Research Article

Identification of an Inactivating Cysteine Switch in Protein Kinase CE, a Rational Target for the Design of Protein Kinase CE–Inhibitory Cancer Therapeutics

Feng Chu,1 John M. Koomen,2 Ryuji Kobayashi,2 and Catherine A. O’Brian1

Departments of 1Cancer Biology and 2Molecular Pathology, The University of Texas M.D. Anderson Cancer Center, Houston, Texas

Abstract sion by selectively inhibiting the protein-tyrosine kinase activity of Critical roles played by some protein kinases in neoplastic BCR-ABL (1). In addition, gefitinib (Iressa) selectively inhibits the transformation and progression provide a rationale for devel- protein-tyrosine kinase activity of the epidermal growth factor oping selective, small-molecule kinase inhibitors as antineo- receptor (EGFR) and is effective against a small fraction of non– plastic drugs. Protein kinase CE (PKCE) is a rational target small cell lung cancers that express various somatic EGFR muta- for cancer therapy, because it is oncogenic and prometastatic tions (3). Both imatinib and gefitinib inhibit their protein kinase in transgenic mouse models. PKCE is activated by sn-1,2- targets by competing with the substrate ATP (1, 2). Similarly, selec- diacylglycerol (DAG). Attempts to develop selective PKCE tive inhibitors have been identified for other protein kinases in inhibitors that block activation by DAG or compete with ATP recent years by screening libraries of chemicals designed to compete have not yet met with success, suggesting a need for new with ATP (5, 6). However, this heavily mined approach has failed to identify selective inhibitors of some protein kinases that are strategies. We previously reported that cystamine and a meta- q q bolic cystine precursor inactivate PKCE in cells in a thiol- rational targets for cancer therapy, such as protein kinase C (PKC ). PKC is a family of 10 isozymes in the AGC group of serine/ reversible manner. In this report, we first determined that q PKCE became resistant to inactivation by disulfides when threonine protein kinases (7, 8). PKC is a rational target for cancer Cys452 was replaced with alanine by site-specific mutagenesis therapy, based on its oncogenic activity in fibroblasts and epithelial of human PKCE or a constitutively active PKCE mutant. These cells (9–11) and prometastatic activity in transgenic mouse models E of chemical carcinogenesis (12, 13). ATP-competitive inhibitors results showed that the disulfides inactivated PKC by thiol- q disulfide exchange, either upon Cys452 S-thiolation or by re- that have been identified for PKC are nonselective; this strategy arrangement to an intra-protein disulfide. Mass spectrometric has also failed to yield selective inhibitors for most of the other PKC isozymes (8, 14). Similarly, allosteric binding site targeting analysis of peptide digests of cystamine-inactivated, carbami- q q domethylated PKCE detected a peptide S-cysteaminylated at has not yielded selective PKC inhibitors. PKC and seven other 452 452 PKC isozymes are activated by phosphatidylserine-dependent Cys , indicating that Cys S-cysteaminylation is a stable 2+ modification. Furthermore, PKCE inactivation by N-ethyl- binding of sn-1,2-diacylglycerol (DAG) to cysteine-rich Zn fingers 452 in the regulatory domain (8). Efforts to develop selective inhibitors maleimide was Cys dependent, providing corroborative evidence that PKCE inhibitors can be designed by targeting of PKC isozymes by targeting the DAG-binding site have not Cys452 with small molecules that stably modify the residue. met with success, and enthusiasm for this strategy has waned as Cys452 is an active site residue that is conserved in only 11 other signaling protein families with homologous DAG-binding human protein kinase genes. Therefore, the PKCE-inactivating sites have been identified (e.g., RAS-GRPs and PKDs; refs. 15, 16). Cys452 switch is a rational target for the design of antineo- In recent years, we have investigated regulatory responses of plastic drugs that selectively inhibit PKCE. (Cancer Res 2005; PKC isozymes to cysteine modification by cystamine and other 65(22): 10478-85) disulfides (17–20). We initially analyzed purified human recombi- nant PKC isozymes and found that structurally diverse disulfides activated PKCy, which is tumor suppressive (21), and inactivated Introduction PKCq,PKCa, and other PKC isozymes to various extents (18, 19). Protein kinases play influential roles in aberrant cell signaling Similarly, treatment of cells with cystamine or a metabolic cystine events involved in neoplastic transformation and malignant prog- precursor activated PKCy and inactivated PKCq in a concentration- ression of epithelial and other types of cells. This is widely recog- dependent and thiol-reversible manner (17, 18). These results iden- nized as a strong rationale for developing selective, small-molecule tified covalent modification of one or more cysteine residues by inhibitors of protein kinases as antineoplastic drugs (1–4). This thiol-disulfide exchange as a novel mode of PKCq inactivation. approach was recently validated as a modality of antineoplastic In this report, we establish that Cys452 is a cysteine switch in therapy by the efficacy of imatinib (Gleevec) against chronic mye- human PKCq (hPKCq) that inactivates the kinase in cells upon q loid leukemia (CML; ref. 1). Imatinib treatment induces CML remis- modification by disulfides or N-ethylmaleimide (NEM). Cys452PKC is an active site residue (22) that is conserved in only 11 of the >400 genes that encode human protein kinases. Further narrowing the q Note: DNA sequence analysis and peptide synthesis were done at M.D. Anderson field, PKCy conserves Cys452PKC but is resistant to inactivation DNA Analysis and Peptide Synthesis Facilities, which are supported in part by NIH by cysteine modification (17–20). Therefore, the design of small- core grant CA16672. Requests for reprints: Catherine A. O’Brian, Department of Cancer Biology, M.D. molecule inhibitors that bind in the active site cavity in an Anderson Cancer Center, 1515 Holcombe Boulevard, Box 173, Houston, TX 77030. orientation favoring reaction with the side chain of Cys452 may Phone: 713-792-7969; E-mail: [email protected]. I2005 American Association for Cancer Research. offer a new avenue for the development of antineoplastic drugs doi:10.1158/0008-5472.CAN-05-1989 that selectively inhibit PKCq.

