Differential stimulation of PKC phosphorylation of potassium channels by ZIP1 and ZIP2

Science

Volumn 285, Issue 5433, 1999, Pages 1565-1569

  Gong, Jianping ^a   Xu, Jia ^a,b   Bezanilla, Magdalena ^a,c   Van Huizen, Rika ^a   Derin, Rachel ^a   Li, Min ^a  

^a JOHNS HOPKINS UNIVERSITY SCHOOL OF MEDICINE   (United States)

^b Aurora Biosciences Corporation   (United States)

^c SALK INSTITUTE FOR BIOLOGICAL STUDIES   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

CELL PROTEIN; GENE PRODUCT; POTASSIUM CHANNEL; POTASSIUM ION; PROTEIN KINASE C; PROTEIN ZIP1; PROTEIN ZIP2; UNCLASSIFIED DRUG;

ALTERNATIVE RNA SPLICING; ARTICLE; BINDING AFFINITY; CELL STRAIN; ENZYME PHOSPHORYLATION; IMMUNOPRECIPITATION; PRIORITY JOURNAL; PROTEIN ASSEMBLY; PROTEIN DOMAIN; PROTEIN LOCALIZATION; PROTEIN PHOSPHORYLATION; PROTEIN PROCESSING; PROTEIN PROTEIN INTERACTION; REVERSE TRANSCRIPTION POLYMERASE CHAIN REACTION;

ALTERNATIVE SPLICING; AMINO ACID SEQUENCE; ANIMALS; BINDING SITES; CARRIER PROTEINS; CELL LINE; CEREBELLUM; DNA, COMPLEMENTARY; ISOENZYMES; MOLECULAR SEQUENCE DATA; MYELIN BASIC PROTEINS; NERVE GROWTH FACTORS; NEURONS; PHOSPHORYLATION; POTASSIUM CHANNELS; PROTEIN KINASE C; PYRAMIDAL CELLS; RATS; RATS, SPRAGUE-DAWLEY; RECOMBINANT FUSION PROTEINS; RNA, MESSENGER; SUBSTRATE SPECIFICITY; TRANSFECTION;

EID: 0033520412     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.285.5433.1565     Document Type: Article

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40. Antibodies for ZIP and Kvβ2 were generated by immunizing rabbits with GST-fusion proteins corresponding to the full-length cDNAs of rat ZIP1 and Kvβ2. The PKCζ and PKCβII antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
41. The HA-tagged Kvβ2 was expressed by transient transfection and cell tysates from HA-Kvβ2 and mock-transfected cells were prepared according to the protocol described in Yu et al. (10). Affi-gel 10 (Bio-Rad, CA) was used to conjugate GST control, GST-ZIP1 and GST-ZIP2. The binding reactions were performed using 20 μl beads and 100 μl of cell extracts in a buffer containing 20 mM Hepes (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM 2-mercaptoethanol. After incubation on ice for 1 hour, the beads were washed three times, and the bound material was released by boiling the beads in SDS-sample buffer. The KvβZ binding was tested by fractionating the bound material on SDS gel followed by immunoblot with anti-HA antibody.
42. 2.
43. J. Gong et al., unpublished data.
44. 3 and protease inhibitor cocktail (1 mM phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, 2 μg/ml aprotinin, and 1 μg/ml pepstatin). Homogenization was carried out at 4°C using a Dounce homogenizer with a typical tissue to buffer ratio of B ml buffer per gram of tissue. After 20 strokes of homogenization, homogenates were centrifuged at 10,000g for 5 min and the supematants were collected. To conjugate antibodies to Protein A-agarose, 40 μl of Protein A beads (Sigma Chemical, St. Louis, MO) in 1 ml of PBS were first incubated overnight with 20 μl of antiserum at 4°C. After removal of unbound material, the agarose was washed three times with PBS, resuspended in 500 μl of 0.2 M sodium borate (pH 9.0). The cross-linking was initiated by adding dimethyloimelimidate to a final concentration of 20 mM. After 30 min incubation at room temperature, the reaction was stopped by addition of 500 μl of 0.2 M ethanolamine. After 2 hours incubation, the conjugated protein A-agarose was washed three times with PBS. The antibody binding was carried out by incubating 200 μl of soluble lysate with 10 μl of antibody-protein A agarose. After overnight incubation at 4°C, the Protein A agarose was collected by centrifugation at 5,000g for 2 min, and washed three times with PBS. The individual Protein A pellets were treated with 10 μl of 2× SDS sample buffer at 100°C for 5 min. The soluble protein samples were separated by SDS-PAGE. The immunoprecipitated polypeptides were detected by immunoblot using the corresponding antibodies as indicated.
45. 5 cells were seeded in a 3.5-cm dish and allowed to grow for 24 hours in the presence of 10% fetal bovine serum and 5% heat inactivated horse serum. The NGF stimulation was initiated by adding purified NGF to a final concentration of 0.1 μg per ml. The cells were allowed to grow for 72 hours and harvested for analyses.
46. We thank D. Ginty for providing purified NGF, A. Lanahan and P. Wortey for cDNA libraries, J. Baraban for anti-translin antibody, Y. Ono for rat PKCζ cDNA, L Roman for in situ hybridization technique, M. Regan for RT-PCR technique, S. Wang for the control GST fusion protein and R. Butzner for help with manuscript preparation. We also thank C Montell, P. Gillespie, and members of the Li Lab for comments on this manuscript M.B. was a NSF predoctoral fellow. R.H. is supported in part by a postdoctoral fellowship from American Heart Association. M.L. is supported by a grant from NIH (NS33324) and an American Heart Association-Pfizer award.