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p38 Map Kinase Substrate Specificity Differs Greatly for Protein and Peptide Substrates
Archives of Biochemistry and Biophysics Vol. 382, No. 2, October 15, pp. 310 –313, 2000 doi:10.1006/abbi.2000.2005, available online at http://www.idealibrary.com on
RESEARCH REPORT p38 Map Kinase Substrate Specificity Differs Greatly for Protein and Peptide Substrates Julio Hawkins,* Song Zheng,* Betsy Frantz,† and Philip LoGrasso* ,1 *Department of Molecular Design and Diversity and †Department of Immunology and Rheumatology, Merck Research Laboratories, P.O. Box 2000, Rahway, New Jersey 07065
Received May 23, 2000
To understand the molecular features that give rise to p38 substrate specificity, we compared the specificity constants, k cat/K m, and the free energy associated with protein versus peptide substrate binding for a variety of p38 substrates. Our results showed that proteins were up to 1320-fold better substrates for p38 compared to their peptide counterparts. Moreover, comparison of sequence elements revealed that in the context of a peptide substrate, but not a protein substrate, the sequence PXT/SP was preferred for catalysis. Finally, in the context of a peptide, the increased binding affinity ascribed to the p-11 position as seen with peptide inhibitors of protein kinase A (PKA), 2 was not present in p38 peptide substrates containing a p-11 residue. These results taken in the context of the ordered sequential kinetic mechanism for p38 suggest that the choice of substrate for p38 will likely have effects on the measured binding of ATP and inhibitor. p38 is a member of the mitogen-activated protein (MAP) kinase family that is activated in response to stimuli such as lipopolysaccharide (LPS), TNF-␣, and IL-1 (1–3). TNF-␣ and IL-1 are proinflammatory cytokines which play a predominant role in chronic inflammation (4), and agents that block their function have been clinically successful in the treatment of rheumatoid arthritis and Crohn’s disease (5–9). Moreover, selective p38 inhibitors lower plasma TNF-␣ levels and are efficacious in animal models of arthritis (10). Many aspects of p38 activation by MAP kinase kinases (MKKs) (3, 11–13), its kinetic mechanism (14), and its inhibition by compounds that compete with ATP for binding have been described (14 –18). However, with the exception of the description of downstream substrates for p38 (3, 18 –20), there has been no characterization of the molecular features that give rise to p38 substrate specificity. While reports on the peptide substrate specificity of extracellular-regulated kinase (ERK 1 and 2) MAP kinase family members have been
1 To whom correspondence should be addressed. Fax: (732) 5944020. E-mail: [email protected]. 2 Abbreviations used: PKA, protein kinase A; MAP, mitogen-activated protein; MKK, MAP kinase kinase.
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described (21), understanding of the specificity determinants for protein and peptide substrates for other MAP kinase family members such as p38 and c-jun N-terminal kinase (JNK) have not been investigated. Using synthetic peptides that correspond to natural phosphorylation sites in known p38 substrates, we compared k cat/K m values to determine what substrates were best for p38 and what amino acid residues were responsible for the specificity. Furthermore, the binding energy of a group or groups represented by X in a larger substrate such as a protein can be compared to that in a smaller substrate such as a peptide and is given by (22) ⌬G X ⫽ RT ln关共k cat/K m兲 peptide /共k cat/K m兲 protein 兴,
[1]
