Table V

Preincubation Mixtures Employed to Measure Stoichiometry of Diamide-Induced PKCa 35S-Glutathiolation

Component (final concentration/amount)" (in order of addition)

Method of addition

1.0.01-1.0 mM diamide

Added as 10 fil of a diamide stock solution in 20 mM

Tris-HCl, pH 7.5 Added as 10 /il of a 1.2 m M [35S]GSH stock solution. The stock solution is prepared by adding 50 ¡A of [35S]GSH (0.5 mCi; specific activity, ~404 Ci/mmol) (DuPont-NEN) per 1 ml of 1.26 m M cold GSH in 20 mM Tris-HCl, pH 7.5 To 120 ¡X1 (final volume) Present in 100 /il of equilibration buffer a Components are presented in order of addition.

weak p emissions to light, Amplify increases the efficiency of 35S-glutathiolated PKCa detection and reduces the exposure time needed for detection to 6-48 hr. Next, the gel is vacuum dried onto filter paper. The filter paper is then spotted at its corners with a few drops of 35S-labeled ink, to allow orientation of the autoradiogram to the gel for molecular weight calculations based on Coomassie-stained molecular weight markers. The gel is exposed to Amersham Hyperfilm MP, using an intensifying screen in a film cassette at —10°.

To determine the stoichiometry of the covalent incorporation of [35S]GSH into PKCa that is sufficient to induce GSH-dependent, oxidative PKCa inactiva-tion, PKCa (with refreshed thiols; see Section II,A) is preincubated for 5 min at 30° with 100 ijlM [35S]GSH and 0.01-1.0 mM diamide, as described in Table V; either the full range of diamide concentrations or a single diamide concentration sufficient for >90% GSH-dependent PKCa inactivation may be employed in the analysis. A preincubation mixture containing 100 fiM [35S]GSH and PKCa alone (no diamide) is required to measure background radioactivity. Preincubations are terminated by adding an equal volume (120 /il) of ice-cold TCA-PPi solution [20% (w/v) trichloroacetic acid-1% (w/v) pyrophosphate] to the samples, which are then vortexed briefly and placed on ice. Nitrocellulose filter circles (25 mm, 0.2 fim GSWP; Millipore, Bedford, MA) are placed on a vacuum-filtration manifold (Pharmacia/Hoeffer) and premoistened with TCA-PPi solution. The TCA-precipitated samples (240 /il) are pipetted onto the filters with a Pipetman, and the filters are washed with TCA-PPi solution (three times, 5 ml each) by vacuum filtration and counted in 5 ml of scintillation fluid. To calculate the stoichiometry of the covalent binding of [35S]GSH to PKCa (mole per mole), the background counts per minute are subtracted from the total filter-bound counts per minute. The counts per minute-to-picomole conversion can be made by simultaneously counting an [35S]GSH standard, for example, 1 nmol of [35S]GSH from the 1.2 mM [35S]GSH stock solution. By this method, we have determined that an S-glutathiolation stoichiometry of approximately 1 mol of GSH per mole of PKCa is sufficient for >90% inactivation of human recombinant PKCa.4

III. Methods for Analyzing PKCa Inactivation by S-thiolation in Mammalian Cells

A. Demonstrating Dithiothreitol-Reversible, Oxidative PKCa Inactivation in Mammalian Cells

1. Principle. To focus on oxidant-induced effects on PKCa activity in mammalian cells that may involve PKCa S-glutathiolation, DTT-reversible changes in PKCa activity produced by diamide treatment of cells are measured. The focus on DTT-reversible changes in PKCa activity distinguishes effects of oxidant-induced PKCa S-glutathiolation on kinase activity4 from effects stemming from oxidant-mediated stabilization of phosphotyrosine residues in the catalytic domain of PKCa, which induces PKC isozyme activation.10 Furthermore, diamide treatment of cells limits the types of oxidative modifications that may be introduced into PKC isozymes to those involving disulfide linkages and thus simplifies the parallel analysis of the oxidative modification of cellular PKCa. NIH 3T3 cells are convenient for the analysis of oxidative PKCa regulation, because PKCa activity can be directly assayed in DEAE-Sepharose-extracted cell lysates owing to the lack of expression of other Ca2+-dependent PKC isozymes in the cells.4 Diamide treatment of NIH 3T3 cells offers an excellent model of potent PKCa inactivation by S-thiolation.4 Methods used to demonstrate DTT-reversible inactivation of PKCa by diamide treatment of NIH 3T3 cells, which occurs in association with PKCa S-thiolation4 (see Section III,B), are described below.

