©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Proteasome Activator Subunit Binds Calcium (*)

(Received for publication, September 12, 1995; and in revised form, October 20, 1995)

Claudio Realini Martin Rechsteiner

From the Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, Utah 84132

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We recently cloned a cDNA encoding the 29-kDa subunit of human red blood cell regulator (REG), a potent activator of the multicatalytic protease (Realini, C., Dubiel, W., Pratt, G., Ferrell, K., and Rechsteiner, M.(1994) J. Biol. Chem. 269, 20727-20732). The sequence of this subunit contains 28 ``alternating'' lysine and glutamic acid residues (a KEKE motif). Similar regions are present in a number of Ca-binding proteins, and using standard filter assays, the recombinant protein is shown to bind Ca and ruthenium red. Ca is also bound to a ubiquitin extension protein containing the 28-residue KEKE region from the 29-kDa REG subunit. Thus, the 29-kDa REG subunit is a Ca-binding protein, and its KEKE region is able to bind divalent cations. Ca reversibly inhibits the enhanced peptidase activity of complexes between the multicatalytic protease and recombinant REG. This raises the possibility that multicatalytic protease activity is regulated by calcium in vivo.


INTRODUCTION

The multicatalytic protease (MCP) (^1)or 20 S proteasome is a large (700 kDa) multimeric enzyme found in eukaryotes, prokaryotes, and archaebacteria (for reviews see (1, 2, 3) ). MCP subunits range in molecular mass from 20 to 30 kDa and can be placed in two families based on their homology to unique alpha- and beta-subunits present in the archaebacterium, Thermoplasma(4) . The assembled enzyme consists of four stacked rings that form a hollow cylinder(5) . The outer rings consist of 7 subunits of the alpha family, and the two inner rings each contain 7 beta-type subunits(6, 7) . It has been suggested that the enzyme's active sites line a central aqueous channel(3) , and the recent x-ray structure of MCP from Thermoplasma confirms this suspicion(8) . Site-directed mutagenesis (9) and covalent labeling with a novel protease inhibitor (10) identify the N-terminal threonine residues of beta-subunits as active site nucleophiles. Thus, MCP is the first example of a threonine protease.

In eukaryotes, MCP serves as the proteolytic core for two larger complexes. In an ATP-dependent reaction, MCP can associate with a regulatory complex that contains at least 15 subunits with apparent molecular masses between 25 and 110 kDa(11, 12, 13) . This generates the ATP-dependent 26 S protease responsible for the degradation of ubiquitinated proteins (14) and unmodified ornithine decarboxylase(15) . Alternatively, in the absence of nucleotides MCP can associate with an 11 S protein complex that we call the regulator (REG), thereby producing a markedly activated peptidase(16, 17) . REG binds to each end of MCP (18) and stimulates hydrolysis of selected fluorogenic peptides as much as 50-fold. SDS-polyacrylamide gel electrophoresis of regulator from human red blood cells revealed 2 subunits with apparent molecular masses of 31 and 29 kDa(17) . We have recently cloned and expressed the gene for the 29-kDa subunit of human regulator (rREG), and we have shown that the recombinant species activates MCP in a manner very similar to the molecule purified from human red blood cells(19) .

A stretch of 28 alternating lysines and glutamate residues is a striking feature of the amino acid sequence of the 29-kDa subunit. We call such regions KEKE motifs and have proposed that these highly charged regions represent association domains(20) ; Perutz has also suggested that alternating positive and negative amino acids or ``polar zippers'' play a role in protein-protein association(21) . KEKE motifs are found in a variety of proteins including subunits of the 26 S protease and MCP, microtubule-associated protein 1B, myosin phosphatase, triadin, and chaperonins such as hsp70 and hsp90(20) . They are also present in the calcium-binding proteins calreticulin, calnexin, endoplasmin, and Ca-dependent adenosine triphosphatase (Ca-ATPase). Calreticulin contains two distinct Ca-binding regions, one of which is a KEKE motif that binds calcium with high capacity and low affinity(22) . Because KEKE motifs are present in Ca-binding proteins and because Ca is an important regulator of cellular processes(23, 24) , we asked whether the 29-kDa REG subunit is capable of binding Ca. Here we report that rREG binds Ca, ruthenium red, and the cationic dye, carbocyanine. Using recombinant technology, we show that, when appended to ubiquitin, the KEKE motif from rREG confers Ca binding to the chimeric protein. Furthermore, concentrations of Ca in the mid-micromolar range reversibly inhibit the peptidase activity of rREG-MCP complexes.


