©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
PEST Sequences Do Not Influence Substrate Susceptibility to Calpain Proteolysis (*)

(Received for publication, October 6, 1994; and in revised form, November 14, 1994)

Maurizio Molinari John Anagli Ernesto Carafoli (§)

From the Institute of Biochemistry, Swiss Federal Institute of Technology, 8092 Zurich, Switzerland

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Mutations lowering the PEST score of domains surrounding the calmodulin (CaM)-binding region of the plasma membrane Ca-ATPase failed to influence the susceptibility of the enzyme to µ-calpain (µ-CANP). Synthetic peptides corresponding to the high PEST score C-terminal sequences A18 and B28 had no effect on the rate of pump proteolysis by µ-CANP, i.e. the peptides did not compete for a putative high PEST score recognition site for µ-CANP in the pump molecule. An accessible CaM-binding region appears to be critical for substrate (i.e. the Ca pump) proteolysis and probably also for its recognition by µ-CANP; phosphorylation of the CaM-binding domain of the pump or its occupation by CaM significantly decreased the rate of proteolysis.


INTRODUCTION

Rogers et al.(1, 20) have shown that proteins with intracellular half-lives of less than 2 h are unusually rich in PEST regions (i.e. sequences rich in proline (P), glutamic and aspartic acid (E), serine (S), and threonine (T)) flanked by clusters containing positively charged amino acids. PEST sequences were found to be common in a number of proteins rapidly degraded by a non-ubiquitin-mediated process. They proposed that the PEST sequences sequestered Ca, thus creating a microenvironment of higher Ca concentration favorable to the attack by CANP (see also (2) ).

The activation mechanism of CANP is still poorly understood. A widely accepted model (3) claims that partial CANP autolysis is an obligatory step in the activation of the enzyme and in the cleavage of substrates. One problem with this model is that in vitro experiments have shown that even µ-CANP (^1)(the ``low Ca-requiring'' form of the protease) needs 140-150 µM Ca for half-maximal rate of controlled autolysis(4) , i.e. Ca concentrations that cells presumably never experience, at least during their normal functional cycle. These problems made the PEST sequence suggestion of Ca sequestration very attractive since it potentially explained CANP action in vivo, i.e. in the presence of physiological Ca concentrations.

However, work in this and other laboratories (4, 5, 6, 7, 8) has shown that µ-CANP activation in vivo did not necessarily require autolysis of the protease and occurred at very low Ca concentrations. The activation pathway of µ-CANP may thus consist in the reversible Ca-dependent translocation of the unautolyzed form of the enzyme to the plasma membrane, where the protease could attack its preferred targets (membrane and cytoskeletal proteins like the Ca-ATPase, band 3, and spectrin). In this model the PEST sequences would have no important role in the activation of µ-CANP in vivo.

Studies by Wang et al.(9) led to the conclusion that the presence of sequences with high PEST score and of CaM-binding regions are good indications that the protein is a preferred CANP substrate. The plasma membrane Ca-ATPase indeed is the preferred µ-CANP substrate in vivo, at least in erythrocytes(10) , i.e. it is partially proteolyzed by the unautolyzed 80-kDa form of the protease at very low Ca concentrations(8) . µ-CANP cleaves both the plasma membrane inserted and the isolated pump as well as a number of expressed or synthesized peptides corresponding to its C-terminal portion in the CaM-binding domain. The Ca-ATPase contains 2 sequences with high PEST score (A18, residues 1079-1096, score = 14.0, and B28, residues 1153-1180, score = 8.3) surrounding the CaM-binding domain (C28, corresponding to amino acids 1101-1128) (see Table 1for the amino acid sequences of the peptides used). These findings made the plasma membrane Ca-ATPase an ideal tool to investigate the importance of the PEST sequences in the substrate susceptibility to µ-CANP digestion.