where R is the gas constant (0.0019893 kcal/mol) and T is the absolute temperature. This relationship holds true when X is involved in the binding interactions only and not directly in the chemical step. This is particularly true for PKA, where it has been shown that product release is rate-limiting (23, 24), and is likely true for p38 and most other kinases as well. Our data showed that proteins were up to 1320-fold better substrates for p38 compared to their peptide counterparts. Moreover, comparison of sequence elements revealed that in the context of a peptide substrate, but not a protein substrate, the sequence PXT/SP was preferred for catalysis. Finally, in the context of a peptide, the increased binding affinity ascribed to the p-11 position as seen with peptide inhibitors of protein kinase A (PKA) (25) was not present in p38 peptide substrates containing a p-11 residue. Our results show that protein substrates (1, 6, 8, 11) for p38 have higher k cat/K m values than their corresponding peptide counterparts (2, 7, 9, 12) (Table I). For ATF2, k cat/K m for the protein substrate (8) is 1320-fold greater that that for the peptide counterpart (9) (Table I). The extremely low k cat/K m for the peptide 9 was a strong indication that in the context of peptide substrates, a recognition sequence of PXT/SP was needed in order for the peptide to be a viable p38 substrate. This observation was further supported with the PHAS I peptide 12 (Table I), which showed undetectable function as a p38 substrate and lacked the signature PXT/SP sequence. 0003-9861/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
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p38 SUBSTRATE SPECIFICITY TABLE I
Protein/Peptide Substrate Sequences, Specificity Constants (k cat/K m), and Free Energy of Binding Attributed to Groups in a Protein for p38 MAP Kinase Compound MAPKAP3 1 2 3 4 5 MAPKAP2 6 7 ATF2 8 9 10 PHAS-1 11 12
Protein/peptide sequence
k cat/K m (1/(M 䡠 s))
⌬G X (kcal/mol)
...QFMNHPWINQSMVVPQTPLHTARVLQ... LCB-SMVVPQTPLHTARVLQ-NH 2 LCB-PQTPLHTARVLQ-NH 2 LCB-TPLHTARVLQ-NH 2 LCB-QFMNHPWINQSMVVPQTPLH-NH 2
27,510 ⫾ 4950 830 ⫾ 200 170 ⫾ 30 60 ⫾ 7 150 ⫾ 20
⫺2.11 ⫺3.08 ⫺3.69 ⫺3.10
...EFMNHPWIMQSTKVPQTPLHTSRVLK... LCB-STKVPQTPLHTSRV-NH 2
34,000 ⫾ 2,480 220 ⫾ 4
⫺3.04
...GPARNDSVIVADQTPTPTRFLK... LCB-VIVADQTPTPTRFLK-NH 2 LCB-GPARNDSVIVADQTPTPTRFLK-NH 2
13,220 ⫾ 2,380 10 ⫾ 2 50 ⫾ 10
⫺4.08 ⫺3.38
...LMECRNSPVAKT... LCB-LMECRNSPVAKT-NH 2
2,490 ⫾ 200 0
Note. Initial velocity studies were carried out under k cat/K m conditions such that [S] Ⰶ K m. Under these conditions, velocities were linear up to 4 M for GST-biotin-ATF2(1–109) and PHAS I, and 0.5 M for GST-biotin-MAPKAP3 and GST-MAPKAP2 CT-myc, and less than 10% of product was generated. k cat/K m was calculated by measuring the velocity at 2 M for GST-biotin-ATF2(1–109) and PHAS I, and 0.2 M for GST-biotin-MAPKAP3 and GST-MAPKAP2 CT-myc. Reactions were carried out in 50-L volumes containing the final concentrations of the following: 25 mM Hepes, pH 7.4; 10 mM MgCl 2; 2 mM DTT; 20 mM -glycerophosphate; 0.1 mM Na 3VO 4; 4 Ci of [␥- 33P]ATP (2000 Ci/mmol); 500 M ATP for peptide substrates and 200 M ATP for protein substrates; and 1 mg/mL bovine serum albumin (BSA). The reactions were initiated with the addition of 10 nM mouse p38 (final concentration) and incubated for 60 min at 30°C. Assays were stopped with 50 L of 100 mM EDTA/15 mM pyrophosphate for protein substrates and 25 L of 8 M guanidine hydrochloride for peptide substrates. For protein substrates, Immobilon-P 96-well plates were pretreated with 100 L of methanol followed by 100 L of 15 mM sodium pyrophosphate. Forty microliters of the stopped reaction was spotted in duplicate on the Immobilon-P 96-well plate. The samples were vacuum-filtered and washed three times each with 100 L of 75 mM H 3PO 4 to remove unincorporated [␥- 33P]ATP. After the third H 3PO 4 wash and a final filtration step to remove H 3PO 4, 50 L of Microscint-20 was added to each well and samples were analyzed on a Packard Topcount liquid scintillation counter. Data are from the average of three experiments each filtered in duplicate. For peptide substrates, 15 L of the stopped reaction was captured on 1-cm 2 SAM membrane and washed two times with 1 M NaCl, two times with 1 M NaCl/1% phosphoric acid, one time with water, and one time with 100% ethanol. Membranes were dried, added to 3 mL of Packard Ultima Gold scintillation fluid, and counted on a Packard 2500TR liquid scintillation counter. Data are from the average of three experiments each spotted once. p38 and GST-biotin-ATF2(1–109) were expressed and purified as described (14). A DNA fragment of MAPKAP 3 (Accession No. U43784) coding for amino acids 1–382 was isolated from universal human