Journal: Molecular neurobiology
Article Title: Synaptic ERK2 Phosphorylates and Regulates Metabotropic Glutamate Receptor 1 In Vitro and in Neurons
Figure Lengend Snippet: Phosphorylation of mGluR1a-CT2 by ERK2. a Amino acid sequence analysis of mGluR1a-CT2(P1001-L1199). Eight ERK2 phosphorylation motifs (T/SP) are highlighted in red . Potential phosphorylation sites include T1064 (1), S1098 (2), T1143 (3), S1147 (4), T1151 (5), S1154 (6), S1169 (7), and T1178 (8). b A representative phosphoamino acid analysis showing GST-mGluR1a-CT2 phosphorylation at serine ( pSer ), but not threonine ( pThr ) and tyrosine ( pTyr ), residues. c, d Phosphorylation of mGluR1a-CT2 at pS/TP ( c ) and pTP ( d ) motifs by active ERK2. e Five mutants (M1–5) derived from mGluR1a-CT2(P1001-L1199). Site-directed mutants include mutations of T1064/S1098 to alanines (M1), T1064/S1098/T1143/S1147 to alanines (M2), T1064/S1098/T1143/S1147/T1151/S1154 to alanines (M3), four serines (S1098/S1147/S1154/S1169) to alanines (M4), and four threonines (T1064/T1143/T1151/T1178) to alanines (M5). e Phosphorylation of mGluR1a-CT2 mutants and WT at pS/TP by active ERK2. Note that no signal was detected in the M4 mutant. g Phosphorylation of GST-mGluR1a-CT2 and Elk-1 at PXpSP by active ERK2. Phosphorylation of CT2 and Elk-1 was detected by immunoblot ( IB ) with a phosphomotif antibody against pS/TP ( c, f ), pTP ( d ), or PXpSP ( g )
Article Snippet: To dephosphorylate GST-fusion proteins, we incubated GST-fusion proteins with Histagged active ERK2 (Millipore, 25 ng) in 25 µl reaction buffer containing 10 mM HEPES pH 7.4, 10 mM MgCl2 , 1 mM Na3 VO4 , 1 mM DTT, 50 µM ATP, and 2.5 µCi/tube [γ-32 P]ATP (~3000 Ci/mmol) for 30 min at 30 °C.
Techniques: Sequencing, Phosphoamino Acid Analysis, Derivative Assay, Mutagenesis