The correlation between the PEST score and the susceptibility of the ATPase to µ-CANP digestion was initially studied by deleting the high PEST score sequences in synthetic C-terminal portions of the Ca-ATPase and then by inserting mutations lowering the PEST scores in the sequences surrounding the CaM-binding domain of the ATPase expressed in Escherichia coli. The results have shown that the PEST sequences near the µ-CANP cleavage site in the substrates play no significant role in CANP susceptibility.


MATERIALS AND METHODS

µ-CANP Purification

The µ-CANP used for these experiments was isolated from freshly collected human erythrocytes by a modified version of the procedure described by Melloni et al.(11) .

One unit of freshly drawn venous human blood in citrate buffer was filtered through a Pall(TM) filter (Pall Schweiz AG, Muttenz, Switzerland) to eliminate the white cells. The filtrate was centrifuged at 800 times g for 15 min to obtain the erythrocytes. All subsequent steps were performed at 4 °C. After three washings with phosphate-buffered saline, pH 7.2, 1 mM EDTA, the packed erythrocytes (250 ml) were lyzed in 5 volumes of 10 mM sodium acetate containing 2.5 mM EDTA and 0.1 mM phenylmethylsulfonyl fluoride, pH 7.2. The hemolysate was centrifuged at 13,000 times g for 20 min to remove the cell membranes. The membrane-free lysate was incubated with 165 g of DEAE-Sepharose CL-6B (Pharmacia Biotech, Inc.) previously equilibrated with a 50 mM sodium acetate buffer, pH 6.7, containing 1 mM EDTA, 1 mM EGTA, and 0.5 mM 2-mercaptoethanol (buffer A). The pH was adjusted to 6.7, and the suspension was stirred for 30 min. The lysate-loaded DEAE-Sepharose was washed in a Buchner funnel with 10 liters of buffer A containing 50 mM NaCl and packed in a glass column (5 times 10 cm). The peak (400 ml) containing the µ-CANP activity was eluted in a single step with 0.2 M NaCl in buffer A. The protein was precipitated with 45% saturated ammonium sulfate. The pellet was resuspended in 30 ml of 50 mM Tris buffer, pH 7.5, 1 mM EDTA, 1 mM EGTA, 0.5 mM 2-mercaptoethanol, and 50 mM NaCl (buffer B) and dialyzed overnight against the same buffer. The dialyzed sample was centrifuged at 20,000 times g, and the supernatant was rechromatographed on a DEAE-Sepharose column with a linear gradient of 0.05-0.2 M NaCl in buffer B. The fractions containing µ-CANP activity were pooled, concentrated, dialyzed against buffer B, and further purified on a butyl-agarose column as described by Melloni et al.(11) .

Peptide Synthesis by Solid Phase

The peptide substrates used for the assays (see Table 1) were derived from the C-terminal portion of isoform 1CI (12) of the plasma membrane Ca-ATPase (hPMCA1CI). The portion consists of about 100 residues(1079-1180) and follows the 10th intramembrane domain of the ATPase. µ-CANP cleaves the pump both in vivo and in vitro in the CaM-binding site (residues 1101-1128).

The peptide substrates A18 (PEST sequence 1), B28 (PEST sequence 2), C28W (CaM-binding domain), C24W (a shortened version of the CaM-binding domain lacking the first 4 N-terminal residues), C24W-P (a phosphothreonine derivative of the same peptide), and C49 (PEST sequence 1 + the CaM-binding domain) were synthesized on an Applied Biosystems (Foster City, CA) Peptide Synthesizer model 431A using the standard scale FastMoc(TM) chemistry according to the manufacturer's instructions. Their syntheses have been described elsewhere: peptides A18 and C28W in Vorherr et al.(13) , peptide B28 in James et al.(14) , peptide C49 in Falchetto et al.(15) , and peptide C24W-P in Hofmann et al.(16) . The purity of all synthetic products was confirmed by electrospray mass spectrometry.

Expression of the C-terminal Fragments in E. coli

The expression of portions of the C-terminal domain of the hPMCA1CI (Met-A18-B28) and the purification of the expressed fragment are described in(17) . The mutated Met-A18*-B28* fragment was expressed and purified by the same procedure.