2. Method. NIH 3T3 fibroblasts (60-80% confluent) cultured at 37° under standard conditions [Dulbecco's modified Eagle's medium (DMEM) plus 10% (v/v) bovine serum] (Life Technologies, Rockville, MD) are incubated with cyclohex-imide (50 /xg/ml) for 5 hr at 37°, to match conditions used to analyze cellular PKCa 35S-thiolation in parallel (see Section III,B). The cells are washed with 10 ml of Hanks' balanced salt solution (HBSS; Life Technologies) and then incubated with diamide (0.1-5.0 mM) under serum-free conditions for 10 min at 37° (8-12 x 106 cells per treatment group, e.g., one 100-mm-diameter dish at 75% confiuency); potent DTT-reversible PKCa inactivation is achieved by >2.5 mM diamide.4 At the end of the treatment period, the cells are washed with ice-cold phosphate-buffered saline (PBS; Life Technologies), and lysed with 1% Triton X-100 in equilibration buffer (defined in Section II,A) (1.5 ml per treatment group) by stirring in 1,5-ml capped tubes for 15 min at 4°. The samples are spun in a microcentrifuge at 14,000g for 2 min at 4° to remove debris and then loaded onto 0.5-ml DEAE-Sepharose columns equilibrated in equilibration buffer (without Triton). After the columns are washed with 2 ml of equilibration buffer, equilibration buffer with 0.3 MNaCl (1 ml) is used to elute PKCa activity, and the protein concentration of the eluted sample is determined. To measure DTT-reversible PKCa inactivation, the eluted PKC is divided into two portions, which are preincubated with/without 30 mM DTT for 15 min at 30° in capped tubes, placed on ice, and assayed immediately. Assays are done as delineated in Table III, except that 5 fig of partially purified PKCa is included per assay mixture, and the assay mixtures that measure background kinase activity include Ca2+ and the PKC inhibitor Go6976 (100 nM) (Calbiochem), which selectively inhibits Ca2+-dependent PKC isozymes and, thus, PKCa in NIH 3T3 cells. PKCa activity is calculated as total minus background kinase activity. The DTT-reversible PKCa inactivation produced by diamide treatment is calculated as the percent inactivation observed with DEAE-extracted PKCa samples preincubated without DTT minus the percent inactivation observed when the samples are preincubated with DTT. Because DTT reversal of disulfide bridge formation is time and concentration dependent, the ratio of DTT-reversible to DTT-irreversible inactivation observed by this method may be increased by prolonging the preincubation with DTT or by increasing the DTT concentration. Thus, disulfide bridge formation cannot be ruled out as the mechanism for the minor DTT-irreversible component of inactivation that is observed in the analysis.

B. Demonstrating Oxidant-Induced PKCa 35S-Thiolation in Mammalian Cells

1. Principle. Because diamide by itself only weakly inactivates purified PKCa across a broad range of diamide concentrations but supports potent inactivation of the purified isozyme by S-glutathiolation,4 demonstration that potent diamide-induced inactivation of PKCa in NIH 3T3 cells (see Section III,A) is associated with induction of cellular PKCa S-thiolation and is not associated with the formation of disulfide-linked complexes between PKCa and other cellular proteins provides compelling evidence that the inactivation mechanism is PKCa S-thiolation.4 Therefore, NIH 3T3 cells are analyzed for diamide-induced, DTT-reversible PKCa inactivation (see Section III,A) and PKCa 35S-thiolation in parallel, under conditions that achieve potent PKCa inactivation. Complex formation between PKCa and other proteins in the cells is analyzed on the basis of the migration position of PKCa in nonreducing SDS-polyacrylamide gels as measured by Western analysis; full recovery of cellular PKCa at its normal migration position is indicative of negligible complex formation with other proteins 4 Measurement of PKCa 35S-thiolation in NIH 3T3 cells entails selective, metabolic 35S labeling of cellular GSH and other cysteine-derived LMW thiols, oxidative induction of PKCa 35S-thiolation by diamide treatment of the metabolically labeled cells, im-munoprecipitation of 35S-thiolated PKCa from the cell lysates, and analysis of 35S-thiolated PKCa (±DTT) by nonreducing SDS-PAGE/autoradiography.4

The general method of 35S metabolic labeling of LMW thiols for analysis of oxidative induction of protein 35S-thiolation in cells is described in Thomas et al.2

The method entails culturing the cells in the absence of sulfur-containing amino acids to deplete GSH and other LMW thiols, followed by labeling of the cells with [35S]cysteine under conditions in which protein synthesis is inhibited, so that the predominant 35S-labeled species are cysteine, cystamine, and GSH. The 35S-labeled cells are then exposed to an oxidative stimulus and lysed under two sets of conditions: one that preserves the oxidatively produced 35S-thiolated protein species present in the cells and one that dethiolates the proteins.2 Cellular protein 35S-thiolation is preserved by lysing cells in the presence of the thiol-modifying agent ¿V-ethylmaleimide (NEM), which blocks free thiols and thus prevents the migration of the 35S-thiolating species to other protein thiols during cell lysis. Cellular protein 35S-thiolation is reversed by lysis of the cells in the presence of DTT.2 Protein S-thiolation is operationally defined in this system as DTT-reversible 35S labeling of cellular proteins detected by nonreducing SDS-PAGE/autoradiography. Because the method of sample preparation for SDS-PAGE/autoradiography entails boiling samples in the presence of SDS, >10 m M DTT fully reverses protein 35S-thiolation in this system. Any protein labeling observed in the DTT-treated samples by SDS-PAGE/autoradiography is generally due to residual protein backbone labeling and may be minimized by more stringent protein synthesis inhibition.