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Materials and Methods Western blot analysis of FLAG-immunoprecipitated PKCq samples was done using a PKCq mAb from BD Biosciences (San Jose, CA) to detect Cell culture and reagents. COS-7 cells obtained from the American the wt/mut hPKCq species and a PKCq mAb (sc-214) from Santa Cruz Type Culture Collection (Manassas, VA) were maintained in DMEM Biotechnology (Santa Cruz, CA) to detect the mPKCq-CAT species. supplemented with 10% fetal bovine serum and penicillin-streptomycin Enhanced chemiluminescence (Amersham Biosciences, Piscataway, NJ) in humidified air with 5% CO at 37jC. The media, antibiotics, and serum 2 was employed as the detection system. were from Invitrogen (Carlsbad, CA). Protein kinase CE inactivation by N-ethylmaleimide. To measure Plasmids and mutagenesis. Murine PKCq (mPKCq) catalytic domain Cys452-dependent PKCq inactivation in NEM-treated cell lysates, wt hPKCq cDNA encoding a constitutively active PKCq mutant (11) and hPKCq cDNA and C452A hPKCq transfectants were prepared and lysed as described were provided by Dr. I. Bernard Weinstein (Columbia University, New York) in Cellular PKCq Inactivation by Disulfides, except that the cells were and Dr. A.C. Newton (University of California-San Diego), respectively. harvested with Buffer B [0.65 mL/plate; 50 mmol/L HEPES (pH 7.0), Sequence analysis confirmed that the cDNAs encoded the catalytic domain 1 mmol/L EDTA, 1 mmol/L EGTA, 10 Ag/mL leupeptin, 250 Amol/L PMSF, of mPKCq (residues 395-737; Genbank accession no. P16054) and hPKCq 1 mmol/L Na VO , 20 mmol/L NaF, 10 nmol/L microcystin]. The cell (residues 2-737; Genbank accession no. Q02156). 3 4 lysates were briefly centrifuged, assayed for protein content, and nor- To generate cDNAs encoding site-specific mutants of mPKCq and hPKCq malized to 1 mg/mL protein. Next, the lysates were treated with NEM with alanine substituted for cysteine, we used the PCR-based, site-directed for 10 minutes at 30jC; the alkylation reaction was terminated by adding mutagenesis kit QuikChange (Stratagene, La Jolla, CA). pcDNA3-PKCq 2 mmol/L DTT (final concentration) to scavenge unreacted NEM. NEM- expression plasmids served as template DNA and contained the coding induced hPKCq inactivation was measured by immunoprecipitating hPKCq sequence for hPKCq with an NH2-terminal FLAG-epitope tag or the coding sequence for the mPKCq catalytic domain with a COOH-terminal FLAG (wt/mut) with FLAG M2 mAb and analyzing the immunoprecipitated q tag (mPKCq-CAT). Oligonucleotides were commercially synthesized and isozyme as described in Cellular PKC Inactivation by Disulfides, except j purified (Sigma-Genosys, The Woodlands, TX). Mutagenic primers were that the 15-minute incubation at 30 C was omitted. 452 q employed in conjunction with complementary primers to generate To measure Cys -dependent PKC inactivation in NEM-treated cells, q site-specific PKCq mutants. The following are mutagenic primers for wt and C452A hPKC transfectants were cultured in DMEM supplemented j hPKCq:5V-GATGATGACGTGGACGCCACAATGACAGAG-3V for C452A with 10% serum for 16 to 20 hours at 37 C and treated with NEM for j hPKCq generation, 5V-CCTTACCCAACTCTACGCCTGCTTCCAGACCAAG-3V 20 minutes at 37 C. To terminate NEM treatment, the cells were rinsed j for C474A hPKCq,5V-CCCAACTCTACTGCGCCTTCCAGACCAAGGAC-3V for with PBS/2 mmol/L DTT, harvested in Buffer B/2 mmol/L DTT at 4 C, and q C475A hPKCq,5V-GATGCAGAAGGTCACGCCAAGCTGGCTG-3V for C546A lysed. hPKC was extracted from the cell lysates by FLAG immunoprecip- hPKCq,5V-GCTGACTTCGGGATGGCCAAGGAAGGGATTCTG-3 for C554A itation and analyzed for NEM-induced inactivation by the methods used hPKCq,5V-GACCACCACGTTCGCTGGGACTCCTGAC-3V for C568A hPKCq, in the analysis of NEM-treated cell lysates. and 5V-CACAAGCGCCTGGGCGCTGTGGCATCGCAG-3V for C652A hPKCq. Disulfide inactivation of the catalytic domain fragment of human E Introduction of the desired mutation unaccompanied by any fortuitous protein kinase C . To prepare a constitutively active, catalytic domain q q q mutation was established for each site-specific PKCq mutant cDNA by fragment of hPKC (hPKC -CDF), hPKC sulfhydryls were refreshed as q sequencing the full-length cDNAs. previously described by incubating purified recombinant hPKC (5 Ag; j Transfection. COS-7 cells (1 Â 106 per 10-cm dish) were expanded for Invitrogen) with DTT for 30 minutes at 4 C followed by removal of DTT q 48 hours, switched to serum-free media for 30 minutes, and transfected