Vol.
187,
No.
September
2, 1992
BIOCHEMICAL
Pontremoli,
Institute Received
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
MODULATION OF INHIBITORY
S.
AND
16, 1992
EFFICIENCY OF RAT SKELRTAL MUSCLR CALPASTATIN BY PHOSPHORYLATION
P.L.
Viotti,
of Biological
July
15,
751-759
M. Michetti, and E. Melloni
Chemistry
F. Salamino,
, University
B. Sparatore
of Genoa, Genoa, Italy
1992
SUMMARY. Rat skeletal muscle calpastatin form is markedly modified in its inhibitory properties by means of a reverse reaction which involves both phosphorylation and dephosphorylation. Dephospho-calpastatin shows greater inhibitory efficiency versus p-calpain, whereas phospho-calpastatin shows maximal inhibition versus m-calpain. Both forma are present in fresh rat muscle. Phosphorylation has been reproduced "in vitro" using a homologous C&Z'+ independent protein kinase and found to result in the incorporation of approximately one mole of 32P per mole of protein. Dephosphorylation was induced by treatment with alkaline phosphatase and 32P release shown found to correlate with modifications of the inhibitory properties. This reversible covalent modification of calpastatin is considered en important advancement in the understanding of how different calpain isoforms can be more efficiently controlled by a single inhibitor isozyme form. 0 1992AcademicPress, 1°C.
The
INTRODUCTION. present the
in all
animal
calpain
of the metal
ion.
protein,
compartment
(l-3,
16,
in a single
form,
containing
for
interaction
activity
predominant
(8).
17).
calpain
from skeletal
chromatography inhibitory
with
the
form
localized
At difference
with calpain,
in
(6,7).
We have recently into
calpastatin
is on
molecular
form (termed calpastatin
by a cell
is present
consensus
shown that
two peaks following
to the two homologous
751
is regulated
the same soluble
four domains with an identical
The two forms show similar
calpastatin
activity
calpastatin,
with respect
calpain,
of two isozymes distinguishable
2+ to Ca , the proteinase
muscle separates
efficiency
proteinase,
and accordingly definedp-calpain and m2+ at yM Ca the latter at mM concentration
is active
In addition
inhibitor
in
dependent neutral
requirement,
The former
(l-5).
natural
cells 2+ Ca
basis of their
2+ Ca
soluble
sequence
calpastatin ion
exchange
mass, but a different calpain
isoforms.
I) shows maximum
The
efficiency
0006-291X/92 $4.00 Copyright 0 1992 by Academic Press. Inc. All rights of reproduction in any form reserved.
Vol.
187,
versus tin
No.
2, 1992
p-calpain,
II)
on
suggested
the basis
rat
skeletal
single
calpastatin
mental
evidences
phosphorylation decrease inhibition calpastatin line
versus
evidence muscle
indicating catalyzed
in
form.
of two
calpastatins
was
modifications, phosphorylation
of the inhibitor
was In this
protein.
the two calpastatin
the dephospho-
calpasta-
species
present
and the phospho-form
is also
supported
of a
by experi-
that
the predominant calpastatin I form undergoes 2+ by a Ca resulting in a independent protein kinase, efficiency
m-calpain.
Conversely
is restored
form (termed
in which
This observation
the inhibitory of
(9,lO)
that
COMMUNICATIONS
post-translational
modification
are presumably
isozyme
calpastatin
of
reports
indicating
RESEARCH
The presence
as the result
covalent
BIOPHYSICAL
abundant
m-calpain.
of preliminary
as a possible
AND
the less
interpreted
we present
paper, in
whereas
is more active
temptatively also
BIOCHEMICAL
following
versus
p-calpain
the original
dephosphorylation
and
in
inhibitory of
calpastatin
an
increased
specificity
of
II by alka-
phosphatase.