cDNA (Clontech) by PCR amplification utilizing Platinum Taq HiFi (GIBCO–BRL) and expressed as a GST-biotin fusion in M9 minimal medium. The protein was purified by glutathione affinity chromatography and size exclusion chromatography. GST-MAPKAP2 CT-myc (Upstate Biotechnology) is a partially purified fusion protein of MAPKAP2 containing residues 46 – 400 of MAPKAP2. PHAS I protein was purchased from Stratagene. The “. . .” notation before and after compounds 1, 6, 8, and 11 indicates additional protein sequence for these substrates. The boxed portion indicates the PXTP peptide recognition sequence and the boldfaced residues are the phosphorylation sites. Peptides 2– 4, 9, and 12 were synthesized on an ABI Model 433 peptide synthesizer employing standard FastMoc chemistry on a 250-mol scale. Peptides were purified by reverse-phase HPLC using a 25⫻ 400 mm DeltaPak C 18 column. Peptides 5, 7, and 10 were purchased from PeptidoGenic Research. The molecular weight of all peptides was confirmed by mass spectral analysis and peptides were determined to be ⬎95% pure by RP-HPLC ( A 214 nm). LCB, long-chain biotin.
It can be seen, however, that in the context of the PHAS I protein substrate 11, there was no requirement for this sequence (Table I). The same was true comparing the protein sequences of MAPKAP-3 (1) and the truncated peptide 4 that only contained the TP portion of the recognition sequence. For MAPKAP-3, like ATF2, there is a 460-fold greater k cat/K m for the protein substrate 1 compared to peptide 4. Other reported phosphorylation sites in the MAPKAP-2 (26) (SHNSLTTPCYTPYYVA) and MAPKAP-3 (20) (QNALQTPCYTPYYVA) proteins that did not contain a PXT/SP sequence were not phosphorylated by p38 in the context of a peptide (data not shown). The same was true for the PKA peptide substrate (LRRASLG), kemptide (25), which does not contain a PXT/SP signature sequence and was not a p38
substrate (data not shown). Thus, like ERK 1 and ERK 2 (21), p38 had a consensus sequence for peptide substrates that had the signature sequence PXT/SP. However, this signature sequence was not required in the context of a protein, suggesting other sites have significant contributions to binding. Applying Eq. [1] to various protein/peptide substrate pairs showed that there was a range in ⌬G X values associated with stabilization of the transition state by the binding energy of the total protein (Table I). For example, the MAPKAP-3 peptide (2) had 2.11 kcal/mol for destabilization of the transition state whereas MAPKAP-2 (7) and ATF2 (9) showed 3.04 and 4.08 kcal/mol, respectively. Since the ATF2 peptide (9) had Asp at p-2 whereas MAPKAP-3 and MAPKAP-2 peptides (2 and 7, respectively) have a Pro residue at p-2, this
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HAWKINS ET AL.
is suggestive of the importance of Pro at the p-2 position in a peptide substrate. It is interesting to speculate that Pro at position p-2 in peptides may be responsible for up to 1 kcal/ mol in binding energy, although it is likely that other sequence elements also contribute to substrate binding. To investigate whether p38 peptide substrates were influenced by the p-11 site as was seen with the inhibitor of PKA (25), where Knighton et al. reported a 7000-fold increase in binding with a peptide (PKI) that contained a p-11 residue compared to a peptide without the p-11 residue, we synthesized peptides 5 and 10. Peptide 5 is a MAPKAP-3 p-11 analog and peptide 10 is an ATF2 p-11 analog. In both cases, the addition of residues out past p-11 had little effect on increasing the binding of these peptides compared to their shorter counterparts (peptides 2 and 9) (Table I). Since the ␣-helical structure shown for the PKI peptide (25) is responsible for the added binding to PKA, either the p38 p-11 peptide substrates do not have this ␣-helical contribution to binding or elements elsewhere in the protein are responsible for the additional binding energy seen for p38 protein substrates. Finally, it should be emphasized that the large differences in protein and peptide substrate specificity for p38 will likely have effects on the second substrate, ATP, binding as well as inhibitor binding. Since