by gel filtration (19). Half of the hPKC sample was incubated with 1 Ag/mL j with the designated pcDNA3-PKCq expression-plasmid or pcDNA3 (empty trypsin (12.7 BAEE units/Ag; Sigma) for 5 minutes at 30 C, and the other vector) for 5 hours at 37jC using LipofectAMINE PLUS (Invitrogen). half was incubated similarly but without trypsin; trypsinolysis was Cellular protein kinase CE inactivation by disulfides. After incubation terminated with 1 mmol/L PMSF. q q in sulfur amino acid–free DMEM supplemented with 10% dialyzed serum To measure cystine-induced hPKC -CDF inactivation, the hPKC -CDF q q (Invitrogen) for 16 to 20 hours after transfection at 37jC, COS-7 cells were and control hPKC preparations, each at 6 Ag hPKC /mL, were incubated treated with cystamine, cystine dimethyl ester (CDME) or disulfiram (Sigma, with 2 mmol/L cystine in 50 mmol/L Tris-HCl (pH 8.0), 200 mmol/L NaCl, St. Louis, MO) in the presence of 5% dialyzed serum for the time interval 1 mmol/L EDTA, and 1 mmol/L EGTA for 20 minutes at 30jC and further specified at 37jC (17, 18). The disulfides were not cytotoxic under the incubated with/without 30 mmol/L DTT for 10 minutes at 30jC. hPKCq- conditions employed in this report. PKCq was extracted from the cells after CDF and hPKCq were assayed as described in Cellular PKCq Inactivation disulfide treatment as previously described (17, 18). Briefly, the cells were by Disulfides, except that assays contained 50 Ag/mL histone III-S (Sigma) rinsed with PBS, harvested with Buffer A [20 mmol/L Tris-HCl (pH 7.5), as phosphoacceptor substrate and 60 ng hPKCq or hPKCq-CDF. 1 mmol/L EDTA, 1 mmol/L EGTA, 10 Ag/mL leupeptin, 250 Amol/L Statistical analysis. Statistical analysis of the results was done with the phenylmethylsulfonyl fluoride (PMSF), 1 mmol/L Na3VO4, 20 mmol/L NaF, Student’s t test using SigmaPlot software. 10 nmol/L microcystin], and lysed by sonication at 4jC. Lysates were Liquid chromatographic-tandem mass spectrometry. To identify the cleared by centrifugation, and protein concentrations measured. To structural modification at Cys452 in cystamine-inactivated PKCq by liquid immunoprecipitate the PKCq transgene product, 400 Ag cell lysate protein chromatographic-tandem mass spectrometry (LC-MS/MS), cystamine- (1.2-1.5 mg/mL) was incubated with 5 Ag FLAG M2 monoclonal antibody inactivated PKCq was prepared as follows. First, sulfhydryls of purified (mAb; Sigma) overnight at 4jC in IP buffer (17) and further incubated human recombinant PKCq (10 Ag; Invitrogen) were refreshed as described for 2 hours after the addition of protein A-Sepharose. Beads were washed in Disulfide Inactivation of hPKCq-CDF, and hPKCq was incubated with thrice with 1 mL IP buffer and resuspended in 20 mmol/L Tris-HCl (pH 7.5), 1 mmol/L cystamine in 50 mmol/L Tris-HCl (pH 8.0), 200 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA (17). 1 mmol/L EDTA, and 1 mmol/L EGTA for 30 minutes at 30jC (750 AL). This DTT-reversible PKCq inactivation was measured as previously described was immediately followed by the addition of 20 mmol/L iodoacetamide (17, 18). Briefly, each immunoprecipitated PKCq sample was incubated in (final concentration) and incubation in the dark for 30 minutes at 37jC. the absence and presence of DTT for 15 minutes at 30jC, and 25 AL Excess cystamine and iodoacetamide were removed from the hPKCq aliquots were added to PKCq assays (final volume, 120 AL). PKCq assays sample by gel filtration chromatography at 4jC using a foil-wrapped, 2 mL contained [g-32P] ATP and [Ser25]PKC 19–31 (Bachem Bioscience, King of G-25 Sephadex column equilibrated in 50 mmol/L Tris-HCl (pH 8.0). Prussia, PA) as substrates and phosphatidylserine and sn-1,2-dioleoylgly- An aliquot of the cystamine-modified, carbamidomethylated hPKCq sample cerol (DAG), each at 30 Ag/mL as lipid cofactors (17). DAG was omitted was digested by incubation with lysyl endopeptidase (Wako Chemicals, from mPKCq-CAT assays. Background activity was measured in control Richmond, VA) for 20 hours at 37jC (hPKCq/protease ratio = 5:1). A second assays that contained the PKC inhibitor GF109203X (1 Amol/L; Alexis hPKCq sample that was thermally denatured by incubation at 95jC for Laboratories, San Diego, CA). PKCq activity is calculated as total minus 7 minutes before digestion was also analyzed. Following digestion, the ex- background (17). tracted peptides were cleaned with C18 reverse-phase ZipTips (Millipore, www.aacrjournals.org 10479 Cancer Res 2005; 65: (22). November 15, 2005

