Anthrax Lethal Factor Proteolysis and Inactivation of MAPK Kinase

Anthrax lethal toxin produced by the bacteriumBacillus anthracis is the major cause of death in animals infected with anthrax. One component of this toxin, lethal factor (LF), inactivates members of the mitogen-activated protein kinase kinase or MEK family through proteolysis of their NH2 termini. However, neither the substrate requirements for LF cleavage nor the mechanism by which proteolysis inactivates MEK have been demonstrated. By means of deletion mutant analysis and site-directed mutagenesis, we have identified an LFIR (LFinteracting region) in the COOH-terminal kinase domain of MEK1 adjacent to the proline-rich region, which is essential for LF-mediated proteolysis of MEK. Point mutations in this region block proteolysis but do not alter the kinase activity of MEK. Similar mutations in MEK6 also prevent proteolysis, indicating that this region is functionally conserved among MEKs. In addition, NH2-terminal proteolysis of MEK1 by LF was found to reduce not only the affinity of MEK1 for its substrate mitogen-activated protein kinase but also its intrinsic kinase activity, indicating that the NH2-terminal end of MEK is important not only for substrate interaction but also for catalytic activity. Anthrax lethal toxin produced by the bacteriumBacillus anthracis is the major cause of death in animals infected with anthrax. One component of this toxin, lethal factor (LF), inactivates members of the mitogen-activated protein kinase kinase or MEK family through proteolysis of their NH2 termini. However, neither the substrate requirements for LF cleavage nor the mechanism by which proteolysis inactivates MEK have been demonstrated. By means of deletion mutant analysis and site-directed mutagenesis, we have identified an LFIR (LFinteracting region) in the COOH-terminal kinase domain of MEK1 adjacent to the proline-rich region, which is essential for LF-mediated proteolysis of MEK. Point mutations in this region block proteolysis but do not alter the kinase activity of MEK. Similar mutations in MEK6 also prevent proteolysis, indicating that this region is functionally conserved among MEKs. In addition, NH2-terminal proteolysis of MEK1 by LF was found to reduce not only the affinity of MEK1 for its substrate mitogen-activated protein kinase but also its intrinsic kinase activity, indicating that the NH2-terminal end of MEK is important not only for substrate interaction but also for catalytic activity. The lethal effects of Bacillus anthracis have been attributed to an exotoxin, which it produces (1Smith H. Keppie J. Nature. 1954; 173: 869-870Google Scholar). This exotoxin is composed of three proteins: protective antigen (PA), 1The abbreviations used are: PA, protective antigen; ERK, extracellular signal-regulated kinase; LF, lethal factor; LFIR, LFinteractingregion; MAPK, mitogen-activated protein kinase; MEK, MAPK/extracellular signal-regulated kinase kinase; NT, NH2terminus; CD, common docking; MOPS, 4-morpholinepropanesulfonic acid; D, docking 1The abbreviations used are: PA, protective antigen; ERK, extracellular signal-regulated kinase; LF, lethal factor; LFIR, LFinteractingregion; MAPK, mitogen-activated protein kinase; MEK, MAPK/extracellular signal-regulated kinase kinase; NT, NH2terminus; CD, common docking; MOPS, 4-morpholinepropanesulfonic acid; D, docking edema factor, and lethal factor (LF) (for recent reviews see Refs. 2Mourez M. Lacy D.B. Cunningham K. Legmann R. Sellman B.R. Mogridge J. Collier R.J. Trends Microbiol. 2002; 10: 287-293Google Scholar and 3Bodart J.-F. Chopra A. Liang X. Duesbery N. Cell Cycle. 2002; 1: 10-15Google Scholar). PA binds to a cell surface receptor (4Bradley K.A. Mogridge J. Mourez M. Collier R.J. Young J.A. Nature. 