2. Method. To analyze PKCa 35S-thiolation in diamide-treated NIH 3T3 cells under conditions that produce potent oxidative inactivation of PKCa, the cells are cultured under the conditions employed in the analysis of PKCa inactivation (see Section III,A), except that LMW thiols are depleted by culturing the cells with dialyzed serum and DMEM lacking sulfur-containing amino acids for 16 hr at 37°, before the addition of the protein synthesis inhibitor cycloheximide (50 /xg/ml). After the cells are incubated with cycloheximide for 1 hr at 37°, [35S]cysteine (Amersham, Arlington Heights, IL) is added to the medium (SOSO /.xCi/ml medium), and the cells are further incubated for 4 hr at 37° so that selective metabolic labeling of GSH and other LMW thiols can ensue. Next, the radiolabeled cells are treated with diamide, and cell lysates are prepared as described in Section III,A, with the following modification. For each treatment condition, two plates (~107 cells per plate) are needed for the analysis; one is for cell lysis in the presence of NEM, and the other is for cell lysis in the presence of DTT. Equilibration buffer (defined in Section II,A) with 1% (v/v) Triton X-100 and either 50 m M NEM or 25 m M DTT is employed to lyse the cells (1 ml/plate).

A fraction of each cell lysate (about 50 ¡i\) should be reserved for analysis of whether diamide induces disulfide-linked complexes between PKCa and other proteins in the cells. This is done by Western analysis with a PKCa monoclonal antibody (mAb) (Transduction Laboratories, Lexington, KY) under nonreducing conditions, that is, by nonreducing SDS-PAGE. Either the appearance of DTT-sensitive PKCa-immunoreactive bands at retarded migration positions and/or the loss of PKCa immunoreactivity at its normal migration position will occur if a substantial amount of PKCa forms complexes with other proteins; we have observed neither phenomenon.4

To detect PKCa 35S-thiolation, PKCa is immunoprecipitated from the radiolabeled cell lysates with a polyclonal PKCa antibody available from Santa Cruz Biotechnology (Santa Cruz, CA); PKCa S-thiolation does not interfere with the binding interactions between PKCa and this antibody.4 Because the analysis entails immunoprecipitation of PKCa, it is necessary to add 50 mM NEM to the DTT-treated cell lysates before proceeding, to prevent reduction of the antibody to its subunits. Cell lysates are precleared with a 50% (v/v) slurry of protein A-Sepharose (100 fil of slurry/ml cell lysate) by end-over-end rotation for 30 min at 4°, followed by a brief spin in a microcentrifuge (10 min, 4°) to recover the supernatant. Sample protein concentrations are normalized by adjusting sample volumes with equilibration buffer, followed by incubation of the samples with equal volumes of the polyclonal PKCa antibody (300-500 fig of lysate protein per 5 fig of antibody; the recommended total volume is 1 ml). The antibody is prepared in 2x IP buffer [IP buffer includes 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1 mMEGTA, 150 mMNaCl, 1% (v/v) NonidetP-40 (NP-40), 10% (v/v) glycerol, leupeptin (10 /ig/ml), and 0.2 mM PMSF]. After incubating the precleared cell lysates with the PKCa antibody for 2 hr at 4° with end-over-end rotation, the incubation is continued for at least 30 min to 1 hr in the presence of protein A-Sepharose [ 100 fi 1 of a 50% (v/v) slurry], and the Sepharose beads are washed three times with IP buffer. The washed beads are resuspended with 100 fi 1 of 2x nonreducing SDS-PAGE sample buffer (defined in Section II,C) and boiled for 5 min. The supernatants are analyzed for PKCa 35S-thiolation, as described in Section II,C. To demonstrate diamide-induced PKCa 35S-thiolation, the analysis must include samples corresponding to (1) untreated cells/NEM-lysate; (2) diamide-treated cells/NEM-lysate, (3) untreated cells/DTT-lysate; and (4) diamide-treated cells/DTT-lysate. Western analysis of the PKCa samples is done in parallel to verify equivalent recovery of immunoprecipitated PKCa from the treatment groups analyzed. Demonstration of diamide-dependent, DTT-sensitive 35S labeling of PKCa by autoradiography and confirmation of equivalent recovery of each PKCa sample by Western analysis are indicative of PKCa 35S-thiolation.

Acknowledgments

This work was supported by NCI Grant CA74831 and by Robert A. Welch Foundation Award G-1141.

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