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Billerica, MA) to remove glycerol and then concentrated by vacuum CDF by a DTT-reversible mechanism (Fig. 1, white columns) that centrifugation to remove the organic solvent. In preparation for LC-MS/MS resembled cystine-induced hPKCq inactivation (Fig. 1, black analysis, the peptides were desalted with a reverse-phase precolumn and columns). These results provide evidence that cystine inactivates a switching valve (Switchos, LC Packings/Dionex, Sunnyvale, CA). The hPKCq by undergoing thiol-disulfide exchange with one or more peptides were separated in the LC-MS/MS analysis using a C18PepMap100 A Â A ˚ cysteine residues in the catalytic domain. column (75 m inner diameter 15 cm, 3 m particle size, 100 A pore size; q q LC Packings/Dionex). The solvent system employed was (A) aqueous 2% hPKC and mPKC express identical cysteine residues and share acetonitrile with 0.01% trifluoroacetic acid (TFA) and (B) aqueous 80% 95% amino acid sequence identity. We previously reported that acetonitrile with 0.01% TFA. The gradient was ramped from 2% B to 40% B the cell-permeable disulfides cystamine and CDME induce DTT- over 10.5 minutes and from 40% B to 90% B over 2.5 minutes. Tandem reversible mPKCq inactivation in COS-7 cells (17). mPKCq-CAT is a mass spectra were acquired for two precursors from each MS1 scan (mass- constitutively active truncation mutant of mPKCq, which consists to-charge ratio, m/z 500-1,400) with an electrospray ion trap mass spec- of the catalytic domain and has a COOH-terminal FLAG-epitope trometer (LTQ, ThermoElectron, San Jose, CA) set at 3.2 kV (spray voltage) tag (11). To corroborate the evidence shown in Fig. 1 that disulfides and 25% normalized collision energy. Data were searched against the inactivate PKCq by covalently modifying cysteine residues in the SwissProt database using Mascot (http://www.matrixscience.com) and allowing for variable cysteine modifications (carbamidomethylation or catalytic domain, we investigated whether cystamine and CDME q cysteaminylation) and methionine oxidation as well as one missed cleav- could induce mPKC -CAT inactivation in COS-7 cells. COS-7 cells age per peptide. Sequence assignments were verified by direct inspection transfected with mPKCq-CAT were treated with the disulfides for of the tandem mass spectra. 30 minutes at 37jC, and mPKCq-CAT was immunoprecipitated from the cell lysates with FLAG mAb. Western analysis with a Results catalytic domain–directed PKCq mAb verified that the amount of q Cys452 is a disulfide-regulated, protein kinase CE–inactivating mPKC -CAT (43 kDa) recovered by FLAG-immunoprecipitation cysteine switch. hPKCq is an 84-kDa polypeptide comprised of an was not altered by disulfide treatment (Fig. 2). Each immunopre- q NH -terminal regulatory domain containing 17 cysteine residues, an cipitated mPKC -CAT sample was assayed after a 15-minute 2 j regulatory/catalytic domain–linking hinge region lacking cysteine, incubation at 30 C in the absence and presence of 30 mmol/L DTT. q and a COOH-terminal catalytic domain containing seven cysteine Cystamine induced >90% inactivation of mPKC -CAT in the cells in residues (8). We recently established that cystine inactivates hPKCq a concentration-dependent and DTT-reversible manner (Fig. 2A). 35 q by thiol-disulfide exchange in association with an [ S] S-cysteiny- In addition, 1 mmol/L CDME abrogated mPKC -CAT activity in lation stoichiometry of f1 mol cysteine/mol hPKCq (19). Based COS-7 cells, with measurable reversal of inactivation by DTT on the abrogation of hPKCq activity by this mechanism, we hypo- (Fig. 2B). thesized that cystine-induced hPKCq inactivation was triggered by The robust reversal of cystamine-induced mPKCq-CAT inactiva- S-cysteinylation of a critical cysteine residue in the vicinity of the tion by DTT (Fig. 2A) clearly indicated that covalent modification active site. of cysteine residues in mPKCq-CAT by thiol-disulfide exchange was To test the hypothesis, we initially investigated whether cystine a major component of the inactivation mechanism in COS-7 cells. could inactivate a constitutively active, tryptic hPKCq-CDF. The However, the reversal of inactivation by DTT was incomplete. Western blot analysis in Fig. 1 shows that limited trypsinolysis of Incomplete reversal could reflect the strength and stability of purified recombinant hPKCq (lane 1) produced an f50-kDa frag- inactivating disulfide bonds introduced into mPKCq-CAT by thiol- ment that was immunoreactive with a catalytic domain–directed disulfide exchange or an inhibitory mechanism independent of PKCq antibody (lane 2). The trypsinized hPKCq species was fully thiol-disulfide exchange. To evaluate whether an inhibitory active in the absence of the regulatory domain–binding cofactors mechanism other than thiol-disulfide exchange contributed to phosphatidylserine/DAG (Fig. 1, white columns), which stimulated the loss of mPKCq-CAT activity in the disulfide-treated cells, the hPKCq activity about 10-fold (black columns). This established that cells were also treated with cell-permeable sulfhydryl agents the trypsinized hPKCq preparation contained constitutively active structurally related to cystamine and cystine. Figure 2B shows hPKCq-CDF. that neither 1 mmol/L N-acetylcysteine nor 1 mmol/L cysteamine The analysis of disulfide-induced hPKCq-CDF inactivation in attenuated mPKCq-CAT activity, contrasting with the strong Fig. 1 shows that 2 mmol/L cystine profoundly inactivated hPKCq- inactivation achieved by the disulfides. Collectively, the results in