Introduction of Mutations Lowering the PEST Score

Mutations lowering the PEST score of sequences A18 and B28 surrounding the CaM-binding site of the expressed C-terminal peptide were introduced by polymerase chain reaction amplification of the cDNA coding for the C terminus of the pump with mutated oligonucleotides. The 5`-primer was 37 nucleotides long and contained a NcoI restriction site and a methionine codon followed by the codons of the first 10 amino acids of the fragment to be expressed: 5`-GGCC ATG GAA CAA ATA CCT CAG CAG CAA TTA GCA CAG-3`. The mutations of the DNA-sequence (in boldface) coding for the A18 region changed the protein sequence from EEIPEEELAE to EQIPQQQLAQ, lowering the PEST score from 14.0 to -3.2. The 3`-primer was 33 bases long and contained the complementary sequence of the B28 region to be mutated: 3`-CGG GTT TTA TTA CGA GGA TGT TTT GCA TTG AGG-5`. The mutations in the DNA-sequence (in boldface) coding for the B28 region changed the protein sequence from AEDDAPTKRNS to AQNNAPTKRNS lowering the PEST score from 8.3 to -2.7 (the PEST scores were calculated with the algorithm proposed by Rogers et al.(1) ). A MaeIII restriction site permitted the addition of the missing DNA portion coding for the last 6 amino acids of the A18*-B28* sequence: 5`-GT AAC TCC AGT CCT CCA TAG G-3`, 3`- AGG TCA GGA GGT ATC CCT AG- 5`. The introduction of the mutations was confirmed by DNA and amino acid sequencing.

Phosphorylation of the Expressed Fragment A18-B28

The fragment was phosphorylated by the cAMP-dependent protein kinase following the procedure described by James et al.(18) .

µ-CANP Proteolysis of the Peptide Substrates

The substrate susceptibility to µ-CANP was examined at various free Ca concentrations (Fig. 1) and substrate to protease ratios (Fig. 2). 1-4 nM substrate-peptides were dissolved in an incubation buffer containing 50 mM Tris, 1 mM EDTA. The proteolysis rate at 25 °C was followed upon the addition of Ca and purified µ-CANP. The free Ca concentration was calculated using a computer program(19) . Additional assays were performed in the presence of equimolar amounts of CaM or synthetic A18 and B28 peptides. The proteolysis was stopped by the addition of excess EDTA. The reaction mixtures were examined by reversed phase high performance liquid chromatography (Nucleosil C(18), 300-5 column) using the following gradient: 0-5 min 20% B, 5-50 min to 75% B, 50-60 min to 100% B. A contained 0.1% trifluoroacetic acid, and B contained 0.085% trifluoroacetic acid, 70% acetonitrile. The disappearance of the peaks corresponding to the unproteolyzed peptides and the concomitant appearance of the proteolytic-fragments was recorded.


Figure 1: Ca dependence of substrate proteolysis by µ-CANP. The figure shows the susceptibility of expressed (, A18-B28; box, A18*-B28*; times A18-B28-P) and synthetic peptides (, C49; + C28; bullet, C24; m, C24-P; , B28) to µM-µ-CANP in the presence of different Ca-concentrations. Additional details are found under ``Materials and Methods.''




Figure 2: Susceptibility of peptides to cleavage by µ-CANP. The susceptibility of substrate-peptides (expressed: , A18-B28; box, A18*-B28*; times, A18-B28-P; synthetic: +, C28; bullet, C24; circle, C24-P) to µ-CANP proteolysis was investigated at different protease to peptide ratios. Additional details are found under ``Materials and Methods.''




RESULTS AND DISCUSSION

Fig. 1offers a general view of the Ca dependence of the µ-CANP proteolysis of the substrate peptides; as mentioned, µ-CANP cleaves the intact ATPase as well as its expressed and synthetic C-terminal portion in the CaM-binding region (i.e. peptide C28).