EXPERIMENTAL PROCEDURES

Materials

Calmodulin was obtained from Boehringer Mannheim and ubiquitin from Sigma. The fluorogenic peptide succinyl-Leu-Leu-Val-Tyr-MCA (sLLVY-MCA) was obtained from Peninsula Laboratories. Ruthenium red and Stains All were from Spectrum Chemicals (New Brunswick, NJ); Ca (specific activity, 10 mCi/mg) was purchased from DuPont NEN.

Preparation of REG, Ubiquitin-KEKE, Ub-KEKE, and Multicatalytic Protease

Recombinant REG was purified to homogeneity from Escherichia coli cells induced with isopropyl-1-thio-beta-D-galactopyranoside(19) . Briefly, recombinant cells were lysed using a French press, and the cell lysate passed over an anion exchange column. Fractions from the peak of activity were pooled, and the regulator was further purified by sizing chromatography. The recombinant regulator was greater than 95% pure as determined by electrophoresis on a SDS-polyacrylamide gel followed by silver staining. Ub-KEKE (Ub-DPVKEKEKEERKKQQEK) and Ub-KEKE (Ub-DPVKEKEKEERKKQQEKEDKDEKKKGEDEDK) were expressed and purified according to published procedures(25) . Multicatalytic protease was purified from outdated human red blood cells as described(26) .

Fluorometric Protease Assays

Spectrofluorometric assays consisted of 100 µM sLLVY-MCA incubated in the presence of MCP and various amounts of recombinant REG in a final volume of 50-100 µl of 10 mM phosphate, pH 7.4. Reactions were initiated by the addition of fluorogenic peptide and terminated by adding 200 µl of cold 100% ethanol. Fluorescence was measured on a Perkin-Elmer fluorometer using an excitation wavelength of 380 nm and an emission wavelength of 440 nm. Iminodiacetic acid was used to eliminate contaminating cations from protein solutions and buffers.

Binding of REG, Ub-KEKE, Ub-KEKE, Ubiquitin, and Calmodulin to Ruthenium Red, Carbocyanine, and Ca

Purified rREG, ubiquitin, ubiquitin-KEKE fusion proteins, and calmodulin were applied to a nitrocellulose filter (0.2 µM, Schleicher and Schuell) using a slot blot apparatus and tested as described in (27) for their ability to bind 2 µMCa (1 µCi/ml) and ruthenium red (28) in the presence of 0, 1, or 5 mM MgCl(2). Interaction of soluble proteins with the cationic carbocyanine dye, Stains All, was measured as described(29) .


RESULTS

The 29-kDa REG Subunit Is a Calcium-binding Protein

There are three generally accepted methods for measuring calcium binding to proteins. Some calcium-binding proteins interact with the cationic carbocyanine dye, Stains All, and produce a characteristic absorption peak at 615 nm(29) . Similarly, ruthenium red has been shown to bind many proteins capable of forming complexes with Ca(28) . Finally, direct association of Ca to filter-bound proteins is considered diagnostic for Ca-binding proteins(27) . We used all three methods to determine whether the rREG subunit binds calcium. Because we suspected that the KEKE region in the subunit confers calcium binding, we constructed two ubiquitin peptide extensions. One fusion protein consists of ubiquitin followed by 31 amino acids (DPVKEKEKEERKKQQEKEDKDEKKKGEDEDK); the other is ubiquitin to which the first 17 amino acids of the KEKE motif (DPVKEKEKEERKKQQEK) are appended. These chimeric proteins were also assayed for their ability to bind Ca.

rREG was purified from E. coli lysates as described under ``Experimental Procedures'' and mixed with 0.001% Stains All; equivalent solutions containing calmodulin or ubiquitin were prepared for comparison. The absorption spectra obtained from the three proteins are presented in Fig. 1A. A peak at 615 nm, characteristic of Ca-binding proteins, is present in the spectra from calmodulin and rREG but absent in the spectrum from ubiquitin. According to the Stains All assay, rREG qualifies as a Ca-binding protein. This conclusion is supported by both ruthenium red and Ca binding assays. The slot blots in Fig. 1B show that rREG, calmodulin, and Ub-KEKE fusion proteins bind radioactive calcium and ruthenium red. Neither Ca nor ruthenium red was bound by ubiquitin, and there was only minimal binding of Ca to Ub-KEKE.