Figure 1. DTT-reversible inactivation of hPKCq-CDF by cystine. The ability of cystine to induce DTT-reversible inactivation of a constitutively active, tryptic hPKCq-CDF was analyzed. Left, hPKCq-CDF (white columns) and hPKCq (black columns) were incubated with/without 2 mmol/L cystine (20 minutes, 30jC), further incubated with/without 30 mmol/L DTT (10 minutes, 30jC), and assayed in the presence or absence of phosphatidylserine/DAG. Columns, mean PKCq activity of triplicate measurements; bars, SD. The activity of phosphatidylserine/DAG-activated hPKCq (1.3 pmol 32P transferred/min) is defined as 100% activity. Single asterisk, P < 0.01, statistically significant difference versus the 2nd and 10th columns (above 4th and 12th columns, respectively). Double asterisk, P < 0.01, significant difference versus the 4th and 12th columns (above the 6th and 14th columns, respectively). Right, Western analysis of hPKCq (90 kDa; lane 1) and hPKCq-CDF (50 kDa; lane 2) with the catalytic domain–directed PKCq polyclonal antibody sc-214 (Santa Cruz Biotechnology). Reproduced in an independent analysis.

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and the wt/mut hPKCq species were immunoprecipitated from the cell lysates with FLAG mAb. Western blot analysis with a PKCq mAb (BD Biosciences) verified that CDME treatment did not alter the amount of the wt/mut hPKCq species recovered by immuno- precipitation (Fig. 3). Each immunoprecipitated wt/mut hPKCq sample was assayed after incubation in the absence and presence of 10 mmol/L DTT. Both 5 and 10 mmol/L CDME induced >80% inactivation of wt hPKCq in COS-7 cells (Fig. 3, black columns), and exposure to DTT restored the original level of activity (Fig. 3, gray columns). Similarly, the five site-specific hPKCq mutants containing alanine substitutions at C474, C475, C546, C568, and C652 were profoundly inactivated by 5 and 10 mmol/L CDME in a DTT-reversible manner (Fig. 3). C554A hPKCq was also inactivated by 5 and 10 mmol/L CDME in a DTT-reversible manner, although it was not as sensi- tive as wt hPKCq. In contrast, C452A hPKCq was resistant to inactivation by 5 mmol/L CDME and modestly inactivated by 10 mmol/L CDME (black columns). Furthermore, C452A hPKCq was resistant to inactivation by disulfiram, a structurally dissim- ilar disulfide, at 100 and 200 Amol/L (45 minutes, 37jC). In con- trast, wt hPKCq and C554A hPKCq were inactivated by 100 and 200 Amol/L disulfiram in a concentration-dependent and DTT- reversible manner, with >80% inactivation of C554A hPKCq achieved at 200 Amol/L disulfiram (Fig. 4A). Collectively, the q results in Figs. 3 and 4A show a critical role for Cys452hPKC in the inactivation of cellular hPKCq by disulfides disparate in structure and potency. When C452A hPKCq was immunoprecipitated from lysates of disulfide-treated cells, its activity was increased by incubation with DTT (Figs. 3 and 4A). In an effort to eliminate this com- plication from our analysis, we generated the mutant C452A mPKCq-CAT, reasoning that elimination of the 17 regulatory domain-cysteine might abolish the effect. Figure 4B shows that C452A mPKCq-CAT was resistant to cystamine-induced inactiva- Figure 2. Inactivation of a constitutively active PKCq mutant in disulfide-treated tion in COS-7 cells, and that its activity was unaffected by expo- cells. mPKCq-CAT transfectants were treated for 30 minutes at 37jC with the specified disulfide or thiol reagent at the concentrations indicated (A)orat sure to DTT after immunoprecipitation from cystamine-treated 1.0 mmol/L (B) and lysed. mPKCq-CAT was immunoprecipitated from cell cells. Taken together with the analysis of hPKCq in Figs. 3 lysates with FLAG mAb, incubated with/without 30 mmol/L DTT, and assayed. q and 4A, these straightforward results establish the identity of Columns, mean mPKC -CAT activity of triplicate measurements; bars, SD. 452 q 100% activity = the activity of mPKCq-CAT immunoprecipitated from untreated Cys as a disulfide-regulated PKC -inactivating cysteine switch. cells and assayed without DTT exposure. A mock analysis conducted with an Stable conjugation of a small molecule at Cys452 inactivates empty vector transfectant. A, single asterisk, P < 0.01, significant difference protein kinase CE. The requirement for Cys452 and the dispens- versus the 100% activity value (3rd column); double asterisk, P < 0.01, significant DTT reversal of inactivation at 5 mmol/L cystamine (11th versus ability of other catalytic domain cysteine residues in the mech- 12th column). B, double asterisks, P < 0.01, significant difference between anism of disulfide-induced PKCq inactivation suggested that Cys452 the PKC activity values for CDME and NAC (5th versus 7th columns) and for cystamine and cysteamine (9th versus 11th columns). Western blot analysis S-thiolation (e.g., disulfide conjugation of cysteamine) was the of immunoprecipitated mPKCq-CAT, which migrated at 43 kDa, was done inactivating modification. This supports the notion that small with a catalytic domain–directed PKCq mAb (sc-1681). Reproduced in an modifying groups at Cys452 would be sufficient to abrogate PKCq independent analysis. activity. However, the possibility that inactivation required rearrangement of S-thiolation modification(s) to intra-protein di- Figs. 1 and 2 establish that disulfide inactivation of PKCq involves sulfides involving Cys452 was also consistent with the results. We covalent modification of one or more cysteine residues in the cata- therefore conducted mass spectrometric analysis of cystamine- lytic domain. inactivated hPKCq to identify the disulfide modification at Cys452. To determine if any of the cysteine residues in the catalytic Exposure of purified recombinant hPKCq to 1 mmol/L cystamine domain of PKCq can function as a disulfide-regulated, PKCq- for 30 minutes at 30jC, which induced 87 F 1% inactivation, was inactivating switch, we generated cDNAs encoding site-specific immediately followed by the addition of 20 mmol/L iodoacetamide hPKCq mutants with alanine substituted for cysteine that to prevent disulfide rearrangement in the cystamine-inactivated corresponded to each cysteine residue in the catalytic domain. hPKCq sample. The sample was then digested with lysyl endo- hPKCq (full length) and the seven derived site-specific mutants peptidase for LC-MS/MS analysis of the Cys452-containing peptide exhibited similar activity and expression levels in COS-7 cells and fragment. LC-MS/MS detected the peptide DVILQDDDVDCTMTEK migrated at 90 kDa in SDS PAGE (data not shown), validating (hPKCq 442-457) with an S-cysteaminyl modification at Cys452. The comparison of their responses to disulfide in the cells. For this pur- m/z ratios of the parent ion and the Cys452-containing fragments j pose, the cells were treated with CDME for 30 minutes at 37 C, in the y ion series (y6-y14) and the b ion series (b13 and b15) in the www.aacrjournals.org 10481 Cancer Res 2005; 65: (22). November 15, 2005