the kinetic mechanism for p38 is ordered sequential with protein substrate, ATF2, binding before ATP and since the protein substrate and ATP binding sites are interactive (14), the choice of substrate for p38 will likely influence the ATP K m . Indeed, for p38 the K m of ATP is 20 M (14) when ATF2 is the substrate and 200 M when epidermal growth factor receptor (EGF) peptide is used as substrate (17). Our laboratory has also observed that the K m of ATP is in the 60 –200 M range when various peptides are used as p38 substrates (LoGrasso et al., unpublished). These observations, along with a recent report (27) suggesting MAPKAP-2 binds p38 prior to ATP binding, further emphasize the interactive nature for substrate and ATP binding in p38 and may suggest different kinetic mechanisms for protein and peptide substrates. Indeed, a recent report by Chen et al. (28) shows p38 to proceed by an ordered sequential mechanism with ATP binding before a peptide substrate. In addition, since most p38 inhibitors are ATP competitive (14 –17), the effects seen on the ATP K m with differing substrates will likely affect measurements of inhibition potency since the IC 50 of ATP competitive inhibitors will be affected by the ATP K m . Thus, the same inhibitor may show considerably different IC 50 values depending on what substrate is chosen. This line of reasoning may be extended to the in vivo situation as well, where different ATP K m values and different compound IC 50 values might be observed depending on which physiological substrate is chosen as a measure of p38 catalytic activity. In summary, we have shown that protein substrates for p38 are much more specific than their peptide counterparts, with a signature sequence of PXTP suggested for peptide substrates. Moreover, the choice of substrate for p38 will likely have profound affects on both ATP and inhibitor binding.
ACKNOWLEDGMENTS We are grateful to Drs. Alice Marcy, Brian McKeever, and Jed Thompson for critical review of our manuscript.
REFERENCES 1. Han, J., Lee, J.-D., Tobias, P. S., and Ulevitch, R. J. (1993) J. Biol. Chem. 268, 25009 –25104. 2. Han, J., Lee, J.-D., Bibbs, L., and Ulevitch, R. J. (1994) Science 265, 808 – 811. 3. Raingeaud, J., Gupta, S., Rogers, J. S., Dickens, M., Han, J., Ulevitch, R. J., and Davis, R. J. (1995) J. Biol. Chem. 270, 7420 –7426. 4. Dinarello, C. A. (1991) Curr. Opin. Immunol. 3, 941–948. 5. Elliot, M. J., Maini, N. M., Feldmann, M., Kalden, J. R., Antoni, C., Smolen, J. S., Leeb, B., Breedveld, F. C., Macfarlane, J. D., Bijl, H., and Woody, J. N. (1994) Lancet 344, 1105–1110. 6. Elliot, M. J., Maini, N. M., Feldmann, M., Long-Fox, A., Charles, P., Bijl, H., and Woody, J. N. (1994) Lancet 344, 1125–1127. 7. Rankin, E. C. C., Choy, E. H. S., Kassimos, D., Kingsley, G. H., Sopwith, A. M., Isenberg, D. A., and Panayi, G. S. (1995) Br. J. Rheumatol. 34, 334 –342. 8. Van Dullemen, H. M., Van Deventer, S. J. H., Hommes, D. W., Bijl, H., Jansen, J., Tytgat, G. N. J., and Woody, J. N. (1995) Gastroenterology 109, 129 –135. 9. Moreland, L. W., Baumgartner, S. W., Schiff, M. H., Tindall, E. A., Fleischmann, R. M., Weaver, A. L., Ettlinger, R. E., Cohen, S., Koopman, W. J., Mohler, K., Widmer, M. B., and Blosch, C. M. (1997) N. Engl. J. Med. 337, 141–147. 10. Badger, A. M., Bradbeer, J. N., Votta, B., Lee, J. C., Adams, J. L., and Griswold, D. E. (1996) J. Pharmacol. Exp. Ther. 279, 1453– 1461. 11. Han, J., Lee, J.-D., Jiang, Y., Li, Z., Feng, L., and Ulevitch, R. J. (1996) J. Biol. Chem. 271, 2886 –2891. 12. Raingeaud, J., Whitmarsh, A. J., Barrett, T., Derijard, B., and Davis, R. J. (1996) Mol. Cell. Biol. 16, 1247–1255. 13. Han, J., Wang, X., Jiang, Y., Ulevitch, R. J., and Lin, S. (1997) FEBS Lett. 403, 19 –22. 14. LoGrasso, P. V., Frantz, B., Rolando, A. M., O’Keefe, S. J., Hermes, J. D., and O’Neill, E. A. (1997) Biochemistry 36, 10422– 10427. 15. Tong, L., Pav, S., White, D. M., Rogers, S., Crane, K. M., Cywin, C. L., Brown, M. L., and Pargellis, C. A. (1997) Nat. Struct. Biol. 4, 311–316. 16. Wilson, K. P., McCaffrey, P. G., Hsiao, K., Pazhanisamy, S., Galullo, V., Bemis, G. W., Fitzgibbon, M. J., Caron, P. R., Murcko, M. A., and Su, S.-S. M. (1997) Chem. Biol. 4, 423– 431. 