From the
A calmodulin-binding motif is a common structural feature of a
number of calpain substrates(1) . Since a calmodulin-like domain
has been identified in both subunits of the calpain molecule, the
proposal was made that the domain(s) would recognize the
calmodulin-binding motifs of the substrates prior to the enzymatic
modification by calpain. In keeping with the proposal, a succesful
attempt to purify µ-calpain from human erythrocytes was made by
using an affinity chromatography approach in which the synthetic
peptide C49, containing the calmodulin-binding domain of the plasma
membrane Ca
The cytosolic Ca
A striking
characteristic of a number of CANP substrates is the presence of
CaM-binding domains and of highly hydrophilic motifs (PEST sequences)
near the cleavage site. Proteins with intracellular half-lives of less
than 2 h are unusually rich in PEST regions, defined as sequences rich
in proline (P), glutamic (and aspartic) acids (E), serine (S), and
threonine (T), flanked by domains containing positively charged amino
acids. PEST sequences are common in a number of proteins rapidly
degraded by a non-ubiquitin-mediated process, which could involve CANP.
This led to the proposal that PEST sequences would sequester
Ca
However, work in this laboratory (13) has shown that
mutations lowering the PEST score of domains surrounding the
CaM-binding region of the plasma membrane Ca
One obvious component of the proposal was the
presence in both CANP subunits of Ca
The proposed mechanism for the activation of calmodulin-like
domain protein kinase could also fit CANP; in the presence of
Ca
The chemicals used were of the highest purity grade
commercially available. µ-CANP was partially purified as described
in Ref. 13. The CANP inhibitor Cbz-Leu-Leu-Tyr-CHN
Since CANP rapidly autolyzes
in the presence of Ca
Fig. 3compares the stimulation of the
Ca
This contribution describes the purification of µ-CANP by
a novel affinity chromatography approach. The results were obtained by
taking advantage of the ability of CANP, and more precisely of its
CaM-like domains, to interact with the CaM-binding region of the plasma
membrane Ca
The co-purification of both
the catalytic and the regulatory subunits of CANP was a remarkable
accomplishment. As reported recently(45) , the two subunits of
CANP dissociate in the presence of Ca
Previous work had
established that CANP migrated from the cytosol to the membranes in the
presence of
Ca
The results of the
experiments on the stimulation of the plasma membrane
Ca
The underlined amino acids, also
reported in the canonical sequence at the top of the table, have been
shown to be responsible for the coordination of Ca
We thank Dr. Paola Dainese for the sequence analysis
of the affinity purified CaMLD and Carmela Galli for the drawing of Fig. 4.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-ATPase, was coupled to a Sepharose
matrix. The calmodulin-like domain of the catalytic subunit of human
µ-calpain expressed in Escherichia coli was also retained
by the C49-Sepharose column. Both µ-calpain and the calmodulin-like
domain interacted with C49 in a Ca
-dependent way and
were eluted from the column by Ca
-chelating agents.
The finding confirmed the interaction between the calmodulin-binding
domain of the plasma membrane Ca
-ATPase and the
calmodulin-like domain of µ-calpain. Experiments were performed to
establish whether irreversibly inactivated µ-calpain or its
expressed C-terminal portion containing the calmodulin-like domain
could activate the hydrolysis of ATP by the plasma membrane
Ca
pump, in keeping with the evident ATPase
stimulation of the same pump by calmodulin. A stimulation was observed,
but it was much weaker than that induced by calmodulin.
-activated neutral protease
calpain (CANP,
(
)EC 3.4.22.17) appears to be
involved in physiologically important processes such as cell division
(2), signal transduction(3, 4) , and long term
potentiation(5) . Its role in the pathological modifications of
cells and tissues (e.g. degenerative diseases of muscle and
nerve(3, 6, 7, 8, 9, 10) ,
development of hypertension, cataract formation) is also frequently
discussed. In spite of the intense interest of CANP as an object of
study, several fundamental questions on its structure (e.g. the role of the heterodimer), its mechanism of activation, and its
recognition of target proteins remain obscure. The latter point, i.e. the identification of the CANP region responsible for the
recognition of substrates, appears of particular interest, also
considering that it could lead to the development of inhibitors
alternative to those directed to the active site.