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2005 American Association for Cancer Research. Cancer Research tandem mass spectrum were consistent with S-cysteaminylation ments, cell lysates were treated with NEM for 10 minutes at 30jC; (+75 Da) at Cys452 and clearly distinguishable from carbamidome- 2 mmol/L DTT was added to terminate the alkylation reaction; thylation of the residue (+57 Da), because the database search and the wt/mut hPKCq species were extracted by immunopre- errors are limited to F2 Da for parent ions and F0.8 Da for frag- cipitation with FLAG mAb and assayed. NEM treatment had no ments (Fig. 5). In addition, the residue mass for S-cysteaminylated effect on the recovery of wt hPKCq and C452A hPKCq by immu- cysteine was observed to be the difference in m/z between the noprecipitation (Fig. 6A). NEM (0.2-1.0 mmol/L) potently inacti- 452 y6 and y5 fragment ions, consistent with the assignment of Cys as vated wt hPKCq in a concentration-dependent manner with a the modified amino acid (Fig. 5). S-cysteaminylated hPKCq 442-457 maximal effect of f80% inactivation (Fig. 6A, black columns), was similarly detected when cystamine-modified, carbamidome- whereas C452A hPKCq was resistant to inactivation by NEM thylated hPKCq was thermally denatured before digestion (data across this concentration range (white columns). not shown), establishing the stability of the disulfide modification. Cys452-dependent hPKCq inactivation in NEM-treated cell lysates These results provide structural evidence that cystamine inacti- supported the notion that Cys452 can be exploited as a target for vates hPKCq by introducing a stable S-cysteaminylation modifica- cancer therapeutics that inhibit hPKCq by permanent modification tion at Cys452. of the sulfhydryl. To test the principle that an alkylating drug could We next sought to corroborate the mass spectrometric evidence inactivate cellular hPKCq by a Cys452-dependent mechanism under that conjugation of a small modifying group at Cys452 inactivates homeostatic conditions, wt hPKCq and C452A hPKCq transfectants PKCq. We reasoned that one way to accomplish this would be to cultured in DMEM supplemented with 10% serum were treated show resistance of C452A hPKCq to inactivation by a small with NEM (1.25-2.0 mmol/L) for 20 minutes at 37jC, lysed in sulfhydryl-alkylating agent, because alkylation of Cys452 in wt PKCq HEPES buffer containing 2 mmol/L DTT to quench unreacted would be an inactivating modification that could not rearrange. NEM, immunoprecipitated, and assayed. Figure 6B shows that For this purpose, we investigated the effects of NEM on wt hPKCq C452A hPKCq was resistant to inactivation by 1.25 mmol/L NEM versus C452A hPKCq activity. (white columns), whereas wt hPKCq was inactivated >70% (black We first examined the effects of treating COS-7 cell lysates with columns). These results indicate the availability of Cys452 in hPKCq NEM on wt hPKCq and C452A hPKCq activity. In these experi- for targeting by alkylating drugs in the cellular milieu.