17. Young, P. R., McLaughlin, M. M., Kumar, S., Kassis, S., Doyle, M. L., McNulty, D., Gallagher, T. F., Fisher, S., McDonnell, P. C., Carr, S. A., Huddleston, M. J., Seibel, G., Porter, T. G., Livi, G. P., Adams, J. L., and Lee, J. C. (1997) J. Biol. Chem. 272, 12116 –12121. 18. Rouse, J., Cohen, P., Trigon, S., Morange, M., Alonso-Llamazares, A., Zamanillo, D., Hunt, T., and Nebreda, A. (1994) Cell 78, 1027–1037. 19. Cuenda, A., Rouse, J., Doza, Y. N., Meier, R., Cohen, P., Gallagher, T. F., Young, P. R., and Lee, J. C. (1995) FEBS Lett. 364, 229 –233. 20. McLaughlin, M. M., Kumar, S., McDonnell, P. C., Van Horn, S., Lee, J. C., Livi, G. P., and Young, P. R. (1996) J. Biol. Chem. 271, 8488 – 8492.
p38 SUBSTRATE SPECIFICITY 21. Gonzalez, F. A., Raden, D. L., and Davis, R. J. (1991) J. Biol. Chem. 266, 22159 –22163. 22. Wilkinson, A. J., Fersht, A. R., Blow, D. M., and Winter, G. (1983) Biochemistry 22, 3581–3586. 23. Adams, J. A., and Taylor, S. S. (1992) Biochemistry 31, 8516 – 8522. 24. Madhusdan, E. A., Trafny, N.-H. X., Adams, J. A., Ten Eyck, L. F., Taylor, S. S., and Sowadski, J. M. (1994) Protein Sci. 3, 176 –187.
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25. Knighton, D. R., Zheng, J., Ten Eyck, L. F., Xoung, N.-H., Taylor, S. S., and Sowadski, J. M. (1991) Science 253, 414 – 420. 26. Stokoe, D., Caudwell, B., Cohen, P. T. W., and Cohen, P. (1993) Biochem J. 296, 843– 849. 27. Gum, R. J., and Young, P. R. (1999) Biochem. Biophys. Res. Commun. 266, 284 –289. 28. Chen, G., Porter, M. D., Bristol, J. R., Fitzgibbon, M. J., and Pazhanisamy, S. (2000) Biochemisty 39, 2079 –2087.
RESEARCH REPORT p38 Map Kinase Substrate Specificity Differs Greatly for Protein and Peptide Substrates Julio Hawkins,* Song Zheng,* Betsy Frantz,† and Philip LoGrasso* ,1 *Department of Molecular Design and Diversity and †Department of Immunology and Rheumatology, Merck Research Laboratories, P.O. Box 2000, Rahway, New Jersey 07065
Received May 23, 2000
To understand the molecular features that give rise to p38 substrate specificity, we compared the specificity constants, k cat/K m, and the free energy associated with protein versus peptide substrate binding for a variety of p38 substrates. Our results showed that proteins were up to 1320-fold better substrates for p38 compared to their peptide counterparts. Moreover, comparison of sequence elements revealed that in the context of a peptide substrate, but not a protein substrate, the sequence PXT/SP was preferred for catalysis. Finally, in the context of a peptide, the increased binding affinity ascribed to the p-11 position as seen with peptide inhibitors of protein kinase A (PKA), 2 was not present in p38 peptide substrates containing a p-11 residue. These results taken in the context of the ordered sequential kinetic mechanism for p38 suggest that the choice of substrate for p38 will likely have effects on the measured binding of ATP and inhibitor. p38 is a member of the mitogen-activated protein (MAP) kinase family that is activated in response to stimuli such as lipopolysaccharide (LPS), TNF-␣, and IL-1 (1–3). TNF-␣ and IL-1 are proinflammatory cytokines which play a predominant role in chronic inflammation (4), and agents that block their function have been clinically successful in the treatment of rheumatoid arthritis and Crohn’s disease (5–9). Moreover, selective p38 inhibitors lower plasma TNF-␣ levels and are efficacious in animal models of arthritis (10). Many aspects of p38 activation by MAP kinase kinases (MKKs) (3, 11–13), its kinetic mechanism (14), and its inhibition by compounds that compete with ATP for binding have been described (14 –18). However, with the exception of the description of downstream substrates for p38 (3, 18 –20), there has been no characterization of the molecular features that give rise to p38 substrate specificity. While reports on the peptide substrate specificity of extracellular-regulated kinase (ERK 1 and 2) MAP kinase family members have been
1 To whom correspondence should be addressed. Fax: (732) 5944020. E-mail: [email protected]. 2 Abbreviations used: PKA, protein kinase A; MAP, mitogen-activated protein; MKK, MAP kinase kinase.