, thus creating the microenvironment of higher
Ca
concentration necessary for CANP activation (11,
12).
-ATPase
failed to influence the susceptibility of the latter to µ-CANP. By
contrast, modifications of the CaM-binding domain of the substrate, e.g. by phosphorylation or its ``occupation'' by
CaM, dramatically decreased the susceptibility of the ATPase to the
protease. The proposal was thus made that an accessible CaM-binding
region is critical for substrate (i.e. the Ca
pump) proteolysis, i.e. the CaM-binding domain of the
substrate would be important for recognition and interaction with the
protease(13) .
-binding domains
with strong sequence similarity to CaM(14, 46) . The
striking resemblance on the amino acid level between the
Ca
-binding domains of CANP and those of CaM, troponin
C, and parvalbumin had already been emphazised 10 years
ago(38) . CANP is not the only example of a protein derived from
the fusion of a gene for a CaM-like protein and a gene controlling the
expression of a protein having another activity (an enzyme). The first
description of the primary structure of a kinase described as an hybrid
of a Ser/Thr protein kinase catalytic site and a CaM-like domain
(calmodulin-like domain protein kinases) appeared in 1991(39) .
In the meantime, other enzymes sharing the same features have been
characterized in plants (39, 40) and protists like Parameciumtetraurelia(41) . They share a
number of properties with CANP. They bind Ca
through
a regulatory domain on the same polypeptide, which contains the
catalytic domain, and they exhibit micromolar dependence on
Ca
for activity. The C-terminal portion of this
family of kinases includes four Ca
binding motifs
similar to those of CaM and the C-terminal portion of both CANP
subunits (domains IV and IV`). shows a comparison of the
sequences of the Ca
-binding loops of CaM, of the
catalytic and regulatory subunits of different CANP isoforms, and of
the calmodulin-like domain protein kinase. The underlined amino acids
coordinate Ca
in CaM; clearly, the canonical CaM
sequence is conserved in both CANP subunits and in the hybrid protein
kinase.
, the CaM-like domain of the former interacts with
a positively charged amphipatic
-helical domain next to or within
the autoinhibitory region. The interaction would remove the
autoinhibition and thus activate the kinase(40) . The CaMLD of
CANP could, however, be important not only in the activation pathway of
the protease, but also for the substrate recognition and targeting
process; for example, the CaMLD of CANP interacts with the endogenous
inhibitor CALST(42, 47) . As shown in this contribution,
it also interacts with substrates, particularly those containing
CaM-binding domains; the CaM-binding site of the preferred CANP
substrate in erythrocytes (the plasma membrane
Ca
-ATPase) indeed interacts with the CaM-like domain
of the protease.
was
synthesized as described previously(15) . SDS-polyacrylamide gel
electrophoresis was carried out according to Laemmli(16) .
Peptide Synthesis
The peptides used for the experiments
were derived from the C-terminal portion of isoform 1CI (17) of
the plasma membrane Ca-ATPase (hPMCA1CI). Peptides
A18 (EEIPEEELAEDVEEIDHA, PEST sequence 1) (18) and C49
(EEIPEEELAEDVEEIDHAERELRRGQILWFRGLNRIQTQIRVVNAFRSS, PEST sequence 1
+ the CaM-binding domain) (19) were synthesized on an
Applied Biosystems (Foster City, CA) peptide synthesizer model 431A
using the standard-scale FastMoc
chemistry according to the
manufacturer's instructions. The purity of all synthetic products
was confirmed by electrospray mass spectrometry.