Figure 3. Resistance of C452A hPKCq to inactivation in disulfide-treated cells. COS-7 cells transfected with wt or mut hPKCq were treated with CDME for 30 minutes at 37jC and lysed. hPKCq species (wt/mut) were immunoprecipitated from the lysates with FLAG mAb, incubated for 15 minutes at 30jC without (black columns) or with 10 mmol/L DTT (gray columns), and assayed. Columns, mean PKCq activity of triplicate measurements; bars, SD. 100% activity = the activity of the hPKCq species (wt or mut) immunoprecipitated from untreated cells and assayed without DTT exposure. Single asterisks, P < 0.01, significant inactivation versus the 100% control value. Double asterisks, P < 0.01, significant resistance of C452A hPKCq to inactivation, based on the comparison of equivalent columns in the wt and C452A hPKCq. Western blot analysis of immunoprecipitated hPKCq was done with BD Biosciences PKCq mAb; wt and mut hPKCe species comigrated at 90 kDa. Reproduced in an independent analysis.

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Figure 4. A, resistance of C452A hPKCq to inactivation in disulfiram-treated cells. The effects of disulfiram treatment on the activity of wt hPKCq, C452A hPKCq, and C554A hPKCq were ascertained as described in Fig. 3 legend, except that the cells were treated with 100 and 200 Amol/L disulfiram for 45 minutes at 37jC. Reproduced in an independent analysis. B, resistance of C452A mPKCq-CAT to inactivation in disulfide-treated cells. The effects of cystamine treatment (30 minutes, 37jC) on the activity of mPKCq-CAT and C452A mPKCq-CAT in COS-7 cells were compared. Western blot analysis was done as described in Fig. 2 legend; nonmutated and C452A mPKCq-CAT comigrated at 43 kDa. Single asterisks, P < 0.01, significant inactivation versus the 100% control value. Double asterisks, P < 0.01, significant resistance of the C452A mutant versus PKCq to inactivation, based on the comparison of equivalent columns in the C452A mutant versus wt hPKCq (A)or mPKCq-CAT (B). For other details, see Fig. 3 legend. Reproduced in an independent analysis.

Eleven human protein kinase genes conserve protein kinase The significance of this to pharmacologic targeting of PKCq is CE residue Cys452. The recent elucidation of the crystal structure that it offers active site binding as a strategy to selectively tar- q of the PKCu catalytic domain has established that the side chain get Cys452hPKC versus cysteine residues on the surfaces of other q of Cys452hPKC is in the active site cavity of PKC isozymes (22). proteins or in binding pockets other than protein kinase active

Figure 5. Tandem mass spectrum of hPKCq 442-457 S-cysteaminylated at Cys452. Purified recombinant hPKCq was inactivated by cystamine, carbamidomethylated, and digested with lysyl endopeptidase. Arrow, y-type fragment ions separated by the residue mass corresponding to S-cysteaminylated cysteine. Reproduced in multiple experiments. In addition, this modified peptide was also observed in tryptic digests and even when the protein was thermally denatured before enzymatic digestion.

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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2005 American Association for Cancer Research. Cancer Research sites. To assess whether Cys452 targeting could serve as a strategy to selectively inactivate PKCq versus other human protein kinases, we searched the human protein kinase sequence database for pro- q tein kinases that conserve Cys452hPKC . The proximity of Cys452 to Glu456 in the sequence of hPKCq pro- q vided a rational basis to investigate the conservation of Cys452hPKC q in human protein kinases, because Glu456hPKC is nearly invari- q ant in human protein kinases (23). Furthermore, Cys452hPKC and q Glu456hPKC both occur in subdomain III, which is one of the 12 conserved catalytic domain subdomains in human protein kinases that fold into a common catalytic core (23). Using a comprehen- sive database of aligned human subdomain III sequences (7), we eliminated sequences corresponding to catalytically dead kinases and analyzed the remaining 443 sequences for CXXXE, where E q denotes E456hPKC . The search revealed that conservation of q Cys452hPKC is restricted to PKCq and 10 other genes that encode q human protein kinases. The genes conserving Cys452hPKC encode DAG-responsive PKC isozymes (PKCa,PKCh,PKCg,PKCy,PKCq, PKCu,PKCD), myotonic dystrophy protein kinase (DMPK), and myotonic dystrophy kinase-related Cdc42-binding protein kinase (MRCK) isozymes (MRCKa, MRCKh, and MRCKg; ref. 7). The rare q conservation of C452hPKC in protein kinases and its residence in the active-site cavity may allow the design of a new class of antineoplastic drugs that selectively inhibit PKCq by binding at the active site and permanently modifying the side chain of Cys452.

