Regulation of NMDA receptors by an associated phosphatase-kinase signaling complex

Science

Volumn 285, Issue 5424, 1999, Pages 93-96

  Westphal, Ryan S ^a   Tavalin, Steven J ^a   Lin, Jerry W ^b   Alto, Neal M ^a   Fraser, Iain D C ^a   Langeberg, Lorene K ^a   Sheng, Morgan ^b   Scott, John D ^a  

^a OREGON HEALTH AND SCIENCE UNIVERSITY   (United States)

^b MASSACHUSETTS GENERAL HOSPITAL   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

GREEN FLUORESCENT PROTEIN; N METHYL DEXTRO ASPARTIC ACID RECEPTOR; PHOSPHATASE; PHOSPHOTRANSFERASE; PROTEIN KINASE;

ARTICLE; BINDING AFFINITY; ENZYME ACTIVITY; ENZYME REGULATION; EXON; PRIORITY JOURNAL; RECEPTOR AFFINITY; RECEPTOR BINDING; SIGNAL TRANSDUCTION; SYNAPTIC TRANSMISSION;

ADAPTOR PROTEINS, SIGNAL TRANSDUCING; AMINO ACID SEQUENCE; ANIMALS; BINDING SITES; CARRIER PROTEINS; CELL LINE; CYCLIC AMP; CYCLIC AMP-DEPENDENT PROTEIN KINASES; CYTOSKELETAL PROTEINS; ENZYME INHIBITORS; HOLOENZYMES; HUMANS; MOLECULAR SEQUENCE DATA; OKADAIC ACID; PATCH-CLAMP TECHNIQUES; PEPTIDE FRAGMENTS; PHOSPHOPROTEIN PHOSPHATASE; PHOSPHORYLATION; RATS; RECEPTORS, N-METHYL-D-ASPARTATE; RECOMBINANT FUSION PROTEINS; SIGNAL TRANSDUCTION; THIONUCLEOTIDES; TRANSFECTION;

EID: 0033516701     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.285.5424.93     Document Type: Article

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52. Rat brain extracts were prepared by Dounce homogenization of frozen brains in phosphate-buffered saline (PBS) containing 1 mM EDTA, 1 mM EGTA, 1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride, 1 mM benzamidine, pepstatin (2 μg/ml), and leupeptin (2 μg/ml), followed by centrifugation at 40,000g for 1 hour. Supernatant (1 ml) was incubated with 20 μl of preimmune or immune serum overnight at 4°C. After addition of protein G-agarose (30 μl) for 1 hour at 4°C, the precipitated complexes were washed five times with homogenization buffer and proteins were eluted with 2X SDS sample buffer. Yotiao was detected by RII overlay. For detection of PKA C subunit activity, immune complexes were incubated with 10 mM cAMP. PKA activity was measured with the substrate Kemptide as described [J. D. Corbin and E. M. Reimann, Methods Enzymol. 38, 287 (1974)]. PKA activity was defined as the activity inhibited by the PKI 5-24 inhibitor peptide (15). Immunoprecipitations in Fig. 2 were performed as described [J. Luo et al., Mol. Pharmacol. 51, 79 (1997)].
53. Rat brain extracts were prepared by Dounce homogenization of frozen brains in phosphate-buffered saline (PBS) containing 1 mM EDTA, 1 mM EGTA, 1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride, 1 mM benzamidine, pepstatin (2 μg/ml), and leupeptin (2 μg/ml), followed by centrifugation at 40,000g for 1 hour. Supernatant (1 ml) was incubated with 20 μl of preimmune or immune serum overnight at 4°C. After addition of protein G-agarose (30 μl) for 1 hour at 4°C, the precipitated complexes were washed five times with homogenization buffer and proteins were eluted with 2X SDS sample buffer. Yotiao was detected by RII overlay. For detection of PKA C subunit activity, immune complexes were incubated with 10 mM cAMP. PKA activity was measured with the substrate Kemptide as described [J. D. Corbin and E. M. Reimann, Methods Enzymol. 38, 287 (1974)]. PKA activity was defined as the activity inhibited by the PKI 5-24 inhibitor peptide (15). Immunoprecipitations in Fig. 2 were performed as described [J. Luo et al., Mol. Pharmacol. 51, 79 (1997)].
54. Designated fragments of yotiao were amplified by PCR and subcloned into pGEX-4T3 (Amersham Pharmacia Biotech) or pet30b (Novagen). Inserted sequences were confirmed by DNA sequencing. GST fusion proteins were purified from bacterial extracts by affinity purification with glutathione-agarose (Amersham Pharmacia Biotech).
55. Bacterial extracts expressing fragments of yotiao as histidine-tagged fusion proteins were separated by SDS-PAGE (4 to 15% gels), transferred to polyvinylidene fluoride membranes (Millipore), and blocked overright in 1% BLOTTO (5% nonfat dry milk, 1% bovine serum albumin, 25 mM tris, and 150 mM NaCl). Blots were then incubated with recombinant PP1 (2 μg/ml) for 2 hours, washed, and incubated with PP1 antisera (1:10,000) for 1 hour. After washing, blots were incubated with horseradish peroxidase-conjugated secondary antibody and washed. PP1 binding was detected by enhanced chemiluminescence (Pierce). Under these conditions PP1 binding is blocked by the Gm peptide, but not by peptides (such as Ht31) that block RII interaction with AKAPs. Protein immunoblots were done as described above with omission of the incubation with recombinant PP1.
56. 32P]ATP and then purified by ammonium sulfate precipitation and fractionation on a D-Salt Excellulose desalting column (Pierce). Labeling of GST alone demonstrated that >80% of the radioactivity was incorporated into the NR1A fragment.
57. We thank V. Coghlan and S. Olsen for isolation and analysis of the original GH4/15 clone; S. Shenolikar, C. Jahr, G. Westbrook, and colleagues at the Vollum Institute for critical evaluation of the manuscript; E. Lee for providing PP1; J. Goldenring for providing a manuscript before publicaton; and A. Westphal, K. Sandstrom, and A. Bishop for expert technical assistance. Supported in part by NIH grants NS10543 (R.S.W.), NS10202 (S.J.T.), GM 48231 (J.D.S.), and NS35050 (M.S.).