Expression of the CaMLD of Human µ-CANP in
Escherichia coli
The cDNA coding for the CaMLD was obtained by
PCR amplification of the corresponding cDNA region of human µ-CANP
large subunit gene(20) . The 0.6-kb BamHI cDNA fragment
was inserted into the BamHI site of the plasmid pET-3d (a
vector for the expression by T7 RNA polymerase, Novagen, Madison,
WI)(47) . The recombinant plasmid pET-L-CaMLD was then used to
transform the protease-poor host strain of E. coli (BL21(DE3)pLysE). The CaMLD was expressed by this T7 RNA
polymerase system using the following procedure. The cultures were
induced by the addition of
isopropyl-1-thio--D-galactopyranoside to a final
concentration of 0.4 mM. After a 4-h incubation, they were
collected by centrifugation and stored overnight at -80 °C.
The cells were thawed, resuspended in 50 mM Tris buffer, 1
mM EDTA, 1 mM NaN
, pH 7.5, and lysed by
sonication on ice (50% pulse for 2 min). The supernatant containing the
expressed protein was cleared by centrifugation and stored at -20
°C. N-terminal sequencing after affinity purification confirmed
that the expressed protein contained, in addition to the CaMLD region
(residues 516-714) of the human µ-CANP catalytic subunit, 11
additional amino acids derived from the expression plasmid.
Coupling of Peptide C49 to CNBr-activated Sepharose
4B
1 g of CNBr-activated Sepharose 4B (Pharmacia Biotech Inc.)
was allowed to swell in 25 ml of 1 mM HCl (15 min). The resin
was washed with 500 ml of 1 mM HCl. 10 mg of peptide C49 (1.7
µmol) were solubilized in 7.8 ml of coupling solution (100 mM NaHCO, 500 mM NaCl, 0.05 mM
CaCl
, pH 8.3), added to the wet resin, and incubated
overnight at 4 °C. The resin was washed with the coupling solution
and finally incubated overnight in 10 ml of blocking solution (1 M ethanolamine). The C49-resin was ready after an additional washing
step with the washing buffer (50 mM Tris-HCl, 0.5 mM
CaCl
, 0.5 mM 2-mercaptoethanol, pH 7.5). The same
procedure was used to couple peptide A18 to the resin.
Partial Purification of µ-CANP and Isolation of
Ghosts from Human Erythrocytes
The crude µ-CANP used for the
experiments described here was isolated from freshly collected human
erythrocytes. One unit (350-500 ml) of freshly drawn venous human
blood in citrate buffer was filtered through a Pall filter (Pall
Schweiz AG, Muttenz, Switzerland) to eliminate the white cells. The red
cells were lysed in hypotonic Tris buffer (10 mM Tris, pH 7.5)
containing 2 mM Na-EDTA, and the cytosolic fraction was
chromatographed on DEAE-Sepharose CL-6B (Pharmacia). The peak
containing the CANP activity was eluted by a NaCl-linear gradient (for
a more precise description of the method used see Ref. 13). The
membrane fraction was collected after the lysis of the red cells,
washed 5 times with 50 mM Tris, 0.5 mM Na-EDTA, pH
7.5, to eliminate the cytosolic proteins (especially endogenous CaM and
µ-CANP), and stored at -80 °C. The ghosts obtained with
this procedure were used to measure the stimulation of the activity of
the plasma membrane Ca
-ATPase by CaM, by the
irreversibly inhibited µ-CANP, and by the purified CaMLD.
Purification of µ-CANP on the C49-Affinity
Column
Affinity purification of CANP based on its interaction
with the endogenous inhibitor (calpastatin) or with a partial sequence
derived from the latter (e.g. the 27-residue peptide,
DPMSSTYIEELGKREVTIPPKYRELLA, encoded by exon 1B of the human
calpastatin gene(25) ) is absolutely
Ca-dependent (i.e. CANP binds to the column
in the presence of Ca
and is eluted with a buffer
containing Ca
-chelating
agents
(
)). The attempt to purify CANP with the
C49 affinity column was similarly based on the assumption of
Ca
-dependence of the interaction of the protease with
the CaM-binding domain of the Ca
-ATPase of the plasma
membrane, the preferred CANP substrate in
erythrocytes(26, 27) .
