Journal of Neurochemistry, 2001, 76, 737±744
Protein kinase C activation induces tyrosine phosphorylation of the NR2A and NR2B subunits of the NMDA receptor David R. Grosshans* and Michael D. Browning*,² *Department of Pharmacology, ²Program in Neuroscience, University of Colorado Health Sciences Center, Denver, Colorado, USA
Abstract The N-methyl-D-aspartate receptor (NMDAR) is an ionotropic glutamate receptor, which plays crucial roles in synaptic plasticity and development. We have recently shown that potentiation of NMDA receptor function by protein kinase C (PKC) appears to be mediated via activation of non-receptor tyrosine kinases. The aim of this study was to test whether this effect could be mediated by direct tyrosine phosphorylation of the NR2A or NR2B subunits of the receptor. Following treatment of rat hippocampal CA1 mini-slices with 500 nM phorbol 12-myristate 13-acetate (PMA) for 15 min, samples were homogenized, immunoprecipitated with anti-NR2A or NR2B antibodies and the resulting pellets subjected to Western blotting with antiphosphotyrosine antibody. An increase in tyrosine phosphorylation of both NR2A
(76 ^ 11% above control) and NR2B (41 ^ 11%) was observed. This increase was blocked by pretreatment with the selective PKC inhibitor chelerythrine, with the tyrosine kinase inhibitor Lavendustin A or with the Src family tyrosine kinase inhibitor PP2. PMA treatment also produced an increase in the phosphorylation of serine 890 on the NR1 subunit, a known PKC site, at 5 min with phosphorylation returning to near basal levels by 10 min while tyrosine phosphorylation of NR2A and NR2B was sustained for up to 15 min. These results suggest that the modulation of NMDA receptor function seen with PKC activation may be the result of tyrosine phosphorylation of NR2A and/or NR2B. Keywords: hippocampus, NMDA, NR2A, NR2B, protein kinase C, tyrosine phosphorylation. J. Neurochem. (2001) 76, 737±744.
Glutamate is the major excitatory neurotransmitter in the mammalian brain where it acts on both metabotropic glutamate receptors and ionotropic glutamate receptors. The N-methyl-d-aspartate receptor (NMDAR) is a member of the group of ionotropic glutamate receptors. This receptor is involved in a wide variety of processes in the central nervous system (CNS) including synaptogenesis and synaptic plasticity. Additionally, the NMDAR has been implicated in excitotoxicity, schizophrenia, various neurodegenerative disorders and in aging (Lipton and Rosenberg 1994; Lee et al. 1999; Mohn et al. 1999; Eckles et al. 2000). Thus, a greater understanding of the modulation of this receptor is likely to be important to the understanding of the physiology and pathophysiology of these processes. The NMDA receptor is a heteromeric protein composed of two classes of subunits, NR1 and NR2. The gene encoding for NR1 undergoes extensive splicing generating up to eight splice variants while four separate genes encode NR2 subunits, NR2A to NR2D. While NR2 subunits cannot form functional channels when expressed alone, they can alter NMDAR channel properties when complexed with
NR1 subunits (Kutsuwada et al. 1992; Meguro et al. 1992; Monyer et al. 1992; Ishii et al. 1993; Sucher et al. 1996). NMDA receptor function is modulated by both tyrosine and serine protein kinases. Kohr and Seeburg (1996) expressed recombinant receptors composed of NR1/NR2A or NR1/NR2B in human embryonic kidney (HEK) 293 cells and demonstrated that intracellular dialysis of the nonreceptor tyrosine kinases Src or Fyn resulted in the potentiation of NR1/NR2A-containing receptors but not of NR1/NR2B-containing receptors. Additionally, it has been Received June 7, 2000; revised manuscript received September 1, 2000; accepted September 4, 2000. Address correspondence and reprint requests to M. D. Browning, Program in Neuroscience, University of Colorado Health Sciences Center, 4200 E. Ninth Ave. Box C236, Denver, CO 80262, USA. E-mail:
[email protected] Abbreviations used: aCSF, arti®cial cerebrospinal ¯uid; BSA, bovine serum albumin; NMDAR, N-methyl-d-aspartate receptor; PKC, protein kinase C; SDS, sodium dodecyl sulphate; DMSO, dimethyl sulfoxide; PMA, phorbol 12-myristate 13-acetate; PMM, phorbol 12-monomyristate.