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described (21), understanding of the specificity determinants for protein and peptide substrates for other MAP kinase family members such as p38 and c-jun N-terminal kinase (JNK) have not been investigated. Using synthetic peptides that correspond to natural phosphorylation sites in known p38 substrates, we compared k cat/K m values to determine what substrates were best for p38 and what amino acid residues were responsible for the specificity. Furthermore, the binding energy of a group or groups represented by X in a larger substrate such as a protein can be compared to that in a smaller substrate such as a peptide and is given by (22) ⌬G X ⫽ RT ln关共k cat/K m兲 peptide /共k cat/K m兲 protein 兴,
[1]
where R is the gas constant (0.0019893 kcal/mol) and T is the absolute temperature. This relationship holds true when X is involved in the binding interactions only and not directly in the chemical step. This is particularly true for PKA, where it has been shown that product release is rate-limiting (23, 24), and is likely true for p38 and most other kinases as well. Our data showed that proteins were up to 1320-fold better substrates for p38 compared to their peptide counterparts. Moreover, comparison of sequence elements revealed that in the context of a peptide substrate, but not a protein substrate, the sequence PXT/SP was preferred for catalysis. Finally, in the context of a peptide, the increased binding affinity ascribed to the p-11 position as seen with peptide inhibitors of protein kinase A (PKA) (25) was not present in p38 peptide substrates containing a p-11 residue. Our results show that protein substrates (1, 6, 8, 11) for p38 have higher k cat/K m values than their corresponding peptide counterparts (2, 7, 9, 12) (Table I). For ATF2, k cat/K m for the protein substrate (8) is 1320-fold greater that that for the peptide counterpart (9) (Table I). The extremely low k cat/K m for the peptide 9 was a strong indication that in the context of peptide substrates, a recognition sequence of PXT/SP was needed in order for the peptide to be a viable p38 substrate. This observation was further supported with the PHAS I peptide 12 (Table I), which showed undetectable function as a p38 substrate and lacked the signature PXT/SP sequence. 0003-9861/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
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p38 SUBSTRATE SPECIFICITY TABLE I
Protein/Peptide Substrate Sequences, Specificity Constants (k cat/K m), and Free Energy of Binding Attributed to Groups in a Protein for p38 MAP Kinase Compound MAPKAP3 1 2 3 4 5 MAPKAP2 6 7 ATF2 8 9 10 PHAS-1 11 12
Protein/peptide sequence
k cat/K m (1/(M 䡠 s))
⌬G X (kcal/mol)
...QFMNHPWINQSMVVPQTPLHTARVLQ... LCB-SMVVPQTPLHTARVLQ-NH 2 LCB-PQTPLHTARVLQ-NH 2 LCB-TPLHTARVLQ-NH 2 LCB-QFMNHPWINQSMVVPQTPLH-NH 2
27,510 ⫾ 4950 830 ⫾ 200 170 ⫾ 30 60 ⫾ 7 150 ⫾ 20
⫺2.11 ⫺3.08 ⫺3.69 ⫺3.10
...EFMNHPWIMQSTKVPQTPLHTSRVLK... LCB-STKVPQTPLHTSRV-NH 2
34,000 ⫾ 2,480 220 ⫾ 4
⫺3.04
...GPARNDSVIVADQTPTPTRFLK... LCB-VIVADQTPTPTRFLK-NH 2 LCB-GPARNDSVIVADQTPTPTRFLK-NH 2
13,220 ⫾ 2,380 10 ⫾ 2 50 ⫾ 10
⫺4.08 ⫺3.38
...LMECRNSPVAKT... LCB-LMECRNSPVAKT-NH 2
2,490 ⫾ 200 0