Figure 1: Calcium binding by recombinant regulator. A, absorption spectra of Stains All in the presence of recombinant regulator (REG), calmodulin (CaM), and ubiquitin (Ub). Samples were 10 µg of protein in 1 ml of 10 mM Tris, pH 8.8, 0.001% Stains All, and 0.1% formamide. B, binding of regulator, Ub-KEKE, Ub-KEKE, calmodulin, and ubiquitin to Ca and ruthenium red (RR). The proteins (5 µg) were applied to a nitrocellulose membrane and probed with either ruthenium red (25 mg/ml) or 2 µMCa (1 µCi/ml) as described under ``Experimental Procedures.'' Filters were stained with Ponceau S to confirm that equal amounts of protein were bound in each slot.



We also measured Ca binding to the test proteins in the presence of increasing concentrations of Mg (Fig. 2). Whereas ubiquitin and bovine serum albumin failed to bind Ca even in Mg-free solution, rREG, Ub-KEKE, and calmodulin bound Ca in the presence of the competing cation. These results indicate that the 29-kDa REG subunit is a Ca-binding protein and that its KEKE motif is likely to confer this ability. Ub-KEKE contains the first 14 residues of the KEKE motif; nonetheless, it failed to bind Ca in the presence of competing Mg (Fig. 2). This suggests that either the entire KEKE motif is required for significant Ca binding or that the second half of the KEKE motif (EDKDDKKKGEDEDK) is responsible for interactions with Ca.


Figure 2: Effect of Mg on Ca binding to test proteins. The test proteins (1 µg each of bovine serum albumin (BSA), Ub, calmodulin (CaM), rREG, Ub-KEKE, and Ub-KEKE) were applied to a nitrocellulose membrane and probed with approximately 2 µMCa as described under ``Experimental Procedures.'' The presence of equal amounts of protein on the nitrocellulose filters was confirmed by Ponceau S staining.



Effect of Ca on the Activity of rREG-MCP Complexes

To address the relevance of Ca binding to rREG, we assayed MCP peptidase activity in the presence of rREG, Ca, and EGTA. A standard peptidase assay was initiated and then rREG was added, followed several minutes later by Ca; EGTA was finally added after another several minutes. A typical spectrofluorometric trace is shown in Fig. 3A. Strong stimulation of peptidase activity was immediately observed upon addition of purified recombinant REG (Phase I Phase II); peptide hydrolysis by the rREG-MCP complex was inhibited by Ca (Phase III), but activity recovered after EGTA was added to chelate the Ca (Phase IV). MCP alone did not respond to the addition of Ca or EGTA (see Fig. 3B). The peptidase activity of rREG-MCP complexes was inhibited by Ca in a dose-dependent manner (Fig. 3C); only 30% of the original activity remained at 300 µM Ca. The inhibitory effect of Ca was not observed in the presence of EGTA, and the activity of MCP was only minimally affected by Ca in the absence of rREG. The reversibility of the Ca-dependent inhibition of rREG-MCP complexes is illustrated by the data in Fig. 3D; rREG-MCP complexes preformed in the presence of 300 µM Ca recovered up to 90% of their activity after increasing amounts of EGTA were added. The dose dependence and reversibility of Ca inhibition of rREG-MCP complexes were observed using two separate preparations of MCP and three different preparations of recombinant REG. These effects were also observed with the 11 S regulator obtained directly from red blood cells. However, the Ca-activated protease, calpain, is present in the partially purified red blood regulator fraction, (^2)which complicates interpretation of calcium effects on MCP peptidase activity.


