©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Irreversible Inactivation of Protein Kinase C by a Peptide-Substrate Analog (*)

(Received for publication, December 13, 1994; and in revised form, January 27, 1995)

Nancy E. Ward Karen R. Gravitt Catherine A. O'Brian (§)

From the Department of Cell Biology, University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Protein kinase C (PKC) is a phospholipid-dependent isozyme family that plays a pivotal role in mammalian signal-transduction pathways that mediate cell growth and differentiation and pathological developments, such as the acquisition of drug resistance by cancer cells. Several peptide-substrate analogs have been shown to reversibly inhibit PKC with high potency and selectivity, but peptide-substrate analogs that antagonize PKC by forming a covalent complex with the enzyme have not been reported. The development of active site-directed irreversible inactivators of PKC could provide new insights into the catalytic mechanism and might ultimately lead to the design of novel therapeutics targeted at PKC.

In this report, we show that the peptide-substrate analog Arg-Lys-Arg-Cys-Leu-Arg-Arg-Leu (RKRCLRRL) irreversibly inactivates PKC in a dithiothreitol-sensitive manner. The inactivation mechanism most consistent with our results is the formation of a covalent linkage between the inhibitor-peptide and the enzyme at its active-site. Limited proteolysis of PKC produces a catalytic-domain fragment that is independent of the phospholipid cofactor. RKRCLRRL antagonized the histone kinase activity of PKC and its catalytic-domain fragment with similar efficacies, achieving >50% inactivation at an RKRCLRRL concentration of 10 µM. In contrast, RKRCLRRL analogs with single amino acid substitutions at Cys were non-inhibitory. The inactivated complex of the catalytic-domain fragment and RKRCLRRL was stable upon dilution, and the inactivation of PKC and the catalytic-domain fragment by RKRCLRRL was quenched by dithiothreitol, providing evidence that the enzyme and the synthetic peptide may be covalently linked in an inactivated complex by a disulfide bond. Substrates and substrate analogs protected the catalytic-domain fragment against inactivation by RKRCLRRL, providing evidence that inactivation entailed binding of RKRCLRRL at the active-site of the enzyme.

S-Thiolation is the formation of mixed disulfides between proteins and low molecular weight thiols. PKC is thought to have a highly reactive Cys residue in its active-site, and Cys residues that are flanked by basic residues, as is the case in RKRCLRRL, display enhanced reactivity. Our results support an inactivation mechanism that entails S-thiolation of the active-site of PKC by RKRCLRRL. This is the first report of irreversible inactivation of PKC by an active site-directed peptide.


INTRODUCTION

Protein kinase C (PKC) (^1)is a phospholipid-dependent isozyme family that plays key roles in signaling mechanisms involved in normal physiological processes such as neurotransmission, muscle contraction, and cell growth and differentiation, and also pathological developments such as tumor promotion and the acquisition of drug resistance by cancer cells in response to chemotherapy(1, 2, 3, 4) . A number of peptide-substrate analogs have been reported to inhibit PKC reversibly, and in some cases with remarkable potency and selectivity(5, 6, 7, 8, 9) . Irreversible inactivation of the cAMP-dependent protein kinase has been achieved with peptide-substrate analogs, some of which have been shown to specifically label a highly reactive active-site Cys residue that is conserved in PKC(10, 11, 12, 13) . However, peptide-substrate analogs that inhibit PKC by forming a covalent complex with the enzyme are still lacking. The development of active site-directed irreversible inactivators of PKC could aid in the identification of active-site residues and in the elucidation of the catalytic mechanism of PKC (12) and could eventually lead to the design of novel therapeutics, e.g. cancer therapeutics for the reversal of drug resistance (14) .