Discussion In this report, we show that Cys452 is a PKCq-inactivating cysteine switch that abrogates PKCq activity when small molecules are conjugated to its side chain, whether by disulfide linkage (i.e., S-cysteaminylation) or alkylation (i.e., modification by NEM). The crystal structure of PKCu, which is the only PKC isozyme with a solved three-dimensional catalytic domain structure, places the q side chain of Cys452hPKC in the active site cavity of PKC isozymes Figure 6. Resistance of C452A hPKCq to inactivation by N-ethylmaleimide. q (22). This suggests a rational strategy for the development of A, analysis of hPKC inactivation in NEM-treated cell lysates. Cell lysates of wt and C452A hPKCq transfectants were treated with NEM for 10 minutes at antineoplastic drugs that selectively inhibit PKCq by permanent 30jC; alkylation was terminated with 2 mmol/L DTT. hPKCq (wt/mut) was modification of Cys452. First, selectivity for protein kinases versus FLAG immunoprecipitated and assayed; 100% activity = the activity of hPKCq (wt or mut) immunoprecipitated from untreated cell lysates. For other details, other classes of proteins could be achieved with agents that bind see Fig. 3 legend. B, analysis of hPKCq inactivation in NEM-treated cells. protein kinase active sites. Second, selectivity for the limited wt and C452A hPKCq transfectants cultured in DMEM supplemented with q number of protein kinases that conserve Cys452hPKC could be 10% serum were treated with NEM for 20 minutes at 37jC and lysed in HEPES buffer containing 2 mmol/L DTT. hPKCq (wt/mut) was immunoprecipitated achieved with agents designed to bind the active site with an and analyzed as described in (A). Single asterisks, P < 0.01, significant affinity below the threshold for inhibition by reversible binding, in inactivation versus the 100% control value. Double asterisks, P < 0.01, q q an orientation that points the reactive group towards the significant resistance of C452A hPKC to inactivation compared with wt hPKC . q Reproduced in an independent analysis. nucleophilic side chain of Cys452hPKC and facilitates inactivation by permanent modification of the sulfhydryl. Third, with respect to q the issue of specificity for PKCq versus other PKC isozymes, PKC~ (22, 23). Cys452hPKC is a component of helix aC, which forms q and PKCL do not conserve Cys452hPKC , and PKCy conserves the another surface in the architecture of protein kinase active sites. residue but is resistant to inactivation by cysteine modification Helix aC faces the activation loop and is crucial for the proper (17–20). Thus, at most, six PKC isozymes other than PKCq could be positioning of key catalytic residues in protein kinase active sites q inactivated by a Cys452hPKC -targeting mechanism. Furthermore, (22, 23). Thus, drugs designed to bind the active site of PKCq in an q distinctions between some of these isozymes and PKCq are already orientation that points a reactive group towards Cys452hPKC would q evident; for example, PKCq is more sensitive than PKCa to not be positioned for reaction with Cys568hPKC and vice versa. This inactivation by cystine but less sensitive than PKCa to inactivation supports the notion that selective PKCq inhibitors may be designed q by disulfiram (18, 19). by targeting Cys452hPKC . Our results distinguish the mechanism of PKCq inactivation by The involvement of protein S-glutathionylation in the regula- disulfide modification from cyclic AMP-dependent protein kinase tion of signaling and metabolic pathways is well established (PKA) inactivation by S-glutathionylation, because the latter is (reviewed in ref. 25). For example, angiotensin II activates Ras by mediated by Cys199PKA (24), which is homologous to Cys568 in PKCq. inducing S-glutathionylation of the small GTPase in smooth muscle q Cys568hPKC is located in the activation loop, which functions as cells (26), and ischemia-reperfusion inactivates glyceraldehyde- a peptide substrate binding surface in protein kinase active sites 3-phosphate dehydrogenase in isolated rat hearts by inducing

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S-glutathionylation of this critical glycolytic enzyme (27, 28). gGCS activity level to that of wt mice (29). Thus, the finding in Interestingly, recent studies suggest that S-cysteaminylation may this report that Cys452 S-cysteaminylation profoundly inactivates also regulate protein function in vivo. PKCq raises the intriguing question of whether PKCq is regulated Cysteamine is produced as a byproduct of pantothenic acid by cystamine in vivo. recycling in a broad spectrum of mammalian tissues (29, 30), where it partially converts to cystamine (31). Recent findings in a Acknowledgments pantetheinase knockout mouse model, which was characterized Received 6/7/2005; revised 7/28/2005; accepted 8/16/2005. by tissues deficient in cysteamine/cystamine, support the notion Grant support: NIH grant RO1 CA108534, Robert A. Welch Foundation grant G- that S-cysteaminylation regulates protein function under homeo- 1141, State of Texas Tobacco Settlement Funds (C.A. O’Brian) and NIH core grant g CA16672 (M.D. Anderson Cancer Center). static conditions in vivo (29). For example, -glutamylcysteine The costs of publication of this article were defrayed in part by the payment of page synthetase (gGCS) is inactivated by thiol-disulfide exchange with charges. This article must therefore be hereby marked advertisement in accordance cystamine (32). In pantetheinase knockout mice, the gGCS activity with 18 U.S.C. Section 1734 solely to indicate this fact. We thank Dr. I. Bernard Weinstein for providing the constitutively active mPKCq- level of the cysteamine/cystamine-deficient hepatic tissue is CAT construct and Dr. Jordan U. Gutterman (M.D. Anderson Cancer Center) for elevated, and oral administration of cystamine attenuated the constructive discussions.

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