(Received for publication, September 12, 1995; and in revised form, October 20, 1995)
From the
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.
The multicatalytic protease (MCP) ()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
- and
-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
family, and the two inner rings each contain 7
-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
-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.
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 µM
Ca
(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 µM
Ca
as described under
``Experimental Procedures.'' The presence of equal amounts of
protein on the nitrocellulose filters was confirmed by Ponceau S
staining.
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.
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'' (
-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
-helix was observed in the
presence of Ca
. (
)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. (
)It is also unclear
that the Ca
effect is mediated only by
rREG
. Native MCP applied to nitrocellulose binds both
Ca
and ruthenium red,
and
-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.