, the crude CANP preparation
obtained as described above was treated with an irreversible, active
site-directed CANP inhibitor (Cbz-Leu-Leu-Tyr-CHN
) (15, 22) prior to incubation with Ca
.
100 µM (final concentration) of the inhibitor was added to
the preparation with gentle mixing at 4 °C. After 30 min,
CaCl
was added to a final concentration of 500
µM, and the reaction mixture was left to incubate on ice
for 3 h. Under these conditions the inhibitor alkylated the essential
cysteine residue at the active site of CANP. The treatment prevented
autoproteolysis and blocked the enzyme in the native 80/30-kDa
heterodimeric form. The inhibitor-modified crude enzyme preparation was
loaded on the C49-affinity column (0.8
6 cm) previously
equilibrated with the washing buffer. The column was washed extensively
with 20 volumes of washing buffer containing 1 M NaCl and then
with 20 volumes of washing buffer to remove nonspecifically bound
proteins. The bound µ-CANP was eluted with 50 mM Tris-HCl,
2 mM Na-EDTA, 0.5 mM 2-mercaptoethanol, pH 7.5
(elution buffer).
Purification of the CaMLD of Human µ-CANP on the
C49-Affinity Column
The crude E. coli extract was
loaded on the C49 affinity column previously equilibrated with the
washing buffer. The column was washed extensively as described above,
and the overexpressed CaMLD was eluted with the elution buffer. The
identity of the protein was confirmed by N-terminal sequencing.
Preparation of µ-CANP and of the CaMLD to Study Their
Stimulation of the Plasma Membrane
Ca
The irreversibly
inactivated catalytic subunit of human µ-CANP and the purified
CaMLD were used to test their ability to stimulate the plasma membrane
Ca-ATPase
-ATPase. The crude µ-CANP was modified with the
irreversible inhibitor Cbz-Leu-Leu-Tyr-CHN
in the presence
of 0.5 mM Ca
and was loaded on a 12%
SDS-Lämmli gel. After gel staining with Coomassie Brilliant Blue,
the band corresponding to the catalytic subunit was cut and
electroeluted from the gel. The denatured, covalently inhibited protein
isolated in this way was inactive as a protease when tested on the
isolated plasma membrane Ca
-ATPase and on a
fluorogenic substrate (Suc-Leu-Tyr-AMC). The fraction containing the
inhibitor-modified catalytic subunit of µ-CANP as well as the
purified CaMLD was dialyzed against the incubation buffer to be used
later for the measurement of the ATPase (30 mM Hepes, 120
mM KCl, 1 mM MgCl
, 0.5 mM EGTA,
pH 7.2(23) ).
Ca
The Ca-ATPase Activity
Assay
-ATPase was measured in
erythrocyte ghosts by following the release of inorganic phosphate by
the colorimetric method of Lanzetta et al.(23) .
CaCl
was added to the reaction mixture to yield a final
free concentration of 7 µM (calculated with the help of a
computer program(24) ). The reaction was performed in the
presence of 1 mM ATP at 37 °C and quantified by measuring
the absorption at 660 nm (Shimadzu dual-wavelength spectrophotometer,
model UV-3000, Shimadzu, Kyoto, Japan). The stimulation of the
Ca
-ATPase by CaM was tested using CaM, µ-CANP,
and CaMLD at concentrations between 0.01 and 2 µM.
Preparation of the C49 Affinity Column
The
synthetic C49 peptide used for the affinity column was derived from the
C-terminal portion of isoform 1CI of the plasma membrane
Ca-ATPase (hPMCA1CI). The peptide contains the PEST
sequence (A18) and the CaM-binding domain of the ATPase. To verify the
accessibility of the latter domain in the peptide coupled to the
Sepharose matrix, a preliminary assay was performed with CaM. As
expected, the protein bound to the column in the presence of
Ca
and could be eluted with 50 mM Tris, 2
mM Na-EDTA, 0.5 mM 2-mercaptoethanol. Thus, the
CaM-binding site of the peptide coupled to the matrix was fully
accessible.