We previously reported that N-myristoylation of the PKC peptide-substrate Arg-Lys-Arg-Thr-Leu-Arg-Arg-Leu (RKRTLRRL) and several nonphosphorylatable analogs of the peptide transformed peptides that lacked inhibitory activity against PKC-catalyzed histone phosphorylation into potent inhibitors of the reaction(9, 15) . The N-myristoylated peptides inhibited PKC and a phospholipid-independent catalytic-domain fragment of the enzyme by a reversible mechanism that involved binding of the inhibitor-peptide at the active-site, according to structure-activity studies and kinetic analysis(9) . Inhibition of PKC also entailed interactions between the N-myristoylated peptides and the phospholipid cofactor of the enzyme(9) . The Cys-containing analog N-myristoyl-RKRCLRRL (NmRKRCLRRL) was by far the most potent of the peptides examined in the inhibition of the catalytic-domain fragment, but its inhibitory potency was reduced by an order of magnitude in the presence of the sulfhydryl-reducing agent dithiothreitol (DTT) (0.5 mM), providing evidence that its inhibitory mechanism may include an irreversible component(9) . NmRKRCLRRL appeared to be an active site-directed inhibitor, because of its structural relatedness to the peptide substrate NmRKRTLRRL, and because its K against the catalytic-domain fragment in the presence of DTT, i.e. under conditions of reversible inhibition (K = 50 ± 2 µM) agreed within a factor of two with the K of NmRKRTLRRL (K = 29 ± 2 µM)(9) .

The Cys residue present in the active-site of cAMP-dependent protein kinase and conserved in PKC isozymes (13) is a likely target for active site-directed irreversible inactivators of PKC. Cys residues surrounded by multiple basic residues have been shown to be highly reactive in studies of disulfide exchange reactions(16) . We therefore hypothesized that the DTT-sensitive inhibitory activity of NmRKRCLRRL against the catalytic-domain fragment of PKC could be due to the reactivity of the Cys residue in the sequence RKRCLRRL at the active site of PKC. In this report, we test the hypothesis by analyzing the effects of the peptide RKRCLRRL on PKC catalysis. We show that RKRCLRRL is a DTT-sensitive, active site-directed irreversible inactivator of PKC and its catalytic-domain fragment. This is the first report of irreversible inactivation of a protein kinase by a peptide-substrate analog composed entirely of naturally occurring, unmodified amino acids (12) , and RKRCLRRL is the first reported active site-directed irreversible inactivator of PKC.


EXPERIMENTAL PROCEDURES

Materials

The peptides RKRCLRRL, RKRALRRL, RKRTLRRL, RKR-D-CLRRL, RKRCLRR, KRCLRRL, RKRCLR, KRCLRR, NmKRCLR, and KRCLR were synthesized using the Vega coupler 250 peptide synthesizer and purified to >98% purity by reverse-phase high performance liquid chromatography using a Vydac C4 column and acetonitrile gradient elution at the M. D. Anderson Cancer Center Synthetic Antigen Facility. Rat brain PKC was purified to near homogeneity according to silver-stained polyacrylamide gels by a previously described method (17, 18) . The purified PKC preparation contains the Ca- and PS-dependent isozymes PKC-alpha, PKC-beta, and PKC- according to immunoblot analysis(19) , and its histone kinase activity was stimulated approximately 10-fold by 0.2 mM Ca and 30 µg/ml PS but was not affected by either Ca or PS alone. A catalytic-domain fragment of PKC was generated from the purified PKC preparation by limited trypsinolysis with a yield of >50%, as described previously(9, 17) . Where indicated, the catalytic-domain fragment was purified from regulatory-domain fragment and residual intact PKC by DEAE ion-exchange chromatography using a 0.0-0.4 M NaCl gradient(9, 20) . The histone kinase activity of the catalytic-domain fragment preparation was stimulated less than 1.5-fold by Ca and PS. The isozymes PKC-alpha and PKC- were purified from the rat brain PKC preparation (17, 18) by hydroxylapatite chromatography(21) , and PKC-beta was purified from R6PKC3 cells, which are engineered to constitutively express abundant amounts of rat brain PKC-beta(1)(22) .

