of South Florida College of Medicine, Tampa, Florida, USA Department of Psychiatry and Behavioral Medicine, University of South Florida College of Medicine, Tampa, Florida, USA

Abstract Studies have suggested that the expression, translocation, and function of a4b2 nicotinic receptors may be modulated by a4 subunit phosphorylation, but little direct evidence exists to support this idea. The objective of these experiments was to identify specific serine/threonine residues on a4 subunits that are phosphorylated in vivo by cAMP-dependent protein kinase and protein kinase C (PKC). To accomplish this, DNAs coding for human a4 subunits containing alanines in place of serines/threonines predicted to represent phosphorylation sites were constructed, and transiently transfected with the DNA coding for wild-type b2 subunits into SH-EP1 cells. Cells were pre-incubated with 32Pi and incubated in the absence or presence of forskolin or phorbol 12,13-dibutyrate. Immunoprecipitated a4 subunits were

subjected to immunoblot, autoradiographic and phosphoamino acid analyses, and two-dimensional phosphopeptide mapping. Results confirmed the presence of two a4 protein bands, a major band of 71/75 kDa and a minor band of 80/ 85 kDa. Phosphoamino acid analysis of the major band indicated that only serine residues were phosphorylated. Phosphopeptide maps demonstrated that Ser362 and 467 on the M3/M4 cytoplasmic domain of the a4 subunit represent major cAMP-dependent protein kinase phosphorylation sites, while Ser550 also contained within this major intracellular loop is a major site for protein kinase C phosphorylation. Keywords: neuronal nicotinic receptor, phosphorylation, protein kinase A, protein kinase C. J. Neurochem. (2007) 103, 456–466.

Neuronal nicotinic receptors represent a family of integral membrane proteins composed of a or a and b subunits that form pentameric, ligand-gated cation channels. Each subunit contains four transmembrane spanning domains with extracellular amino- and carboxy-termini, a small intracellular loop (50% of the relative phosphorylation of the subunit. The idea that the unstimulated human a4 subunit exists in a partially phosphorylated state is supported by similar find-

ings for other nicotinic receptor subunits. Both the b and d subunits of the rat muscle receptor present in myotubes are phosphorylated under basal conditions (Miles et al. 1987), and a3 neuronal nicotinic receptor subunits expressed in chicken ciliary ganglion neurons exhibit a basal level of phosphorylation (Vijayaraghavan et al. 1990). In addition to the phosphorylation of S467 under basal conditions, peptide fragments in both C2 and C3 incorporate most of the remaining radioactivity indicating that serines other than S467 are also basally phosphorylated; these residues remain to be identified. Protein kinase C has also been implicated in controlling a4b2 nicotinic receptor function. For example, phosphorylation of the rat nicotinic a4b2 receptor by PKC has been implicated in its recovery from desensitization and a mutant receptor on a PKC consensus sequence (S336A) exhibited little recovery from inactivation (Fenster et al. 1999). The present study, using phosphopeptide mapping, indicated that the corresponding residue in the human sequence, S334, is not phosphorylated in vivo by PKC in SH-EP1 cells, indicating that phosphorylation of a specific conserved residue is either species or cell type specific. Others have shown indirectly that PKC is also involved in increasing mouse a4b2 nicotinic receptor function that may occur due to surface translocation following treatment of (human embryonic kidney) 293T cells with phorbol 12-myristate 13-acetate for as short of time as 30 min (Nashmi et al. 2003). We tested other conserved residues in human, rat, and mouse predicted to contain consensus PKC phosphorylation sites (T532 and S586) for in vivo phosphorylation by PKC, but none were phosphorylated. As our maps exhibited several phosphopeptide sites that were phosphorylated by PKC, in concert with the finding that the conserved sites we tested were not phosphorylated by PKC, we began mutating non-conserved PKC sites and identified residue S550 on the human a4 subunit as a major site that can be phosphorylated by PKC in vivo. As residue S550 is not conserved in the human, rat, and mouse sequences, its phosphorylation may well be involved in modulating functional differences observed between the human and rat receptors such as the human receptor exhibiting sensitization (Gopalakrishnan et al. 1997; Buisson and Bertrand 2001), whereas the rat receptor exhibiting inactivation following sustained agonist exposure (Hsu et al. 1997; Fenster et al. 1999). Results obtained from the present in vivo study support some, but not all findings from in vitro studies using synthetic peptides and a fusion protein containing the M3/ M4 cytoplasmic domain of the rat a4 protein (Wecker et al. 2001; Guo and Wecker 2002; Wecker and Rogers 2003). Results indicating that Ser362 and 467 on the human a4 subunit are major substrates for PKA are in agreement with studies using the fusion protein demonstrating that Ser365 and 472 on the rat a4 subunit are major substrates for PKA (Guo and Wecker 2002). In contrast, although S491 within

2007 The Authors Journal Compilation 2007 International Society for Neurochemistry, J. Neurochem. (2007) 103, 456–466

Nicotinic receptor a4 subunit phosphorylation 465

the RCRSRCIQYC sequence in the fusion protein was phosphorylated upon PKA stimulation (Guo and Wecker 2002), and an 8-mer synthetic peptide was an excellent substrate for PKA in vitro (Wecker and Rogers 2003), the corresponding Ser486 in the human sequence was not a PKA substrate in vivo. The lack of the PKA-mediated phosphorylation of Ser486 is likely to reflect differences obtained from in vitro versus in vivo experiments. The experiments with the rat fusion protein performed in vitro were lacking the intracellular protein expression machinery, regulatory proteins, and b2 partner subunit, perhaps leading to an easier access of the kinase to its protein substrate. The finding that Ser486 on the human a4 subunit was not phosphorylated in vivo by PKA was somewhat surprising especially as this serine is located in the center of a putative endoplasmic reticulum-retention signal sequence (RXR) that has been shown to play a role in GABA-B receptor release from the endoplasmic reticulum (MargetaMitrovic et al. 2000) and in the surface expression of NMDA receptors (Scott et al. 2001). It is conceivable that in the presence of the b2 subunit, a conformational change in subunits induced by another signal may be required to allow access of the kinase to this residue; alternatively, its phosphorylation may occur by a different kinase. Indeed, Ser486 is located within a consensus sequence for phosphorylation by other kinases such as protein kinase G and calcium/calmodulin-dependent protein kinase. In summary, the present studies provide the first direct evidence for the PKA-mediated in vivo phosphorylation of Ser362 and 467 and the first evidence for the PKCmediated phosphorylation of Ser550 and perhaps 362 within the M3/M4 cytoplasmic domain of human a4 subunits. In addition, these studies also indicate the differential phosphorylation of immature versus mature a4 subunits by PKA and PKC (Pollock et al. 2006). Results (Fig. 2) demonstrate that PKA phosphorylates the immature a4 subunit and not the mature form in the plasma membrane, whereas PKC appears to phosphorylate both forms. Studies identifying additional sites that are phosphorylated in vivo, as well as elucidating the function of each of these sites are underway. Acknowledgements The authors thank Dr Ron Lukas (Barrow Neurological Institute, Phoenix, AZ, USA) for generously providing the SH-EP1 cells. These studies were supported by a grant from the National Institute of Drug Abuse #DA14010 (to LW).

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