[9 Regulation of Protein Kinase C Isozyme Activity by SGlutathiolation

By Nancy E. Ward, Feng Chu, and Catherine A. O' Brian I. Introduction

Protein S-thiolation is a posttranslational modification of select proteins produced by a nonenzymatic, oxidative mechanism, which entails the formation of disulfide linkages between thiols of reactive cysteine residues and low molecular weight (LMW) thiols.1 Common endogenous S-thiolating species are glutathione (GSH), cysteine, and cystamine.2 Mechanisms of protein S-glutathiolation reactions include thiol-disulfide exchange reactions between protein thiols and GSH disulfide (GSSG) and reactions between the protein thiols and GSH that involve the oxidant-produced sulfenic acid intermediates GSOH or PrSOH, or the thiyl radical intermediate PrS'.

Oxidative stimuli that produce protein S-thiolation in cells include physiological stimuli that induce intracellular hydrogen peroxide production and bolus treatment with either the thiol-specific oxidant diamide or hydrogen peroxide.3'4 Protein S-thiolation serves as a line of defense against oxidative damage to cells by reversibly blocking protein thiols that might otherwise be irreversibly oxidized to sulfinic or sulfonic acid under conditions of oxidative stress.3'5 The existence of enzymatic protein dethiolation mechanisms, which are catalyzed by glutaredoxin (thioltransferase) and thioredoxin, provides evidence that protein S-thiolation serves as a regulatory mechanism for certain enzymes and binding proteins.6 Indeed, the demonstrated ability of S-glutathiolation to dramatically and reversibly alter the catalytic activity of some enzymes strongly implicates protein S-glutathiolation as a mechanism of regulation of select redox-sensitive enzymes.1'4'5

In this chapter, we describe methods that have been used to demonstrate that the isozyme protein kinase Ca (PKCa) is exquisitely sensitive to oxidant-induced S-glutathiolation, which fully and reversibly inactivates the kinase activity.4 The methods are likely to be broadly applicable to other PKC isozymes, as preliminary observations indicate that several other PKC isozymes are inactivated by analogous

1 J. A. Thomas, B. Poland, and R. Honzatko, Arch. Biochem. Biophys. 319, 1 (1995).

2 J. A. Thomas, W. Zhao, S. Hendrich, and P. Haddock, Methods Enzymol. 251,423 (1995).

3 D. M. Sullivan, N. B. Wehr, M. M. Fergusson, R. L. Levine, and T. Finkel, Biochemistry 39, 11121 (2000).

4 N. E. Ward, J. R. Stewart, C. G. Ioannides, and C. A. O'Brian, Biochemistry 39, 10319 (2000).

5 E. Cabiscol and R. L. Levine, Proc. Natl. Acad. Sci. U.S.A. 93,4170 (1996).

6 S. A. Gravina and J. J. Mieyal, Biochemistry 32, 3368 (1993).

GSH-dependent, oxidative mechanisms.63 The methods may also be applied to other protein kinases that are potential candidates for regulation by S-thiolation, for example, the cAMP-dependent protein kinase (PKA), which harbors a highly reactive cysteine residue in its active site.7

The methodology for the study of PKC S-glutathiolation is likely to be relevant in investigations of the mechanism of GSH antagonism of tumor promotion,4 and the potential role of PKC S-glutathiolation as a negative feedback loop for PKC-mediated oxidant production in cells.8'9 The methods described will also be valuable in defining the relationship between the opposing effects on PKC activity that have been produced by oxidant treatment of mammalian cells, that is, a GSH-independent PKC activation mechanism that involves PKC phosphorylation at tyrosine10 and the GSH-dependent inactivation mechanism of PKC S-glutathiolation.4

II. Methods for Analyzing the Inactivation of Purified Protein Kinase C Isozymes by S-Glutathiolation

A. Refreshing Protein Kinase C Isozyme Thiols

1. Principle. Refreshing PKC isozyme thiols to their native, reduced state is a necessary preliminary step for studies of the oxidative regulation of purified PKC. Although PKC isozymes are typically stored in the presence of 5-15 mM 2-mercaptoethanol, over long-term storage, the 2-mercaptoethanol slowly oxidizes and may eventually fail to fully protect PKC thiols from oxidation. In the case of commercial purified PKC isozymes supplied in volumes of 30-200 /xl [Cal-biochem (La Jolla, CA), Pan Vera (Madison, WI), Biomol (Hamburg, Germany)], air oxidation of the 2-mercaptoethanol in solution may occur rapidly and cannot be directly measured because of the expense of the material. Instead, it is more practical and cost-effective to routinely refresh PKC thiols before analysis of oxidant effects on PKC activity. Human PKC isozymes contain 16 to 23 cysteine residues, for example, human PKC« contains 20 cysteine residues.4 We have found that omission of the reductive thiol preconditioning step can sometimes result in the production of aberrant and unreproducible effects on PKC activity by a given oxidative insult, for example, if PKC thiols are not refreshed, diamide may occasionally appear to activate a purified rat brain PKC isozyme mixture described in Ward et al.4 We attribute those unreproducible effects to partial oxidation of