Purification of µ-CANP on the C49 Affinity
Column
The crude erythrocyte extract containing the irreversibly
inhibited µ-CANP was loaded on the C49 column previously
equilibrated with the Ca-containing washing buffer (Fig. 1, laneL). The extensive washing allowed
the elimination of the nonspecifically bound material in the
flow-through (Fig. 1, laneFT). The protein
bound to the peptide C49 coupled to the matrix was then eluted with the
elution buffer containing a Ca
-chelating agent (Fig. 1, lanes1-6) and was identified as
CANP using a polyclonal antibody against the human platelet-m-CANP,
which cross-reacted with erythrocytes µ-CANP (data not shown).
Figure 1:
Purification of human µ-CANP using
the C49 affinity column. LaneS, low molecular weight
standard; laneL, crude human erythrocytes lysate
loaded on the affinity column; laneFT, flow-through
(unbound protein); lanes1-6, elution peak
containing the purified µ-CANP. Additional details are found under
``Materials and Methods.''
The possibility was considered that µ-CANP was trapped by the
column by unspecific interactions with the matrix. The synthetic
peptide (A18) from the C-terminal portion of isoform 1CI of the plasma
membrane Ca-ATPase, located immediately upstream of
the CaM-binding domain, was thus coupled to the matrix used for the C49
affinity column (CNBr-activated Sepharose 4B); the A18 column failed to
bind CANP in a Ca
-dependent manner.
Purification of the CaMLD of the Catalytic Subunit of
µ-CANP Using the C49 Affinity Column
Since the heterodimeric
form of µ-CANP could be purified by taking advantage of the
affinity of the protease for the CaM-binding region of the substrate,
it seemed very likely that the regions of the CANP molecule responsible
for the interaction were the CaM-like domains of the protease (domain
IV and/or domain IV`). The crude E. coli extract containing
the expressed CaMLD was loaded on the C49 affinity column, previously
equilibrated with the washing buffer (Fig. 2, laneL). The column was washed extensively (Fig. 2, laneFT) as described above, and the pure CaMLD could
be eluted with the EDTA-containing buffer (Fig. 2, lanes1-3). N-terminal sequencing confirmed that the
isolated protein was indeed the CaMLD of µ-CANP.
Figure 2:
Purification of the CaM-like domain
(CaMLD) of the catalytic subunit of human µ-CANP using the C49
affinity column. LaneS, low molecular weight
standard; laneL, crude E. coli extract
loaded on the affinity column; laneFT, flow-through
(unbound protein); lanes1-3, elution peak
containing the purified CaMLD. Additional details are found under
``Materials and Methods.''
Activation of the Ca
The
Ca-ATPase by
µ-CANP and by Its Isolated CaMLD
-dependent ATP-hydrolysis by the plasma membrane
Ca
-ATPase can be reversibly stimulated by CaM
(28-29) or irreversibly stimulated by CANP
proteolysis(30) . µ-CANP cleaves the purified pump upstream
to the CaM-binding domain, removing its autoinhibitory effect on the
pump. Interestingly, the plasma membrane Ca
-ATPase
could be weakly activated by irreversibly blocked CANP and by its
isolated CaMLD.
-ATPase of erythrocytes ghosts by incubation with
CaM with the irreversibly inactivated µ-CANP and with the CaMLD of
the major CANP subunit; maximal stimulation in the case of CaM was
reached at about 1.7 µM, when the activity of the ATPase
was 3.5 times higher than in the absence of the effector. The
stimulation by µ-CANP was much less pronounced (1.7-fold), and that
by the CaMLD even weaker (1.4-fold). Both reached maximal value at
about 2 µM.