Note. Initial velocity studies were carried out under k cat/K m conditions such that [S] Ⰶ K m. Under these conditions, velocities were linear up to 4 M for GST-biotin-ATF2(1–109) and PHAS I, and 0.5 M for GST-biotin-MAPKAP3 and GST-MAPKAP2 CT-myc, and less than 10% of product was generated. k cat/K m was calculated by measuring the velocity at 2 M for GST-biotin-ATF2(1–109) and PHAS I, and 0.2 M for GST-biotin-MAPKAP3 and GST-MAPKAP2 CT-myc. Reactions were carried out in 50-L volumes containing the final concentrations of the following: 25 mM Hepes, pH 7.4; 10 mM MgCl 2; 2 mM DTT; 20 mM -glycerophosphate; 0.1 mM Na 3VO 4; 4 Ci of [␥- 33P]ATP (2000 Ci/mmol); 500 M ATP for peptide substrates and 200 M ATP for protein substrates; and 1 mg/mL bovine serum albumin (BSA). The reactions were initiated with the addition of 10 nM mouse p38 (final concentration) and incubated for 60 min at 30°C. Assays were stopped with 50 L of 100 mM EDTA/15 mM pyrophosphate for protein substrates and 25 L of 8 M guanidine hydrochloride for peptide substrates. For protein substrates, Immobilon-P 96-well plates were pretreated with 100 L of methanol followed by 100 L of 15 mM sodium pyrophosphate. Forty microliters of the stopped reaction was spotted in duplicate on the Immobilon-P 96-well plate. The samples were vacuum-filtered and washed three times each with 100 L of 75 mM H 3PO 4 to remove unincorporated [␥- 33P]ATP. After the third H 3PO 4 wash and a final filtration step to remove H 3PO 4, 50 L of Microscint-20 was added to each well and samples were analyzed on a Packard Topcount liquid scintillation counter. Data are from the average of three experiments each filtered in duplicate. For peptide substrates, 15 L of the stopped reaction was captured on 1-cm 2 SAM membrane and washed two times with 1 M NaCl, two times with 1 M NaCl/1% phosphoric acid, one time with water, and one time with 100% ethanol. Membranes were dried, added to 3 mL of Packard Ultima Gold scintillation fluid, and counted on a Packard 2500TR liquid scintillation counter. Data are from the average of three experiments each spotted once. p38 and GST-biotin-ATF2(1–109) were expressed and purified as described (14). A DNA fragment of MAPKAP 3 (Accession No. U43784) coding for amino acids 1–382 was isolated from universal human cDNA (Clontech) by PCR amplification utilizing Platinum Taq HiFi (GIBCO–BRL) and expressed as a GST-biotin fusion in M9 minimal medium. The protein was purified by glutathione affinity chromatography and size exclusion chromatography. GST-MAPKAP2 CT-myc (Upstate Biotechnology) is a partially purified fusion protein of MAPKAP2 containing residues 46 – 400 of MAPKAP2. PHAS I protein was purchased from Stratagene. The “. . .” notation before and after compounds 1, 6, 8, and 11 indicates additional protein sequence for these substrates. The boxed portion indicates the PXTP peptide recognition sequence and the boldfaced residues are the phosphorylation sites. Peptides 2– 4, 9, and 12 were synthesized on an ABI Model 433 peptide synthesizer employing standard FastMoc chemistry on a 250-mol scale. Peptides were purified by reverse-phase HPLC using a 25⫻ 400 mm DeltaPak C 18 column. Peptides 5, 7, and 10 were purchased from PeptidoGenic Research. The molecular weight of all peptides was confirmed by mass spectral analysis and peptides were determined to be ⬎95% pure by RP-HPLC ( A 214 nm). LCB, long-chain biotin.