MATERIAL, AND METHODS
a. . p-C&pain and m-calpain were purified from CalDaind-Durlflcatlon rat skeletal muscle as previously reported (11). The specific activities of the purified proteinases were 3950 units/mg and 4000 units& for )1-calpain and mcalpain, respectively. The two calpastatin forms were isolated by ion exchange chromatography as previously described (8). For a better separation, calpastatin II was resubmitted the to same chromatographic step. The specific activities of purified calpastatin I and II were 5600 and 4400 units/mg, respectively. . . p-Calpain, m-calpain and calpastatin BssaYnfcalDaindcalPastatinactlvltv activities were routinely assayed as pieviously reported (12). One unit of calpain activity is defined as the amount that causes release of 1 nmole of free aminogroups per minute in the specified conditions. One unit of calpastatin is defined as the amount that inhibits a unit of calpain activity. a. . Purlflcatlon Qfalkaline~frnmtiDlasmamembraneaf~ ner&rophi&. Neutrophils, prepared from freshly human blood as described in (13), were suspended in 6 ml of 0.25 M sucrose (25 x lo4 /ml) and distrupted with 50 strokes in a glass-teflon homogenizer. The crude lisate was then loaded on a 55% Percoll solution and centrifuged for 20 min at 100,000 x g as described in (15). The membrane fraction was collected and than suspended in 4 ml of 2 mg/ml sodium deoxicolate. The insoluble material was discarded by centrifugation for 10 min at 100,000 x g and the supernatant was loaded on a DE-52 column (1xlOcm) previously equilibrated in 50 mM borate pH 7.5 containing 0.1 mM EDNA. The column was washed with 10 column-volume of the same buffer, and the proteins were eluted with a linear gradient of sodium chloride from 0 to 0.3 M (lOO+lOO ml). The phosphatase activity emerges in a single peak eluted at O-12-0.15 M NaCl. The fractions under the peak were collected and concentrated to 4 ml by ultrafiltration on an Amicon YM-10 membrane. The alkaline phosphatase was then submitted to hydrophobic chromatography on a butyl-agarose column in the same condition as reported in (8). The peak containing the phosphatase activity was collected, concentrated and dialyzed against 50mM borate, pH 7.5, containing 0.1 mM EDTA. The alkaline phosphatase activity was assayed in 0.5 ml of 5OmM sodium borate, pH 8.3, containing 2mM 2-glycerol-phosphate, and an appropiate amount of phos752
Vol. 187, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
phatase. The mixture was incubated for 15 min at 37°C and the inorganic phosphate liberated was determinated by procedure reported in (14). One unit is defined as the amount of enzyme which catalyzes the release of 1 nmol/min of Pi from 2-glycerol-phosphate under the assay conditions. The specific activity for the purified alkaline phosphatase was 4300 (units/mg protein). Alternatively, the alkaline phosphatase was assayed in the conditions reported above, following the dephosphorylation of 32P-histone, previously phosphorylated by Peak II protein kinase. ** . PurlflcatlonQfPeaksIdIILLmkinasefrPmm muscle. Freshly collected rat skeletal muscle (120 g) was minced and suspended in 600 ml of cold 10 mM HEPES, pH 7.5, containing 0.25 M sucrose, 10 mM 2-mercaptoethanol, 5 mM EDTA, 0.1 mM leupeptin, and 2 mM PMSF. The tissues was homogenized on Waring Blendor homogenizer set up at maximum speed and centrifuged at 40,000 x g for 15 and loaded on a DE-52 column (3.0 x 15 min. The clear supernatant was collected cm) previously equilibrated in 10 mM HEPES, pH 7.5, containing 10 mM P-mercaptoethanol and 1 mM PD'A. The column was washed with the same buffer, until the absorbance at 280 nm was below 0.02. The absorbed proteins were then eluted with a linear gredient of sodium chloride from 0 to 0.3 M (200+200 ml) dissolved in the equilibrating buffer. The flow rate was 0.4 ml/min and fractions of 2.5 ml were collected. The histone-phosphorylating activity emerged in two separate pAC3: a first one eluted at a NaCl concentration of 0.08-0.11 M and named Peak I protein kinase, contains a protein kinase activity requiring C&Z+, phosphatidylserine and diacylglycerol for catalytic activity ; the second one named Peak II protein kinase, was eluted at approximately 0.22 M NaCl, and found to contain a protein kinase activity independent by the presence or the absence of any effecters. The two peaks were separately colleted (Peak I proteine kinase, fractions 34-65; Peak II protein kinase, fractions 95-130), concentrated and further purified by affinity chromatography on threonine-sepharose columns equilibrated in 10 mM borate pH 7.5 containing 0.1 mM EDTA, 10 mM 2-mercaptoethanol, and 10% glycerol (13). This procedure increases the specific activity of Peak I protein kinase approximately 250 fold and that of Peak II protein kinase more than 500 fold. The final specific activity of Peak I and II protein kinase was 108 units/mg and 280 units/mg respectively. Protein kinase activity was assayed pH 7.5, containing 0.4 uCi of (J'32-P) ATP, 10 in 0.2 ml of 50 mM sodium borate, and 1 mM PDTA. For Peak I ).JM ATP, 5mM MgC12, 0.1 mg of type III-S histone, protein kinase EIYIA was replaced with 10 )lg of phosphatidylserine, 0.2pg of The mixture was incubated for 10 min at 30°C diacylglycerol and 0.5 mM CaC12. and stopped by addition of 1 ml of 10% (W:V) trichloroacetic acid and 1 mg bovine serum albumin. One unit of protein kinase activity is defined as the amount of enzyme which catalyzes the incorporation of 1 nmol of 32P/min into histone under the assay conditions. v nf c&pa&&& 1. Purified calpastatin I(50 units) was incubated at 30°C for 10 min in 0.4 ml of 50 mM sodium borate, containing 5 mM PH 7.5, Peak II protin kinase. The samples MgC12, 10 JJM ATP with 5 units of purified were then heated for 2 min at 90°C and assayed for their inhibitory efficiency against the calpain isoforms. m af c,&astatj.ns. Purified calpastatins (50 units) were incubated at 37°C for 30 min in 0.8 ml of 50 mM sodium borate pH 8.3 with alkaline phosphatase (90 units). The mixtures were than heated for 2 min at 90°C and assayed for their inhibitory efficiency against calpain isoforms.