2001; 414: 225-229Google Scholar) and, upon proteolytic activation to a 63-kDa fragment, heptamerizes to form a membrane channel that mediates the entry of three molecules of LF or edema factor into the cell (5Milne J.C. Furlong D. Hanna P.C. Wall J.S. Collier R.J. J. Biol. Chem. 1994; 269: 20607-20612Google Scholar, 6Gordon V.M. Leppla S.H. Hewlett E.L. Infect. Immun. 1988; 56: 1066-1069Google Scholar, 7Mogridge J. Cunningham K. Collier R.J. Biochemistry. 2002; 41: 1079-1082Google Scholar). Edema factor is an adenylate cyclase and, together with PA, forms a toxin referred to as edema toxin (8Leppla S.H. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 3162-3166Google Scholar). LF is a Zn2+-metalloprotease, which together with PA forms a toxin referred to as lethal toxin. Lethal toxin is the dominant virulence factor produced by B. anthracis and is the major cause of death in infected animals (9Pezard C. Berche P. Mock M. Infect. Immun. 1991; 59: 3472-3477Google Scholar). Although LF has been shown to cleave the NH2 termini of select members of the mitogen-activated protein kinase kinase or MEK family (10Duesbery N.S. Webb C.P. Leppla S.H. Gordon V.M. Klimpel K.R. Copeland T.D. Ahn N.G. Oskarsson M.K. Fukasawa K. Paull K.D. Vande Woude G.F. Science. 1998; 280: 734-737Google Scholar, 11Pellizzari R. Guidi-Rontani C. Vitale G. Mock M. Montecucco C. FEBS Lett. 1999; 462: 199-204Google Scholar, 12Vitale G. Bernardi L. Napolitani G. Mock M. Montecucco C. Biochem. J. 2000; 352: 739-745Google Scholar), the substrate requirements that determine LF specificity are unknown. Indirect evidence suggests that epitopes distal to the cleavage site are required for LF-MEK interaction. Yeast two-hybrid assays for binding partners of LF have isolated cDNA for MEK2, which lacks the NH2-terminal cleavage site (13Vitale G. Pellizzari R. Recchi C. Napolitani G. Mock M. Montecucco C. Biochem. Biophys. Res. Commun. 1998; 248: 706-711Google Scholar). Moreover, although it has been demonstrated that LF-cleaved MEK1 as well as recombinant MEK1, which lacks the seven NH2-terminal residues that are removed by LF, has reduced kinase activity (10Duesbery N.S. Webb C.P. Leppla S.H. Gordon V.M. Klimpel K.R. Copeland T.D. Ahn N.G. Oskarsson M.K. Fukasawa K. Paull K.D. Vande Woude G.F. Science. 1998; 280: 734-737Google Scholar), it is not clear how the absence of these residues alters MEK activity. Therefore, to identify regions distal to the cleavage site that are required for proteolysis, we have constructed a series of internal and COOH-terminal deletion mutants of MEK1 and have analyzed their cleavability by LF. The results reveal that a functionally conserved COOH-terminal region located adjacent to a proline-rich insert of MEK1 is essential for LF-mediated proteolysis of MEKs. In addition, to determine the mechanism by which LF inactivates MEKs, we have examined the kinase activity of MEK toward ERK at rate-limiting or saturating concentrations. Our results show that proteolysis by LF removes NH2-terminal epitopes of MEK that are important not only for MEK-substrate association but also for intrinsic kinase activity. Constitutively activated MEK (x7, Δn3/S222D) was a kind gift of N. Ahn (University of Colorado, Boulder, CO). Expression vectors encoding His6-tagged wild-type rat ERK2 as well as a chimeric protein consisting of the first two subdomains of p38 MAPK fused to subdomains 2–10 of ERK2 (PIIE) were a kind gift from M. Cobb (University of Texas Southwestern Medical Center, Dallas, TX). Mouse MEK6 cDNA was a kind gift from J. Han (Scripps Institute, La Jolla, CA). CL100, c-Jun, activating factor-2, and B-Raf were obtained from Upstate Biotechnology (Lake Placid, NY). Polyclonal antibodies raised against the NH2 terminus of MEK1 (MEK1(NT)) were obtained from Upstate Biotechnology. Polyclonal antibodies raised against the COOH terminus of MEK1 (C-18) as well as the NH2-terminal His-tag (H-15) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). COOH-terminal deletion mutants of MEK1 were made by digesting a plasmid encoding His6-tagged human wild-type MEK1 (pMKK1) (14Mansour S.J. Resing K.A. Candi J.M. Hermann A.S. Gloor J.W. Herskind K.R. Wartmann M. Davis R.J. Ahn N.G. J. Biochem. (Tokyo). 