Figure 3: Effects of Ca on peptide hydrolysis by rREG-MCP complexes. A, cleavage of sLLVY-MCA by MCP upon serial addition of rREG, Ca, and EGTA. The reaction was performed at room temperature in the presence of 100 µM sLLVY-MCA and 200 ng of MCP (I), MCP + 550 ng of REG (II), MCP + REG + 375 µM Ca (III), MCP + REG + Ca + 1 mM EGTA (IV). B, cleavage of sLLVY-MCA by MCP upon serial addition of Ca and EGTA. The reaction was performed at room temperature in the presence of 100 µM sLLVY-MCA and 200 ng of MCP (I), MCP + 375 µM Ca (II), MCP + Ca + 1 mM EGTA (III). C, effect of Ca on the peptidase activity of MCP and REG-MCP complexes. MCP (100 ng) was incubated with 100 µM sLLVY-MCA alone (solid squares) or in the presence of 75 ng of REG (solid circles). Control samples containing rREG-MCP complexes were incubated in the presence of 1 mM EGTA (open circles). After 20 min the reaction was quenched with ethanol and MCA fluorescence was measured. D, reversibility of the Ca effect on peptidase activity by rREG-MCP complexes. rREG-MCP complexes were preincubated at 37 °C in the presence of 300 µM Ca. Following this preincubation sLLVY-MCA was added to 100 µM, and increasing amounts of EGTA were added (see figure); the incubation continued at 37 °C for 20 min prior to addition of ethanol and measurement of free MCA by fluorescence spectroscopy.




DISCUSSION

A variety of Ca binding motifs have been identified in proteins (for review, see (23) ). The largest family, by far, consists of proteins with EF-hands such as troponin C, calmodulin, or calpain. Other Ca-binding proteins use ``elbows'' (alpha-lactalbumin) or EF-hand-like motifs (annexins). Some Ca-binding proteins do not possess either structure; nonameric repeats (LXGGXGNDX) in E. coli homeolysin (30) and acidic stretches in calsequestrin (31) have been proposed to be Ca-binding sites. Ruthenium red and Ca binding assays were used to show that recombinant fragments from calreticulin (22) and the ryanodine receptor (32) bind calcium in regions containing KEKE motifs. However, the Ca-binding sites were not precisely localized within the expressed peptides, which were generally 100-200 amino acids long. The experiments presented above provide strong evidence that calcium binds directly to a KEKE motif since a Ub-KEKE extension protein bound Ca and ruthenium red but ubiquitin did not (Fig. 2). This conclusion is reinforced by circular dichroism spectra from the free 31-residue KEKE peptide of REG. A significant loss of alpha-helix was observed in the presence of Ca. (^3)KEKE motifs may generally be involved in binding Ca and/or Mg since they are present in triadin (33) , a protein of the sarcoplasmic reticulum thought to bind Ca. Also, calnexin(34) , endoplasmin(35) , and Ca-dependent adenosine triphosphatase (36) contain such regions.

The trace in Fig. 3shows that Ca binding can reverse the increased peptidase activity conferred by rREG. This raises the possibility that calcium regulates proteolytic activity by the multicatalytic protease in vivo. It should be noted, however, that 60 µM Ca was required to suppress peptidase activity by half; this concentration is higher than the accepted values of 0.1-10 µM for intracellular calcium levels(37) .

The experiments presented above do not address the mechanism by which Ca inhibits rREG-MCP peptidase activity. The stimulatory effect of the REG depends on its physical interaction with MCP(17) . Glycerol gradient and native gel analysis of rREG-MCP mixtures suggest that the complexes dissociate in the presence of Ca, but the experiments are not conclusive because of the low affinity of rREG for the multicatalytic protease. (^4)It is also unclear that the Ca effect is mediated only by rREG. Native MCP applied to nitrocellulose binds both Ca and ruthenium red,^4 and alpha-subunits of MCP contain KEKE motifs(20) . Thus, the rREG-MCP interaction could result in conformational changes that activate Ca binding by MCP subunits, and this event might inhibit peptide hydrolysis. Discovering how Ca inhibits rREG-MCP peptidase activity will require further experimentation.