[-P]ATP was purchased from Amersham Corp., frozen Sprague-Dawley rat brains were from Pel-Freez (Rogers, AR), phosphocellulose paper was from Fisher Scientific (Houston, TX), and hydroxylapatite resin was from Bio-Rad. Casein kinase II was from Upstate Biotech, Inc. (Lake Placid, NY). DEAE-Sepharose, G-25 Sephadex, ATP, PS, DTT, phenylmethylsulfonyl fluoride, leupeptin, histone III-S, bovine pancreas L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin, Ellman's reagent, and all other reagents were purchased from Sigma.

Protein Kinase Assay

To remove beta-mercaptoethanol (5 mM) from enzyme preparations prior to their addition to histone kinase assays, purified PKC, catalytic-domain fragment of PKC, and PKC isozymes were subjected to gel-filtration on a 2-ml G-25 Sephadex column equilibrated in 20 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM EGTA, 10 µg/ml leupeptin, 0.4 mM phenylmethylsulfonyl fluoride (fraction volume = 0.4 ml) (23) . This method quantitatively removed beta-mercaptoethanol from the enzyme preparations, according to colorimetric assays of the reactivity of the fractions with Ellman's reagent (2.5 mM).

The histone kinase reaction mixture (120 µl) contained 20 mM Tris-HCl, pH 7.5, 10 mM MgCl(2), 0.2 mM CaCl(2), 30 µg/ml PS, 6 µM [-P]ATP (5,000-8,000 cpm/pmol), 0.67 mg/ml histone III-S, purified PKC (5 ng), and where indicated, the peptide-substrate analog under investigation. In assays of the catalytic-domain fragment, PS was omitted, and CaCl(2) was replaced by 1 mM EGTA(9, 17) . Reactions were initiated by the addition of [-P]ATP, proceeded for 5 to 10 min at 30 °C with linear kinetics, and terminated on phosphocellulose paper(9, 17) . P-Labeled histone was quantitated as described previously (9, 17) . All assays were performed in triplicate and expressed as the mean value ± S.D.


RESULTS

The peptide-substrate analog RKRCLRRL potently inhibited Ca- and PS-stimulated PKC-catalyzed histone phosphorylation (Fig. 1, closed circles), under conditions where the closely related peptides RKRALRRL (Fig. 1, triangles) and RKRTLRRL (15) lacked inhibitory activity. The inhibitory activity of RKRCLRRL was quenched by the sulfhydryl-reducing agent DTT (0.5 mM) (Fig. 1, open circles). Limited trypsinolysis of PKC produces a regulatory-domain fragment and an active catalytic-domain fragment, which is independent of the cofactors Ca and PS(24) . When the catalytic-domain fragment was purified from the regulatory-domain fragment and intact PKC by DEAE ion-exchange chromatography, potent inhibition of its Ca- and PS-independent histone kinase activity was achieved by RKRCLRRL (Fig. 2, closed circles), and the inhibitory activity of RKRCLRRL was abolished by the inclusion of 0.5 mM DTT in the reaction mixtures (Fig. 2, open circles). RKRCLRRL was equally effective against a catalytic-domain fragment preparation that contained the regulatory-domain fragment and residual intact PKC (data not shown). Neither RKRTLRRL (15) nor RKRALRRL (1-40 µM) antagonized the histone kinase activity of the catalytic-domain fragment. The DTT-sensitive nature of the inhibitory activity of RKRCLRRL against PKC and its catalytic-domain fragment and the lack of inhibitory activity observed with RKRCLRRL analogs containing single amino acid substitutions at Cys provide evidence that the inhibitory mechanism may involve the formation of a DTT-sensitive covalent enzyme-inhibitor complex. Comparison of the inhibitory curves generated by RKRCLRRL in Fig. 1and Fig. 2reveals the similarity of the inhibitory potencies of RKRCLRRL against PKC and its catalytic-domain fragment. This supports an inhibitory mechanism that involves interactions between RKRCLRRL and the catalytic domain of PKC.


Figure 1: DTT-sensitive antagonism of the histone kinase activity of PKC by RKRCLRRL. The Ca- and PS-stimulated histone kinase activity of PKC was assayed as described under ``Experimental Procedures'' in the presence of RKRCLRRL (bullet), RKRALRRL (), and RKRCLRRL plus 0.5 mM DTT (circle). 100% activity was 858 ± 34 nmol of P transferred per min/mg.