1994; 116: 304-314Google Scholar) with the indicated restriction enzymes and ligating the resulting fragment into the appropriate pRSET vector as follows: Δc1-(160–392)EcoRI, Δc2-(218–392) NheI/NcoI, and Δc3-(229–392) NheI/AflIII. Additional COOH-terminal deletion mutants of MEK1 were made by PCR amplification of pMKK1 using PCR primers 5′-GGACAGCAAATGGGTCGGG-3′ (corresponding to the multiple cloning site on pRSET) and 5′-CCGTATAAGCTTAGGGGCC-3′ to introduce a novel HindIII site at position 924. The resulting PCR product was digested withBamHI and HindIII, gel-purified, and ligated intoBamHI/HindIII-digested pRSETA yielding a construct encoding Δc5-(292–392) as well as one encoding Δc4-(261–392) because of a fortuitous error. Internal deletion mutants of MEK1 were made by digesting pMKK1 with the indicated restriction enzyme and re-ligating the gel-purified plasmid as follows: Δi1-(20–318) HincI and Δi2-(94–219) MscI. To construct Δi3-(292–318), we used the Stratagene QuikChange site-directed Mutagenesis kit to introduce novel XhoI sites into pMKK1 at position 910 using the primer 5′-GGCCAAGGACCCTCGAGAGGCCCCTTAGC-3′ and its complementary sequence and then at position 988 using the primer 5′-GTTGGATTACATAGTCCTCGAGCCTCCTC-3′ and its complementary sequence. The resulting construct was digested with XhoI and re-ligated following gel purification to generate Δi3-(292–318). All of the sequences were confirmed by direct DNA sequencing. Constructs containing MEK1 mutations in theLF-interacting region (LFIR) were generated by introducing the mutations into pMKK1 with the use of the QuikChange site-directed mutagenesis kit. The primers used were 5′-CCAACAACTCAGCAATTGCCATGGG-3′ for F310A, 5′-CCAACAAGTGAAAAATTGCCATGGG-3′ for E311H, 5′-GGATTACATAGTCAACCACCCTCCTCC-3′ for E319H, 5′-CCCATGGCAATTGCTCACTTGTTGG-3′ for Phe310-Glu311-Glu319 (FEE), 5′-ATTTTTGAGTTGGCGGATTACATAGTC-3′ for L313A, 5′-TTGTTGGATTACGCAGTCAACGAGCCT-3′ for I316A, 5′-TTGGATTACATAGCCAACGAGCCTCCT-3′ for V317A, 5′-AACGAGCCTCCTGCAAAACTGCCCAGT-3′ for P322A, and 5′-CCTCCTCCAAAAGCGCCCAGTGGAGTG-3′ for L324A and their respective complementary sequences. MEK6 was PCR-cloned from a plasmid containing mouse MEK6 cDNA and ligated into a pRSET (NH2-terminal His6-tagged) bacterial expression vector. Constructs containing MEK6 point mutations were generated by introducing the mutations with the use of the QuikChange site-directed mutagenesis kit. The primers used were 5′-cgaaaccctggccttaaagacccaaaagaagcatttg-3′for I15D, 5′-CTCAAACAGGTGGCAGAGGAGCCATCGCCA-3′ for V271A, 5′-GAGGAGCCATCGGCACAACTCCCAGCAGAC-3′ for P276A, and 5′-CCATCGCCACAAGCCCCAGCAGACAAGTTC-3′ for L278A and their respective complementary sequences. ERK(CD) was made by site-directed mutagenesis of the His6-tagged wild-type ERK2 expression vector to introduce aspartate to asparagine substitutions at residues 321 and 324 with the primer 5′-ctggagcagtattataacccaagtaatgagcccattgctgaa-3′and its complementary sequence. All of the sequences were confirmed by direct DNA sequencing. Anthrax lethal factor was produced in a non-toxigenic, sporulation-defective strain ofB. anthracis (BH445) as described elsewhere (15Park S. Leppla S.H. Protein Expression Purif. 2000; 18: 293-302Google Scholar). Recombinant MEK protein was expressed in Escherichia coliand purified by fast pressure liquid chromatography essentially as described earlier (10Duesbery N.S. Webb C.P. Leppla S.H. Gordon V.M. Klimpel K.R. Copeland T.D. Ahn N.G. Oskarsson M.K. Fukasawa K. Paull K.D. Vande Woude G.F. Science. 