The expressed peptides A18-B28, A18-B28-P, and A18*-B28* all contain the CaM-binding domain and were thus good µ-CANP substrates, i.e. they were about 85% proteolyzed in the presence of 14 µM free Ca and completely proteolyzed in the presence of 75 µM Ca after 1 h of incubation. No significant differences in the rate of proteolysis of the three peptides were detected at the Ca concentrations and incubation times used for the experiments. The phosphorylation of peptide A18-B28 (Ser-1178, located in the high PEST score sequence 2, see the Introduction) increased the acid character of the latter and was thus expected to increase its Ca-sequestration ability. This ought to have led to enhanced susceptibility of the peptide to µ-CANP digestion, especially at low Ca concentrations. Similarly, lowering of the PEST score of the region surrounding the CaM-binding domain by mutating some of its acidic residues could have been expected to increase the resistance of the peptides to the protease. However, both expectations proved fallacious; the decrease of the PEST score of the sequence A18 from 14.0 to -3.2 and of the sequence B28 from 8.3 to -2.7, respectively, had no influence on the proteolysis rate. Similarly, no appreciable differences were detected in the rates of digestion of the phosphorylated and nonphosphorylated A18-B28 peptide. By contrast, phosphorylation of the CaM-binding peptide C24 on the Thr, which is the substrate for protein kinase C(21) , decreased its proteolysis rate by more than 50% (Fig. 1). Similar results were obtained by adding CaM to the incubation mixtures; the proteolysis rate of all peptides was strongly reduced (Fig. 3), showing that µ-CANP needed a free CaM-binding domain to efficiently cleave the peptides. Possibly, a free CaM-binding domain is also needed for the proper recognition of the substrates by µ-CANP. Other indications in this direction were provided by experiments in which peptides C28, A18, or B28, were added to the reaction mixture containing µ-CANP and the substrate-peptide A18-B28. Equimolar concentrations of C28 slowed down the proteolysis rate of the latter, while a 10-fold excess of the two high PEST score sequences A18 and B28 failed to influence it (data not shown). These observations thus strongly indicate that the PEST sequences did not compete with the endogenous recognition site for µ-CANP on the pump. Their importance in the induction of µ-CANP proteolysis thus appears to be minor.


Figure 3: Ca dependence of substrate proteolysis by µ-CANP in the presence (dotted lines) and in the absence (solid lines) of CaM. The expressed peptides (, A18-B28; box, A18*-B28*) and the synthetic peptide (+, C28) were incubated with µ-CANP in the presence of equimolar amounts of calmodulin or in its absence. Additional details are found under ``Materials and Methods.''



The results have shown that the larger peptides were more sensitive to µ-CANP, i.e. they were more efficiently proteolyzed not only by lower Ca concentrations (Fig. 1), but also by lower protease/substrate ratio (Fig. 2). The length of the peptide thus appears to be a determining factor for the rate of proteolysis, especially at very low Ca concentrations. Between 81 and 85% of peptides A18-B28, A18*-B28*, and A18-B28-P (all were 103 amino acids in length) was proteolyzed after 1 h of incubation with µ-CANP by 14 µM free Ca. By contrast, only about 70% of C49 (49 residues) was proteolyzed during the same period of time; higher concentrations of free Ca (up to 40 µM) and higher protease/substrate ratio were necessary for an equivalent digestion of the shorter substrate-peptide C28 (28 amino acids). A shorter version of the CaM-binding region (peptide C24, 24 amino acids) was digested less efficiently than the complete CaM-binding region (C28). The two high PEST score sequences (peptide A18, 18 amino acids, and peptide B28, 28 amino acids, both lacking the CaM-binding site where µ-CANP cleaves the other peptides) were practically insensitive to µ-CANP even at very high Ca concentrations (up to 200 µM).


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Inst. of Biochemistry III, Swiss Federal Institute of Technology (ETH), Universitätstrasse 16, 8092 Zurich, Switzerland. Tel.: 41-632-30-11; Fax: 41-632-12-13.

(^1)
The abbreviations used are: µ-CANP, Ca-activated neutral protease (calpain); CaM, calmodulin.