Figure 2: DTT-sensitive antagonism of the histone kinase activity of the catalytic-domain fragment of PKC by RKRCLRRL. The Ca- and PS-independent histone kinase activity of the catalytic-domain fragment of PKC was assayed as described under ``Experimental Procedures'' in the presence of RKRCLRRL (bullet) and RKRCLRRL plus 0.5 mM DTT (circle). The 100% activity value was 678 ± 34 nmol of P transferred per min/mg.



To directly test whether RKRCLRRL forms an irreversible complex with PKC, we determined the effect of dilution of preincubation mixtures containing the catalytic-domain fragment of PKC and RKRCLRRL on the inhibitory potency of RKRCLRRL in histone kinase assays. In these experiments, we preincubated the catalytic-domain fragment of PKC with inhibitory concentrations of RKRCLRRL, and then the preincubation mixture was diluted into histone kinase assay mixtures so that the final RKRCLRRL concentration would be weakly inhibitory or non-inhibitory. The ability of RKRCLRRL to inhibit the catalytic-domain fragment in the histone kinase assay following preincubation with the catalytic-domain fragment was compared with its inhibitory potency in the absence of preincubation. Table 1shows that following preincubation with the catalytic-domain fragment, RKRCLRRL fully retained its inhibitory activity in the histone kinase assay, even when the final concentration of RKRCLRRL in the assay mixture was non-inhibitory. For example, when the catalytic-domain fragment was preincubated for 10 min at 30 °C with 6 µM RKRCLRRL and then added to histone kinase assay mixtures at a final RKRCLRRL concentration of 0.2 µM, RKRCLRRL inhibited the histone kinase reaction by 83 ± 7%, but 0.2 µM RKRCLRRL was non-inhibitory when it was added directly to the histone kinase assay mixtures (Table 1). Following preincubation of the catalytic-domain fragment with RKRCLRRL, a similar degree of inhibition was observed in the histone kinase assay across a broad range of final RKRCLRRL concentrations (0.2-2.0 µM) (Table 1). These data show that RKRCLRRL inactivates PKC by forming an irreversible complex with the catalytic domain of the enzyme.



The structural similarity between RKRCLRRL and the PKC substrate RKRTLRRL (K(m) = 20 µM) (15) strongly suggests that the PKC inactivator RKRCLRRL is active site-directed. To test this, we examined the ability of PKC substrates to protect the catalytic-domain fragment of PKC against inactivation by RKRCLRRL(12) . The catalytic-domain fragment was preincubated with 6 µM RKRCLRRL as described in the legend to Table 1, except that various PKC substrates and substrate analogs were included in the preincubation mixture. These experiments were designed so that following dilution of the preincubation mixture into histone kinase assay mixtures, the substrate Mg-[-P]ATP was present in the reaction mixtures at its standard concentration (6 µM [-P]ATP), and the peptide-substrate analogs, FARKGALRQ and RKRALRRL, were present at a concentration (10 µM) where they had no inhibitory activity against the histone kinase reaction(9) . Table 2shows that Mg-[-P]ATP, Mg-[-P]ATP plus FARKGALRQ, and Mg-[-P]ATP plus RKRALRRL each afforded protection against inactivation of the catalytic-domain fragment of PKC by RKRCLRRL. The protection of the catalytic-domain fragment of PKC against RKRCLRRL that was afforded by Mg-[-P]ATP alone and in the presence of defined peptide-substrate analogs provides evidence that RKRCLRRL is an active site-directed inactivator of PKC. As another test of whether the peptide operates as an active site-directed PKC inactivator, we examined the ability of the peptide to inactivate casein kinase II. Casein kinase II differs from PKC in that its substrate recognition motif does not include basic residues but instead requires acidic residues. In assays of the phosphorylation of casein by casein kinase II performed under conditions described in (25) , we found that RKRCLRRL did not inhibit casein kinase II activity at peptide concentrations up to and including 100 µM.