Figure 3:
A comparison between the activation
effects by CaM, by the irreversibly inactivated catalytic subunit of
human µ-CANP and by its isolated CaMLD on the plasma membrane
ATPase. A, stimulation of the plasma membrane
Ca-ATPase by CaM; B, stimulation of the
plasma membrane Ca
-ATPase by irreversibly inactivated
µ-CANP; C, stimulation of the plasma membrane
Ca
- ATPase by the CaMLD. Additional details are found
under ``Materials and Methods'' and
``Discussion.''
-ATPase. The general preference of CANP
for CaM-binding proteins as substrates in vivo and in
vitro(1) and previous work showing that changes in the
CaM-binding domains of substrates impaired their susceptibility to CANP
digestion (13) led to the proposal of a direct interaction
between CANP and CaM-like domains. This interaction was established by
the following findings. (a) µ-CANP could be purified by
affinity chromatography on a column of peptide C49, which contains the
CaM-binding domain of the plasma membrane Ca
-ATPase; (b) an expressed peptide containing the CaMLD of the catalytic
subunit of human µ-CANP was isolated from crude E. coli extracts using the same C49 affinity column; (c) the
activity of the Ca
-ATPase of erythrocytes ghosts was
stimulated, albeit only weakly, by irreversibly inactivated µ-CANP
and by the purified CaMLD (subdomain IV of human µ-CANP). Although
the work described here only relates to the plasma membrane
Ca
-ATPase, it appears likely that the CaMLDs of CANP
will interact in a similar way with the CaM-binding regions of other
CANP targets (e.g. spectrin).
. In the
chromatography approach described here, the protease was loaded onto
the column in the presence of 500 µM Ca
,
a concentration inducing the dissociation. In spite of that, both
subunits were retained in the column in a
Ca
-dependent way and eluted with
Ca
-chelating agents. This is further evidence that
both subunits of CANP interacted with the CaM-binding domain of the
ATPase. The finding that the subunits were not eluted in an equimolar
ratio (Fig. 1, lanes1-6) could be traced
back to the loss of a portion of the regulatory subunit during the
preceding purification steps described under ``Materials and
Methods.'' A comparison of lanesL and FT in Fig. 1reveals that nearly all of the loaded small
subunit (band at 30 kDa) was bound to the column, with only a faint
trace of it appearing in the flow through.
(27, 31, 32, 33, 34, 35, 36) .
Although it is not yet conclusively established whether the interaction
occurs with membrane/cytoskeletal proteins or with membrane
phospholipids, recent experiments have shown that trypsinization, but
not the treatment with phospholipase C, greatly reduced µ-CANP
binding to inside out membrane vesicles(34) . Moreover, Kawasaki et al.(37) have reported that the binding of CANP to
membranes was inhibited by a fragment of calpastatin that interacted
with the CaMLDs of CANP. As discussed in this work, this region of the
protease interacts with target proteins as well. The results presented
here are relevant to the problem, i.e. they add weight to the
suggestion that CANP binds to membrane or cytoskeletal substrate
proteins (probably at physiological Ca
concentrations
as well). On the other hand, it must be mentioned that work in this
laboratory has never identified the autolyzed µ-CANP (lacking the
Gly-rich hydrophobic tail of the regulatory subunit, but containing the
intact CaMLDs) bound to the membrane(27) .
-ATPase by CANP are of interest. It was already
known that limited cleavage of the ATPase by CANP led to its
stimulation due to the removal of the autoinhibitory CaM-binding domain
of the pump. The present work is the first report of a stimulation of
the pump by CANP, independent from proteolysis. Since the activation by
inactive CANP or by its CaMLD was far lower than in the case of CaM, a
word of caution on this matter is in order; the results nonetheless
strenghten the proposal of a direct interaction between the CaMLD of
CANP and the CaM-binding regions of substrates. That the stimulation,
however limited, was not due to the presence of traces of proteolytical
activity in the preparation of irreversibly inactivated, electroeluted,
catalytic subunit of µ-CANP was established by appropriate control
experiments using a fluorogenic substrate (Suc-Leu-Tyr-AMC), which is
particularly sensitive to CANP. Another point that could be mentioned
is that the work described here has considered only the catalytic
subunit or the CaMLD located in the C-terminal portion of it. It is in
principle possible that the slightly different CaMLD of the regulatory
subunit is principally responsible for the interaction with substrates.