1998; 280: 734-737Google Scholar). Wild-type ERK2 and ERK(CD) were expressed similarly with the exception that cultures were grown and induced overnight at 30 °C. To measure MEK cleavage in a cell-based assay, lysates of Xenopus laevisoocytes were prepared as described previously (16Shibuya E.K. Boulton T.G. Cobb M.H. Ruderman J.V. EMBO J. 1992; 11: 3963-3975Google Scholar). Recombinant MEK proteins (0.5 μg) were added as indicated in the text to 50 μl of oocyte lysate and diluted to a final volume of 0.5 ml with oocyte extraction buffer (0.25 m sucrose, 0.1 m NaCl, 0.02 m Hepes (pH 7.5), 2.5 mmMgCl2). MEK1(NT) antibody (5 μl) was added, and lysates were incubated overnight on a rotator shaker at 4 °C. Immune complexes were precipitated with protein A-agarose, washed, separated by SDS-PAGE, and immunoblotted using polyclonal antibodies raised against MEK1(NT). Cleavage of MEK proteins in vitro was measured by adding 0.2 μg of MEK and 0.2 μg of LF to 2.5 μl of 4× assay buffer (20 mm NaCl, 20 mm EGTA, 320 mmpotassium phosphate buffer, pH 7.2), and distilled water was added to a final volume of 10 μl. After incubation at 30 °C for 5–10 min, proteins were separated by SDS-PAGE, blotted onto polyvinylidene difluoride membrane, and probed with antibodies to the NH2terminus of MEK1 (1:1000). To measure B-Raf phosphorylation of MEK deletion mutants, 2 μl (0.4 units) of recombinant B-Raf, 0.2 μg of MEK protein, 3 μl of AB (20 mm MOPS (pH 7.2), 25 mm β-glycerophosphate, 5 mm EGTA, 1 mm sodium orthovanadate, 1 mm dithiothreitol), and 3 μl of ATP mixture (γ-32PATP (10 mCi/ml, 3000 mCi/mmol, Amersham Biosciences) diluted 1:3 in 0.5 mm ATP, 75 mm MgCl2) were mixed with distilled water to a final volume of 10 μl and incubated for 15 min at 30 °C. When assaying the ability of B-raf to phosphorylate MEK1 in the absence or presence of LF, B-raf was added last and the reaction was incubated for 5 min at 30 °C. Proteins were then separated by SDS-PAGE upon 10% gels and processed for autoradiography. To measure MEK activity in the presence of LF or inactive LF(E687C) (0.2 μg), samples were prepared in a similar manner with the exception that ERK (wild type, PIIE, or ERK(CD) was added. Proteins were then separated by SDS-PAGE on 14% gels and processed for autoradiography. To identify regions of MEK that are distal to the NH2-terminal cleavage site and are required for interaction with LF, we undertook an analysis of internal and carboxyl-terminal deletion mutants of MEK1. Two internal and five COOH-terminal deletion mutants (Fig.1 A) were generated and analyzed each for their ability to be recognized and cleaved by LF. Of the deletion mutants analyzed, only Δi2 was noticeably cleaved (Fig.1, B–D, data not shown). This finding was surprising considering that Δi2 lacks residues 94–219, corresponding to the amino-terminal kinase domain and suggests that the NH2terminus may form a stable association with the COOH-terminal portion of MEK1. Because neither Δc5-(292–392) nor Δi1-(20–318) was cleaved by LF but Δi2 was, we hypothesized that those regions absent from Δc5 and Δi1 but present in Δi2 must be necessary for binding and/or cleavage. To test this hypothesis, we used site-directed mutagenesis to introduce novel restriction sites in MEK1 to produce a new deletion mutant, Δi3, lacking residues 292–318. Analyses with Δi3 showed that it was resistant to cleavage by LF (Fig.1 E). These results indicate that a region contained either in whole or in part within residues 292–318 is necessary for binding and/or cleavage by LF. We have called this region theLF-interacting region or LFIR. Because not only MEK1 but also MEKs 2–4 and MEK6 and MEK7 are cleaved by LF, we reasoned that critical elements