q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 76, 737±744
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previously reported that NR2A, NR2B and NR2D are phosphorylated on tyrosine residues in the mammalian brain (Moon et al. 1994; Lau and Huganir 1995; Dunah et al. 1998). Activation of PKC has also been shown to enhance NMDA currents in a variety of experimental systems and it is known that the receptor itself is subject to phosphorylation by PKC (Ben-Ari et al. 1992; Chen and Huang 1992; Kelso et al. 1992; Tingley et al. 1993; Leonard and Hell 1997; Tingley et al. 1997; Xiong et al. 1998). However, in collaboration with Dr John MacDonald and colleagues, we have recently shown that the effect of PKC activators on the NMDAR was blocked by inhibitors of tyrosine kinases, including a peptide inhibitor of Src and an antibody selective for Src itself. Moreover, in hippocampal neurones from mice lacking c-Src, PKC-dependent up-regulation was absent (Lu et al. 1999). Thus, PKC-dependent potentiation of NMDA receptor function appears to be mediated via activation of the non-receptor tyrosine kinase (Src) signalling cascade. The goal of the present studies was to determine whether this potentiation was likely due to direct tyrosine phosphorylation of the NMDAR itself. Here we utilize rat CA1 hippocampal mini-slices along with immunoprecipitation and Western blotting to demonstrate an increase in tyrosine phosphorylation of the NR2A and NR2B subunits of the NMDA receptor in response to treatment with phorbol esters. Additionally, we report that PMA leads to a rapid increase of phosphorylation of serine 890 of the NR1 subunit with levels returning to near basal values by 10 min.
Experimental procedures Materials PMA, PMM and chelerythrine were purchased from Sigma (St Louis, MO, USA); lavendustin A, PP2, okadaic acid and Pansorbin Cells were obtained from Calbiochem (San Diego, CA, USA); antiphosphotyrosine clone 4G10 was from Upstate Biotechnology (Lake Placid, NY, USA) and anti-NR1 was from Pharmingen (Franklin Lakes, NJ, USA). Hippocampal slice preparation and drug treatments Hippocampal CA1 mini-slices were prepared as previously described (Nayak et al. 1998). Sprague±Dawley rats (2±4 months old) were used for all experiments. After sacri®cing the animal, the brain was removed and placed in ice-cold oxygenated arti®cial cerebrospinal ¯uid (aCSF: 124 mm NaCl, 4 mm KCl, 1 mm MgSO4, 2.5 mm CaCl2, 10 mm Dextrose, 1 mm KH2PO4, 25.7 mm NaHCO3). Both hippocampi were then dissected out and 400-mm slices prepared on a McIlwain tissue chopper. Following preparation of CA1 mini-slices (Par®tt et al. 1992), slices were incubated at interface in aCSF at 328C and oxygenated with 95%O2/5%CO2. Slices were allowed to recover under these conditions for 90 min with fresh aCSF being added every 20 min. aCSF was then drained from the incubation chamber and aCSF containing the appropriate concentration of drug or vehicle alone
(dimethyl sulfoxide (DMSO) ®nal of 0.05±0.2%) was added. In our previous studies of the potentiation of NMDAR currents in acutely dissociated and in cultured hippocampal neurones PMA was applied at 100 nm (Lu et al. 1999). In the present studies we utilized 500 nm PMA in order to ensure adequate penetration of the hippocampal slice. Slices were removed after 15 minutes, sonicated in 1% sodium dodecyl sulphate (SDS) and frozen until use. Protein concentrations were determined using the BCA protein assay kit from Pierce (Rockford, IL, USA) with bovine serum albumin (BSA) as a standard. Immunoprecipitation Prior to immunoprecipitation, heat-killed Staphylococcus aureus cells (Sacc cells) bearing surface expressed protein A were prepared. Six and a half milliliters of manufacturer's 10% cell suspension was prewashed by pelleting at 2800 g for 10 min followed by resuspension in 3% SDS, 10% BME, 50 mm Tris±HCl pH 7.5, 150 mm NaCl. The suspension was boiled for 5 min, cooled, and pelleted again at 2800 g for 10 min. The process was then repeated. The pellet was then resuspended in 6 mL of TEN buffer (50 mm Tris±HCl pH 7.5, 2 mm EDTA, 150 mm NaCl) and pelleted as described above. This process was repeated three times with the ®nal pellet being resuspended in a