6a F. Chu, N. E. Ward, and C. A. O'Brian, Carcinogenesis 22, 1221 (2001).

7 H. N. Bramson, N. Thomas, R. Matsueda, N. C. Nelson, S. S. Taylor, and E. T. Kaiser, J. Biol. Chem. 257, 10575 (1982).

8 H. M. Korchak, M. W. Rossi, and L. E. Kilpatrick, J. Biol. Chem. 273, 27292 (1998).

9 M. A. Stevenson, S. S. Pollock, C. N. Coleman, and S. K. Calderwood, Cancer Res. 54,12 (1994).

10 H. Konishi, M. Tanaka, Y. Takemura, H, Matsuzaki, Y. Ono, U. Kikkawa, and Y. Nishizuka, Proc. Natl. Acad. Sci. U.S.A. 94, 11233 (1997).

PKC thiols that may occur to variable extents in storage. In addition, we have found that if PKC thiols are not refreshed before analysis, reducing agents such as dithiothreitol (DTT) may activate PKC by as much as 2-fold in the system of analysis. Refreshing PKC thiols before analysis of oxidative regulation of the enzyme achieves a reliable baseline of PKC activity, which remains unchanged (±10%) when the enzyme is subsequently incubated with millimolar DTT in the analysis.

2. Method. The method entails incubation of PKC with DTT followed by chromatography of the PKC sample on a small desalting gel-filtration column to remove excess DTT from the enzyme.4 First, a 2-ml G-25 column (bed height, ~4 cm) is equilibrated in equilibration buffer [20 mM Tris-HCl (pH 7.5), 1 mM EDTA,

I mMEGTA, soybean trypsin inhibitor (20 //g/ml), leupeptin (10 /ig/ml), 250 ¡jlM phenylmethylsulfonyl fluoride (PMSF)] at 4°. Next, in the case of commercial purified human PKCa (available from Pan Vera, Calbiochem, and Biomol), 5 fig of PKCa (this is about 50 /xl of the commercial stock) is diluted into Equilibration buffer containing 2 mM DTT (final volume, 500 /¿l) and then incubated for 20 or 30 min at 4° to refresh the thiols. To remove the excess DTT from the PKCa sample, the sample is then gel filtered on the G-25 column at 4°. The column may be washed and reused numerous times, but before its initial use, the column must be calibrated to identify the elution positions of PKCa and DTT. This can be done by measuring PKC activity and the reactivity of DTT with 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB, Ellman's reagent) (yellow product formation, Amax at 412 nm) in fractions of 200-250 ¡A eluted from the column. Typically, pooling the fractions that span 750 to 1500 /1\ (this includes the eluted volume of the sample) achieves excellent recovery of PKCa activity (>95%) and >90% removal of DTT. PKCa should be routinely recovered from the calibrated column in a single vial that can be capped and that has minimal air space after PKCa collection to minimize air oxidation of thiols. Because only residual levels of the sulfhydryl protectant DTT remain, it is best to use the gel-filtered PKCa sample for analysis of oxidative regulation of the isozyme within a relatively short period of time, that is, <2 hr.

B. Inducing Inactivation of Purified PKCa by S-Glutathiolation

1. Principle. The oxidant activity of diamide is thiol specific and restricted to disulfide bridge formation.11 The defined oxidant activity of diamide facilitates the elucidation of PKC inactivation mechanisms that involve thiol oxidation. Measurement of the DTT reversibility of diamide-induced effects on PKCa activity is a useful control to verify that the effects result from disulfide bridge formation.11 Because diamide only weakly inactivates PKCa,4 diamide can be used as an inducing agent for investigations of the effects of PKCa S-glutathiolation on the activity of the isozyme.

II N. S. KosowerandE. M. Kosower, Methods Enzymol. 251, 123 (1995).

0 0

Post a comment