of the LFIR must be conserved among the MEKs. Residues 292–306 are present at the COOH-terminal end of a proline-rich insert, which is to MEKs 1 and and are not to an important in LF substrate However, an analysis of a sequence of MEKs in the region following the proline-rich insert the presence of conserved elements We used site-directed mutagenesis to determine the of these residues in LFIR the mutations in and nor a in with the ability of LF to cleave MEK1 2 However, the of for or the ability of LF to cleave MEK1, indicating that these residues are critical for binding and/or cleavage by LF. Moreover, the LFIR to be functionally conserved because the of similar mutations at or in MEK6 also proteolysis by LF 2 These results indicate that a conserved COOH-terminal region of MEKs is required for LF-mediated The COOH end of the proline-rich region of MEK1 is necessary for the association of MEK with the MEK1 A. Biol. 1994; 1: Scholar, Biol. Scholar), the that the LFIR and elements in MEK1 may To this we the ability of B-Raf to phosphorylate and Δi3 or Δc5 MEK. with the that LF with MEK in a region that a in assays demonstrated that B-Raf phosphorylate neither Δi3 nor S. of the ability of B-Raf to phosphorylate MEK1 with point mutations in the LFIR indicated that with the exception of substitutions with neither the ability of B-Raf to phosphorylate 2 nor These results indicate that although the LFIR and the region of MEK1 are not are adjacent or the LFIR and the regions of MEK1 do LF activation of MEK. We this by assaying the ability of B-Raf to phosphorylate MEK1 in vitro in the presence of of LF. The presence of an of LF but not was to reduce the ability of B-Raf to phosphorylate MEK1 by these results indicate that adjacent or epitopes of MEK1 are required not only for cleavage by LF but also for The the that LF may the activity of MEK by its activation by However, a inactive LF containing a in the binding site K.R. N. Leppla S.H. Microbiol. 1994; Scholar), was as as wild-type LF to MEK phosphorylation 3 Moreover, we have found that the ability of activated MEK1 to phosphorylate and ERK2 is in the presence of LF 3 Because the kinase activity of MEK1 is not upon the in phosphorylation and activation of ERK with to LF must have from a in the intrinsic kinase activity of MEK1 and/or a in the ability of MEK1 to with To test this hypothesis, we the effects of LF upon the ability of MEK1 to phosphorylate ERK2 protein containing mutations at the common docking domain through which the NH2 terminus of MEK1 binds ERK2 M. Cell Biol. 2000; Scholar). We reasoned that LF reduced the ability of MEK1 to with ERK through its LF not phosphorylation of However, we found that although LF reduced ERK2 phosphorylation by it reduced ERK(CD) phosphorylation by as 3 Similar results were obtained we phosphorylation of PIIE, a chimeric protein consisting of the first two subdomains of p38 MAPK fused to subdomains 2–10 of ERK2 and lacking a docking site on ERK2 3 B. Cobb M.H. J. Biol. Chem. 1999; Scholar). These results indicate that the of MEK activity following proteolysis by LF be attributed to a ability to In the phosphorylation was measured with MEK and ERK present in the of phosphorylation not only the affinity of MEK for ERK but also the intrinsic kinase activity of MEK. However, the of each of these may be by the of substrate to kinase that at of ERK the of phosphorylation is by the affinity of MEK for ERK, at of ERK, the reaction and the of phosphorylation the intrinsic kinase activity of MEK. Therefore, in the following the of MEK1 protein was at μg the of substrate was from to to enzyme a of the of wild-type ERK substrate a activation of MEK, that of an enzyme 3 Similar have been made and were attributed to a of protein and the intrinsic of the kinase Proc. Natl. Acad. Sci. U. S. A. Scholar, Science. 