In summary, we have demonstrated that the 29-kDa subunit of REG is a calcium-binding protein. We have also provided strong evidence that the KEKE motif present in rREG is a Ca-binding site. Although the experiments show that Ca reversibly inhibits peptide hydrolysis by rREG-MCP complexes, the molecular mechanism has not been discovered. Moreover, the physiological significance of this finding remains an open question. Nonetheless, there is a real possibility that intracellular calcium levels regulate proteolysis by the multicatalytic protease.


FOOTNOTES

*
These studies were supported by National Institutes of Health Grant GM37009 and by a grant from the Lucille P. Markey Charitable Trust. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

(^1)
The abbreviations used are: MCP, multicatalytic protease; KEKE, alternating lysine (K) and glutamate (E) amino acids; rREG, recombinant 29-kDa subunit of 11 S regulator; MCA, 7-amino-4-methylcoumarin; sLLVY, succinyl-Leu-Leu-Val-Tyr; Ub, ubiquitin.

(^2)
W. Dubiel, unpublished observation.

(^3)
Z. Zhang, C. Realini, and M. Rechsteiner, manuscript in preparation.

(^4)
C. Realini, unpublished observation.


ACKNOWLEDGEMENTS

We would like to thank Greg Pratt for construction and expression of Ub-peptide fusion proteins. We also thank Laura Hoffman, Greg Pratt, and Vicença Ustrell for helpful comments on the manuscript.