To identify structural features of RKRCLRRL that play important or essential roles in its DTT-sensitive inactivation of PKC, we examined the abilities of RKRCLRRL analogs to inactivate PKC and its catalytic-domain fragment in a DTT-sensitive manner. Among the peptides studied, RKRCLRRL was the most potent inactivator of PKC and its catalytic-domain fragment (Table 3). A truncated analog lacking the C-terminal Leu residue (RKRCLRR) was also a potent inactivator of PKC and its catalytic-domain fragment (Table 3). However, analogs lacking a single basic residue (KRCLRRL and RKRCLR) either failed to inactivate PKC and its catalytic-domain fragment or did so only very weakly, and KRCLR, which lacked two of the basic residues of the parent peptide, was inactive (Table 3). These results indicate the critical importance of the basic residues of RKRCLRRL in its DTT-sensitive inactivation of PKC. The failure of KRCLR to inactivate PKC and its catalytic-domain fragment could be due to weak reactivity of the Cys residue as a consequence of the limited number of basic residues in the peptide compared to RKRCLRRL (16) and/or to a weak affinity for the active-site of the enzyme compared to RKRCLRRL, since the closely related peptides RKRTLRRL and KRTLR serve as PKC substrates with K(m) values of 20 and 300 µM, respectively(15, 26) . The affinity of the N-myristoylated analog NmKRCLR at the active-site of PKC can be expected to be superior to that of KRCLR, because the related peptide NmKRTLR reversibly inhibits the histone kinase activity of the catalytic-domain fragment of PKC (IC = 200 µM), whereas KRTLR does not(27) . Therefore, we analyzed the effects of NmKRCLR on the histone kinase activity of the catalytic-domain fragment of PKC, in order to determine whether the sequence KRCLR was sufficiently basic to provide the Cys residue with DTT-sensitive reactivity at the active-site of the enzyme. NmKRCLR effected 50% antagonism of the histone kinase activity of the catalytic-domain fragment at a concentration of 8 ± 2 µM in the absence of DTT and at a concentration of 27 ± 3 µM in the presence of 0.5 mM DTT. At 20 µM NmKRCLR, the loss of catalytic-domain fragment activity was 95 ± 9% in the absence of DTT and 33 ± 2% in its presence. These results provide evidence that NmKRCLR antagonizes the catalytic-domain fragment of PKC by a mixed mechanism that includes reversible and irreversible components. Thus, three flanking basic residues appear sufficient to endow the Cys residue of the peptide-substrate analog with the ability to undergo a reaction with the catalytic domain of PKC that irreversibly inactivates the enzyme in a DTT-sensitive manner. Table 3shows that substitution of Cys with its D-isomer in RKRCLRRL weakened the peptide in the inactivation of PKC and its catalytic-domain fragment, indicating a degree of stereoselectivity in the inactivation of the enzyme by the peptide.



The studies described above were carried out using a purified preparation of a PKC isozyme mixture that contained PKC-alpha, PKC-beta, and PKC-. These isozymes have closely related peptide and protein substrate specificities(28, 29) . Using preparations of the purified isozymes, we determined that RKRCLRRL effected DTT-sensitive inactivation of each isozyme with comparable efficacy. 50% inactivation of PKC-alpha, PKC-beta, and PKC- was achieved at RKRCLRRL concentrations of 11 ± 2, 6 ± 2, and 4 ± 1 µM, respectively, and in each case, inactivation was quenched by 0.5 mM DTT.