It can be seen, however, that in the context of the PHAS I protein substrate 11, there was no requirement for this sequence (Table I). The same was true comparing the protein sequences of MAPKAP-3 (1) and the truncated peptide 4 that only contained the TP portion of the recognition sequence. For MAPKAP-3, like ATF2, there is a 460-fold greater k cat/K m for the protein substrate 1 compared to peptide 4. Other reported phosphorylation sites in the MAPKAP-2 (26) (SHNSLTTPCYTPYYVA) and MAPKAP-3 (20) (QNALQTPCYTPYYVA) proteins that did not contain a PXT/SP sequence were not phosphorylated by p38 in the context of a peptide (data not shown). The same was true for the PKA peptide substrate (LRRASLG), kemptide (25), which does not contain a PXT/SP signature sequence and was not a p38
substrate (data not shown). Thus, like ERK 1 and ERK 2 (21), p38 had a consensus sequence for peptide substrates that had the signature sequence PXT/SP. However, this signature sequence was not required in the context of a protein, suggesting other sites have significant contributions to binding. Applying Eq. [1] to various protein/peptide substrate pairs showed that there was a range in ⌬G X values associated with stabilization of the transition state by the binding energy of the total protein (Table I). For example, the MAPKAP-3 peptide (2) had 2.11 kcal/mol for destabilization of the transition state whereas MAPKAP-2 (7) and ATF2 (9) showed 3.04 and 4.08 kcal/mol, respectively. Since the ATF2 peptide (9) had Asp at p-2 whereas MAPKAP-3 and MAPKAP-2 peptides (2 and 7, respectively) have a Pro residue at p-2, this
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is suggestive of the importance of Pro at the p-2 position in a peptide substrate. It is interesting to speculate that Pro at position p-2 in peptides may be responsible for up to 1 kcal/ mol in binding energy, although it is likely that other sequence elements also contribute to substrate binding. To investigate whether p38 peptide substrates were influenced by the p-11 site as was seen with the inhibitor of PKA (25), where Knighton et al. reported a 7000-fold increase in binding with a peptide (PKI) that contained a p-11 residue compared to a peptide without the p-11 residue, we synthesized peptides 5 and 10. Peptide 5 is a MAPKAP-3 p-11 analog and peptide 10 is an ATF2 p-11 analog. In both cases, the addition of residues out past p-11 had little effect on increasing the binding of these peptides compared to their shorter counterparts (peptides 2 and 9) (Table I). Since the ␣-helical structure shown for the PKI peptide (25) is responsible for the added binding to PKA, either the p38 p-11 peptide substrates do not have this ␣-helical contribution to binding or elements elsewhere in the protein are responsible for the additional binding energy seen for p38 protein substrates. Finally, it should be emphasized that the large differences in protein and peptide substrate specificity for p38 will likely have effects on the second substrate, ATP, binding as well as inhibitor binding. Since the kinetic mechanism for p38 is ordered sequential with protein substrate, ATF2, binding before ATP and since the protein substrate and ATP binding sites are interactive (14), the choice of substrate for p38 will likely influence the ATP K m . Indeed, for p38 the K m of ATP is 20 M (14) when ATF2 is the substrate and 200 M when epidermal growth factor receptor (EGF) peptide is used as substrate (17). Our laboratory has also observed that the K m of ATP is in the 60 –200 M range when various peptides are used as p38 substrates (LoGrasso et al., unpublished). These observations, along with a recent report (27) suggesting MAPKAP-2 binds p38 prior to ATP binding, further emphasize the interactive nature for substrate and ATP binding in p38 and may suggest different kinetic mechanisms for protein and peptide substrates. Indeed, a recent report by Chen et al. (28) shows p38 to proceed by an ordered sequential mechanism with ATP binding before a peptide substrate. In addition, since most p38 inhibitors are ATP competitive (14 –17), the effects seen on the ATP K m with differing substrates will likely affect measurements of inhibition potency since the IC 50 of ATP competitive inhibitors will be affected by the ATP K m . Thus, the same inhibitor may show considerably different IC 50 values depending on what substrate is chosen. This line of reasoning may be extended to the in vivo situation as well, where different ATP K m values and different compound IC 50 values might be observed depending on which physiological substrate is chosen as a measure of p38 catalytic activity. In summary, we have shown that protein substrates for p38 are much more specific than their peptide counterparts, with a signature sequence of PXTP suggested for peptide substrates. Moreover, the choice of substrate for p38 will likely have profound affects on both ATP and inhibitor binding.
ACKNOWLEDGMENTS We are grateful to Drs. Alice Marcy, Brian McKeever, and Jed Thompson for critical review of our manuscript.
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