REFERENCES

  1. Orlowski, M. (1990) Biochemistry 29, 10289-10297 [Medline] [Order article via Infotrieve]
  2. Rivett, A. J. (1993) Biochem. J. 291, 1-10 [Medline] [Order article via Infotrieve]
  3. Rechsteiner, M., Hoffman, L., and Dubiel, W. (1993) J. Biol. Chem. 268, 6065-6068 [Free Full Text]
  4. Zwickl, P., Grziwa, A., Pühler, G., Dahlmann, B., Lottspeich, F., and Baumeister, W. (1992) Biochemistry 31, 964-972 [Medline] [Order article via Infotrieve]
  5. Grziwa, A., Baumeister, W., Dahlmann, B., and Kopp, F. (1991) FEBS Lett. 290, 186-190 [CrossRef][Medline] [Order article via Infotrieve]
  6. Kopp, F., Dahlmann, B., and Hendil, K. B. (1993) J. Mol. Biol. 229, 14-19 [CrossRef][Medline] [Order article via Infotrieve]
  7. Kopp, F., Kristensen, P., Hendil, K. B., Johnsen, A., Sobek, A., and Dahlmann, B. (1995) J. Mol. Biol. 248, 264-272 [CrossRef][Medline] [Order article via Infotrieve]
  8. Löwe, J., Stock, D., Jap, B., Zwickl, P., Baumeister, W., and Huber, R. (1995) Science 268, 533-539 [Medline] [Order article via Infotrieve]
  9. Seemüller, E., Lupas, A., Stock, D., Löwe, J., Huber, R., and Baumeister, W. (1995) Science 268, 579-582 [Medline] [Order article via Infotrieve]
  10. Fenteany, G., Standaert, R. F., Lane, W. S., Choi, S., Corey, E. J., and Schreiber, S. L. (1995) Science 268, 726-731 [Medline] [Order article via Infotrieve]
  11. Hoffman, L., Pratt, G., and Rechsteiner, M. (1992) J. Biol Chem. 267, 22362-22368 [Abstract/Free Full Text]
  12. Udvardy, A. (1994) J. Biol. Chem. 268, 9055-9062 [Abstract/Free Full Text]
  13. Ma, C.-P., Vu, J. H., Proske, R. J., Slaughter, C. A., and DeMartino, G. N. (1994) J. Biol. Chem. 269, 3539-3547 [Abstract/Free Full Text]
  14. Hough, R., Pratt, G., and Rechsteiner, M. (1987) J. Biol. Chem. 262, 8303-8313 [Abstract/Free Full Text]
  15. Murakami, Y., Matsufuji, S., Kameji, T., Hayashi, S., Igarashi, K., Tamura, T., Tanaka, K., and Ichihara, A. (1992) Nature 360, 597-599 [CrossRef][Medline] [Order article via Infotrieve]
  16. Ma, C.-P., Slaughter, C. A., and DeMartino, G. N. (1992) J. Biol. Chem. 267, 10515-10523 [Abstract/Free Full Text]
  17. Dubiel, W., Pratt, G., Ferrell, K., and Rechsteiner, M. (1992) J. Biol. Chem. 267, 22369-22377 [Abstract/Free Full Text]
  18. Gray, C. W., Slaughter, C. A., and DeMartino, G. N. (1994) J. Mol. Biol. 236, 7-15 [CrossRef][Medline] [Order article via Infotrieve]
  19. Realini, C., Dubiel, W., Pratt, G., Ferrell, K., and Rechsteiner, M. (1994) J. Biol. Chem. 269, 20727-20732 [Abstract/Free Full Text]
  20. Realini, C., Rogers, S. W., and Rechsteiner, M. (1994) FEBS Lett. 348, 109-113 [CrossRef][Medline] [Order article via Infotrieve]
  21. Perutz, M. (1994) Protein Sci. 3, 1629-1637 [Abstract/Free Full Text]
  22. Baksh, S., and Michalak, M. (1991) J. Biol Chem. 266, 21458-21465 [Abstract/Free Full Text]
  23. Clapham, D. E. (1995) Cell 80, 259-268 [Medline] [Order article via Infotrieve]
  24. Berridge, M. J. (1995) BioEssays 17, 491-500 [Medline] [Order article via Infotrieve]
  25. Yoo, Y., and Rechsteiner, M. (1990) Anal. Biochem. 191, 35-40 [Medline] [Order article via Infotrieve]
  26. Dubiel, W., Ferrell, K., Pratt, G., and Rechsteiner, M. (1992) J. Biol. Chem. 267, 22699-22702 [Abstract/Free Full Text]
  27. Maruyama, K., Mikawa, T., and Ebashi, S. (1984) J. Biochem. (Tokyo) 95, 511-519 [Abstract]
  28. Charuk, J. H. M., Pirraglia, C. A., and Reithmeier, R. A. F. (1990) Anal. Biochem. 188, 123-131 [Medline] [Order article via Infotrieve]
  29. Campbell, K. P., MacLennan, D. H., and Jorgensen, A. O. (1983) J. Biol. Chem. 258, 11267-11273 [Abstract/Free Full Text]
  30. Ludwig, A., Jarchau, T., Benz, R., and Goebel, W. (1988) Mol. & Gen. Genet. 214, 553-561
  31. Fliegel, L., Ohnishi, M., Carpenter, M. R., Khanna, V. K., Reithmeier, R. A. F., and MacLennan, D. H. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 1167-1171 [Abstract]
  32. Chen, S. R. W., and MacLennan, D. H. (1994) J. Biol. Chem. 269, 22698-22704 [Abstract/Free Full Text]
  33. Knudson, C. M., Stang, K. K., Moomaw, C. R., Slaughter, C. A., and Campbell, K. P. (1993) J. Biol. Chem. 268, 12646-12654 [Abstract/Free Full Text]
  34. Wada, I., Rindress, D., Cameron, P. H., Ou, W.-J., Doherty, J. J., II, Louvard, D., Bell, A. W., Dignard, D., Thomas, D. Y., and Bergeron, J. J. M. (1991) J. Biol. Chem. 266, 19599-19610 [Abstract/Free Full Text]
  35. Mazzarella, R. A., and Green, M. (1987) J. Biol. Chem. 262, 8875-8883 [Abstract/Free Full Text]
  36. Verma, A. K., Filoteo, A. G., Stanford, D. R., Wieben, E. D., Penniston, J. T., Strehler, E. E., Fischer, R., Heim, R., Vogel, G., Mathews, S., Strehler-Page, M.-A., James, P., Vorherr, T., Krebs, J., and Carafoli, E. (1988) J. Biol. Chem. 263, 14152-14159 [Abstract/Free Full Text]
  37. James, P., Vorherr, T., and Carafoli, E. (1995) Trends Biochem. Sci. 20, 38-43 [CrossRef][Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.