DISCUSSION

In this report, we present data in support of a mechanism of PKC inactivation by the peptide-substrate analog RKRCLRRL that most likely involves the formation of a covalent complex between the active site of the enzyme and the inhibitor peptide. Our evidence for this is as follows. 1) RKRCLRRL antagonized the histone kinase activity of PKC and its catalytic-domain fragment with similar efficacies, and in both cases activity was fully restored by 0.5 mM DTT. In addition, analogs of RKRCLRRL with single amino acid substitutions at Cys (RKRALRRL and RKRTLRRL) were non-inhibitory against the histone kinase activity of PKC and the catalytic-domain fragment. The unique ability of RKRCLRRL compared with other RKRXLRRL peptides to inactivate PKC provides evidence that the high reactivity of the Cys residue in the basic peptide environment compensates for the weak binding affinity of the peptide at the active site in the inactivation mechanism. 2) The inactivated complex of catalytic-domain fragment and RKRCLRRL was stable upon dilution. 3) Substrates and substrate analogs protected the catalytic-domain fragment of PKC against inactivation by RKRCLRRL. 4) The isozymes PKC-alpha, PKC-beta, and PKC- each have a Cys residue that is homologous to a highly reactive active-site Cys residue in the cAMP-dependent protein kinase(13) . RKRCLRRL inactivated PKC-alpha, PKC-beta, and PKC- in a DTT-sensitive manner with similar efficacies. This is the first report of irreversible inactivation of PKC by an active site-directed peptide.

S-Thiolation is the formation of mixed disulfides between proteins and low molecular weight thiols; this reaction has been observed between endogenous low molecular weight thiols and several cellular proteins, including glyceraldehyde-3-phosphate dehydrogenase and creatine kinase(30, 31) . PKC appears to be a good candidate for S-thiolation, because a highly reactive active-site Cys residue present in cAMP-dependent protein kinase is conserved in PKC isozymes(13) . In addition, the peptide-substrate analog RKRCLRRL appears to be a good candidate for an S-thiolation reaction at the active-site of PKC, because the reactivity of Cys residues has been shown to be increased by as much as 10^6-fold by flanking basic residues, in studies of disulfide-exchange reactions (16) . DTT reversal of the inactivation of PKC by RKRCLRRL provides evidence that the enzyme and synthetic peptide are linked in the inactivated complex by a disulfide bond. This supports an inactivation mechanism that entails S-thiolation of the active-site of PKC by the peptide. In view of these results, the possibility that cellular proteins subject to regulation by S-thiolation in vivo(30, 31) may include PKC and perhaps other protein kinases deserves attention.

Attempts to analyze the kinetics of PKC inactivation by RKRCLRRL revealed an extremely rapid reaction that was complete within several seconds, even at 4 °C. (^2)Thus, successful analysis of the kinetics of PKC inactivation by RKRCLRRL will require the development of an applicable continuous assay of PKC activity, e.g. an assay system based on spectral or fluorescent changes associated with product formation. However, even in the absence of kinetic analysis, it is clear that RKRCLRRL, which achieves >50% inactivation of PKC and its catalytic-domain fragment at a concentration of 10 µM ( Fig. 1and Fig. 2), is a highly potent active site-directed peptidic inactivator of PKC. In addition, the K(m) of the closely related peptide RKRTLRRL (K(m) = 20 µM) (15) provides an estimate of the affinity of RKRCLRRL for the active-site of PKC. Recently, peptide-substrate analogs of cAMP-dependent protein kinase that contain an intrachain disulfide bond, e.g. Leu-Arg-Arg-Ala-Cys = Cys-Gly, were shown to affinity label the enzyme by undergoing a disulfide-exchange reaction at its active site(12) . This report shows that the presence of a single cysteine residue at the phospho-acceptor position of a synthetic peptide-substrate analog containing multiple basic residues may result in novel active-site affinity labels of PKC and perhaps other protein kinases that phosphorylate highly basic sequences.


FOOTNOTES

*
This work was supported by Grant G-1141 from The Robert A. Welch Foundation, National Institutes of Health Award CA52460, and an award from the Sid W. Richardson Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 713-792-7969; Fax: 713-792-8747.

(^1)
The abbreviations used are: PKC, protein kinase C; DTT, dithiothreitol; FARKGALRQ, Phe-Ala-Arg-Lys-Gly-Ala-Leu-Arg-Gln; Nm, N-myristoyl; PKC, protein kinase C; PS, phosphatidylserine; RKRCLRRL, Arg-Lys-Arg-Cys-Leu-Arg-Arg-Leu.

(^2)
N. E. Ward and C. A. O'Brian, unpublished observations.


ACKNOWLEDGEMENTS

We thank Patherine Greenwood for expert preparation of the manuscript.


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