(Received for publication, October 6, 1994; and in revised form, November 14, 1994)
From the
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.
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 ()(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.
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 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
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
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
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) .
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.
Figure 1:
Ca dependence of
substrate proteolysis by µ-CANP. The figure shows the
susceptibility of expressed (
, A18-B28;
, A18*-B28*;
A18-B28-P) and synthetic peptides (
, C49; + C28;
, 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;
, A18*-B28*;
, A18-B28-P; synthetic:
+, C28;
, C24;
, C24-P) to µ-CANP proteolysis was
investigated at different protease to peptide ratios. Additional
details are found under ``Materials and
Methods.''
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;
, 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).