1998; 280: Scholar). Because results were obtained in vitro in the presence of recombinant indicate that ERK is of MEK activity. with the that the NH2-terminal docking domain of MEK1 binds with LF the affinity of MEK for ERK However, LF also the of ERK phosphorylation at saturating indicating that the NH2-terminal residues of MEK1 a in intrinsic kinase activity. To this we this assay, ERK(CD) as the substrate E). these LF not alter the affinity of MEK for its substrate but the of ERK(CD) phosphorylation saturating LF MEK activity not only by its affinity for ERK but also by its intrinsic kinase activity. To MEKs are the only identified of LF. of the LF cleavage sites on and MEK7 elements of In the cleavage site is by a series of residues and by an with the exception of MEKs 3 and the cleavage site is by one or these the site for LF cleavage the a is is the and This is similar to a described MAPK binding site or consisting of a which is by residues on one or M. Cell Biol. 2000; Scholar). This the that LF may be a domain which and of However, using in we have been to cleavage of c-Jun, or activating factor-2, of that a domain not shown). In addition, two-hybrid for binding partners of LF have isolated cDNA for MEK2, which lacks the NH2-terminal cleavage site (13Vitale G. Pellizzari R. Recchi C. Napolitani G. Mock M. Montecucco C. Biochem. Biophys. Res. Commun. 1998; 248: 706-711Google Scholar). regions of MEKs, in to the NH2-terminal cleavage must be required for LF substrate we have identified a functionally conserved COOH-terminal region of MEKs that is essential for LF-mediated proteolysis of MEK. The presence of a conserved region distal to the cleavage which is necessary for binding and/or cleavage by LF, may in part the to identify LF MEKs. The that as B-Raf A. Biol. 1994; 1: Scholar, Biol. Scholar), kinase Biol. 2002; Scholar), and the protein MEK 2001; Scholar, Collier A. Science. 1998; Scholar), also with MEK in this region suggests that this region a domain of MEK1 and MEKs. The presence of a domain may have for LF because the presence of proteins may alter the of LF to its LF MEK activity by of MEKs. However, by this mechanism to how LF inactivates MEKs, because as in the LF the activity of activated MEK1. Because phosphorylation these is not only upon kinase activity but also substrate we reasoned that LF MEK by either its intrinsic kinase activity or its affinity for The because MEK1 deletion mutants lacking the NH2-terminal residues are in their ability to ERK B. Cobb M.H. J. Biol. Chem. 1999; Scholar, M. EMBO J. Scholar) and because mutations in the docking domain the with which MEK1 ERK M. Cell Biol. 2000; Scholar). However, we found that as well as the affinity of MEK for its LF also the intrinsic kinase activity of MEK. The was not but is not upon to the of protein kinase and its that NH2-terminal and activation substitutions to MEK1, Ahn and S.J. J.M. Ahn N.G. Biochemistry. Scholar) have hypothesized that regions of the NH2 terminus form with the activation and that of the within this region within the activation that In addition, the of the of the protein kinase which is indicate that a of residues at its NH2 terminus with the activation it in an K. B. D. EMBO J. 1998; Scholar, S. P. J. Biol. Chem. 2002; Scholar). By we that the NH2 terminus of MEK1 with its activation to its activity. NH2-terminal may also protein because we previously that the of MEKs is in with PA and LF (10Duesbery N.S. Webb C.P. Leppla S.H. Gordon V.M. Klimpel K.R. Copeland T.D. Ahn N.G. Oskarsson M.K. Fukasawa K. Paull K.D. Vande Woude G.F. Science. 1998; 280: 734-737Google The of the domain to the activation may interaction and ERK phosphorylation and We D. for a to which we an in this We also A. G. Vande and for as well as J. for on the