The finding that CaM-binding proteins are preferred substrates of CANP in vivo(26, 27, 43, 44) indicates that the
protease may be able to interact with them even in a system containing
high concentrations of CaM, i.e. to compete with the latter
for the same binding site in vivo. It could thus be speculated
that the binding of CANP to the target protein would not be necessarily
followed by its cleavage. In the particular case of the
Ca
-ATPase of the plasma membrane, the in vitro experiments shown here have documented that the irreversibly
inactivated CANP or its CaMLD could qualitatively mimic the action of
CaM, activating the pumping of Ca
out of the cell. It
could then be further speculated that, following the return of the
intracellular Ca
concentration to normal, CANP would
leave the pump and return to the quiescent, soluble form. However,
should the intracellular Ca
concentration rise
excessively, or be consistently maintained at a higher than normal
level, CANP would cleave the (CaM-binding) autoinhibitory domain of the
ATPase, permanently activating the latter. An activation pathway
alternative to that generally accepted (21) thus can be
postulated (Fig. 4). (a) The increased
Ca
-concentration in a limited portion of the cytosol (Fig. 4, a and b) would allow CANP to bind to a
few molecules of CaM-binding proteins (e.g. the
Ca
-ATPase) embedded in the membrane; (b) the
substrate-associated protease could partially digest the target (Fig. 4, b and c). Importantly, this may occur
in the absence of CANP autolysis(27) ; (c) if the
target is the Ca
-ATPase, the binding of CANP to its
CaM-binding domain (Fig. 4b) and/or its partial
proteolysis (Fig. 4c) would induce activation of the
enzyme to restore the physiological intracellular Ca
concentration; (d) at this point, CANP would dissociate
from the substrate and return to the cytoplasm in the native form,
ready to be reactivated in the case of a new increase in Ca
concentration. The partially proteolyzed pump (Fig. 4c) is then in all likelihood further proteolyzed
by other cellular proteases specialized in the elimination of partially
modified proteins(26) .
Figure 4:
A proposal for the effect of CANP on the
plasma membrane Ca-ATPase. a, resting state; b, the increase of the Ca
concentration in
the cytosol allows the binding of CANP to the ATPase molecule;
possibly, CANP acts as ``pseudo CaM'' when the Ca
concentration is insufficient to trigger the activation of the
protease. The binding of CANP, perse, would be
sufficient to partially activate the ATPase; c, CANP (the
intact or the autolytically modified form) cleaves the pump,
eliminating the autoinhibitory function of the CaM-binding domain; the
Ca
-ATPase would be permanently activated and then
further degraded by the cellular proteolytical machinery. This pathway
eventually implies the loss of the ATPase molecule and the irreversible
activation of CANP by autolysis. d, if the Ca
concentration in the cytosol would rapidly decrease, the CANP and
the plasma membrane Ca
-ATPase would both return,
unmodified, to the resting state. Additional details are found under
``Discussion.''
Table: Sequence similarity between some E-F-hand
Ca-binding proteins, the CaM-like domains of the two
subunits of different CANP isoforms, and the CaM-like domain of a
hybrid protein kinase from plants
in
CaM; the amino acids corresponding to X, Y, Z and -Z do so
through the oxygen of their (carboxylic) side chain; -Y through
its peptide-bond carbonyl oxygen; -X through the oxygen of a
water molecule. G represents a conserved glycine in the middle of the
Ca
-binding loops of all EF-hand type proteins (see
``Discussion'').
-activated neutral
protease (calpain); CaM, calmodulin; CaMLD, calmodulin-like domain;
Cbz, benzyloxycarbonyl.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.