Abstract
To examine the phosphorylation of casein kinase II in cells, the enzyme was isolated by immunoprecipitation from metabolically labeled human epidermal carcinoma A431 cells using polyclonal antipeptide antibodies specific for either the alpha subunit or the beta subunit of the enzyme. When isolated from 32P-labeled cells, the beta subunit was found to be significantly labeled on serine residues whereas only minimal labeling was associated with the alpha subunit. In vitro, the beta subunit of purified bovine casein kinase II was autophosphorylated, also on serine residues. Cleavage of the beta subunit, that had been autophosphorylated in vitro, at tryptophan 9 and tryptophan 12 using N-chlorosuccinimide demonstrated that the autophosphorylation site is located near the amino terminus of the protein, most likely at serine 2 and serine 3. Two-dimensional maps of phosphopeptides generated by digestion of the beta subunit with endoproteinase Glu-C indicted that the majority of the phosphate that was incorporated into the protein in cells was at sites that were indistinguishable from the sites that were autophosphorylated in vitro. In addition to phosphorylation at the autophosphorylation site, the beta subunit is also phosphorylated at an additional site, serine 209, in intact cells. This residue, which is near the carboxyl terminus of the protein, can be phosphorylated in vitro by p34cdc2.
Highlights
To examine the phosphorylation of casein kinase I1 in cells, the enzyme was isolated by immunoprecipitation from metabolically labeled human epidermal carcinoma A431 cells using polyclonal antipeptide antibodies specific for either the a subunit or the #J subunit of the enzyme
When isolated from ”P-labeled cells, the #J subunit was found to be significantly labeled on serine residues whereas only minimal labeling was associated with the a subunit
Casein kinase I1 (CKII)’ is a multifunctional messengerindependentproteinserine/threonine kinase. its precise biological functions remain poorly understood, a number of studies have suggested that this enzyme plays an important
Summary
Containing proteinwere neutralized and dialyzed against phosphatebuffered saline. The ~ 3 4 pep~tide~ wa's c~oupl~ed to Sulfolink coupling gel (4 mg/ml gel) according to manufacturer's instructions to Materials prepare a resin for affinity purification of immune serum according. Lysates were precleared by the addition of an equal volume of20% (v/v) Pansorbin that had been saturated with normal rabbit serum according to described procedures (Harlow and Lane, 1988).Following incubation for 1h on ice, these lysates were clarified by centrifugation for 5 min a t 4 "C in a microcentrifuge. Pansorbin was added (50 p1 of 10% (v/v)) and the samples incubated for an additional 30 min on ice. Immune complexes were collected by centrifugation for 30 s in a microcentrifuge and were washed three times by resuspension in RIPA buffer and once by resuspension in 20 mM Tris-C1, pH 7.5. Tide antibodies were raised in New Zealand White rabbits against a After tumbling the suspension for 1h at 4 "C, the immune complexes peptide corresponding to amino acids 198-215 of the deduced amino were collected by centrifugation for 1 min in a microcentrifuge. Lyzed by P81 paper assays (Litchfield etal., 1990a) or by electrophoresis on cellulose plates (20 x 20 cm) for 30 min a t 1000 v using pH
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