ACKNOWLEDGEMENTS

We thank Edy Vilei for collaboration in the molecular biology experiments and Peter James and Werner Staudenmann for the sequence analysis of the synthetic and expressed peptides.


REFERENCES

  1. Rogers, S., Wells, R., and Rechsteiner, M. (1986) Science 234, 364-368 [Medline] [Order article via Infotrieve]
  2. Rechsteiner, M. (1990) Semin. Cell Biol. 1, 433-440 [Medline] [Order article via Infotrieve]
  3. Suzuki, K. (1987) Trends Biochem. Sci. 12, 103-105 [CrossRef]
  4. Cong, J., Goll, D. E., Peterson, A. M., and Kapprell, H. P. (1989) J. Biol. Chem. 264, 10096-10103 [Abstract/Free Full Text]
  5. Cong, J., Thompson, V. F., and Goll, D. E. (1993) J. Biol. Chem. 268, 25740-25747 [Abstract/Free Full Text]
  6. Pontremoli, S., Sparatore, B., Salamino, F., Michetti, M., Sacco, O., and Melloni, E. (1985) Biochem. Int. 11, 35-44 [Medline] [Order article via Infotrieve]
  7. Pontremoli, S., Salamino, F., Sparatore, B., De Tullio, R., Pontremoli, R., and Melloni, E. (1988) Biochem. Biophys. Res. Commun. 157, 867-874 [Medline] [Order article via Infotrieve]
  8. Molinari, M., Anagli, J., and Carafoli, E. (1994) J. Biol. Chem. 269, 27992-27995 [Abstract/Free Full Text]
  9. Wang, K. K., Villalobo, A., and Roufogalis, B. D. (1989) Biochem. J. 262, 693-706 [Medline] [Order article via Infotrieve]
  10. Salamino, F., Sparatore, B., Melloni, E., Michetti, M., Viotti, P. L., Pontremoli, S., and Carafoli, E. (1994) Cell Calcium 15, 28-35 [Medline] [Order article via Infotrieve]
  11. Melloni, E., Sparatore, B., Salamino, F., Michetti, M., and Pontremoli, S. (1982) Biochem. Biophys. Res. Commun. 106, 731-740 [Medline] [Order article via Infotrieve]
  12. Carafoli, E. (1994) FASEB J., (1994) 8, 993-1002
  13. Vorrherr, T., James, P., Krebs, J., Enyedi, A., McCormick, D. J., Penniston, J. T., and Carafoli, E. (1990) Biochemistry 29, 355-365 [Medline] [Order article via Infotrieve]
  14. James, P., Vorrherr, T., Krebs, J., Morelli, A., Castello, G., McCormick, D. J., Penniston, J. T., De Flora, A., and Carafoli, E. (1989) J. Biol. Chem. 264, 8289-8296 [Abstract/Free Full Text]
  15. Falchetto, R., Vorherr, T., and Carafoli, E. (1992) Protein Sci. 1, 1613-1621 [Abstract/Free Full Text]
  16. Hofmann, F., Anagli, J., Carafoli, E., and Vorherr, T. (1994) J. Biol. Chem. 269, 24298-24303 [Abstract/Free Full Text]
  17. Hofmann, F., James, P., Vorherr, T., and Carafoli, E. (1993) J. Biol. Chem. 268, 10252-10259 [Abstract/Free Full Text]
  18. James, P., Pruschy, M., Vorherr, T., Penniston, J. T., and Carafoli, E. (1989) Biochemistry 28, 4253-4258 [Medline] [Order article via Infotrieve]
  19. Fabiato, A., and Fabiato, F. J. (1979) J. Physiol. 75, 463-505
  20. Rechsteiner, M. (1988) Adv. Enzyme Regul. 27, 135-151 [CrossRef][Medline] [Order article via Infotrieve]
  21. Wang, K. K., Wright, L. C., Machan, C. L., Allen, B. G., Conigrave, A. D., and Roufogalis, B. D. (1991) J. Biol. Chem. 266, 9078-9085 [Abstract/Free Full Text]

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