RESULTS Muscle
cells
protein
kinase
contain
a protein
(see Methods),
homologous
calpaatatin
dependent
and
(Fig.
is completed
kinase that
activity,
herewith
defined
catalyzes
the phosphorylation 32 P-groups 1A). The incorporation of when one equivalent 753
of phosphate
as
Peak
of
purified
results
is bound
II
doseto
the
Vol.
187,
No.
2, 1992
BIOCHEMICAL
Eik. L m
AND
ef -
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
I. hu rsak I.I Protein kinase. (A) Calpa-
statin I (50 units) was phosphorylated with 5 units of Peak II protein kinase aa described in Methods, in the presence of 0.5pCi of ATP 732P. At the time indicated aliquots of 0.1 ml of the incubation mixture were collected and the reaction stopped by addition of 1 ml of 10% (W:V) trichloroacetic acid together with 1 mg of bovine serum albumin. The radiolabelled proteins were recovered and washed with 5% trichloroacetic acid on glass microfibre filters and then counted. (B) Calpastatin I (50 units),phosphorylated as described in Fig. lA, was loaded on twoseparate lanes of an 8% SDS-PAGE,gel. The first lane was stained with blue Coamassie, dried and than submitted to autoradiography. The second one aas cut in 0.4 cm slices and the position of calpastatin activity was identified as reported in (8) and in Methods.
calpastatin tor
protein
tion
each
identify to
was incubated
mixture
and
characterize for
one separately
radioactive
for
the migration
at
the radiolabelled
experiments
is
agents
and its
(Peek
I protein
gels.
insensitive nature
activity.
kinase,
see Methods)
the incuba-
in two was
aliquots
utilized
migrate kinase ions,
lB,
the
in the same
posi-
utilized
these
lipids
A second protein isolated
to
and the second one
As shown in Fig.
by metal
was also
divided
bands,
activity
to be elucidated.
the inhibi-
II kinase,
sample
The Peak II protein
to activation
remains
One
protein
band and the peak of calpastatin
on the electrophoretic
90 "C,
to SDS-PAGE.
of calpastatin
process,
Peek protein
2 minutes
submitted
by autoradiography
the phosphorylation
10 min with
was then heated
localize
tion
To
molecule.
in
or
chelating
kinase
from muscle
activity cells
and
2+
found
to be activated
sulted
however
by Ca
to be very
;
its
modest (data
capacity
to phosphorylate
not shown). 754
calpastatin
re-
Vol.
187,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
0’ 0
10 20 30 CALPASTATIN I. UL
* . .
EliLLcJllmw3iIlinhibitorvsPeclflcltv
RESEARCH
COMMUNICATIONS
10 20 30 CALPASTATIN I, uL
QfcalPastatinIinducedbY
m. Calpastatin I (50 units) was incubated in the presence (open symbols) and in the absence (closed symbols) of 5 units of Peak II protein kinase as Calpastatin activity was assayed using 4 units of pdescribed in Methods. calpain (A) or m-calpain (B) as reported in Methods.
Effect
nf
w
previously
skeletal
following
its
muscle is more active
calpastatin
II
efficiency
ef
calDastatin.
(calpastatin
on m-calpain.
skeletal
nf m
order to
calpastatin
acquires properties
muscle calpastatin
the existence
isolated
for
30 min at 37°C with a sample of alkaline
established
to inactivate
then assayed onp-calpain,
to those charac-
II.
II could be converted
reaction.
Purified muscle,
phosphatase.
at 90°C for 2 min,
and m-calpain isoforms (Fig.
The
a condition
phosphatase but not calpastatin
755
its
versus ,u-calpain.
nf w
described from rat skeletal
by heating the mixture
in
In
mechanism of the two calpa-
calpastatin
I by means of a dephosphorylating
in
2,
II.
of an interconversion
II,
terminated
Pig.
increase
similar
monerties
we have explored if native
as previously
present
As shown in
by a remarkable
in j,& m
verify
forms,
I
I)
I undergoes a profound modification
characterized
calpastatin
We have
and that the less represen-
versus m-calpain and a decrease in the inhibition
of native
Bffecf
statin
calpastatin
specificity,
Thus, phosphorylated teristic
onp-calpain,
form is mostly active
phosphorylation
inhibitory
pmpstia
that the major form of calpastatin
reported
in rat ted
nn the inhU2hx
3).
activity,
to
calpastatin was incubated reaction
was
previously which
was
In these conditions
Vol.
187,
No.
BIOCHEMICAL
2, 1992
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
001
50.
15 30 CALPASTATIN
45 I or
O0
60 UL
II.
15 30 CALPASTATIN
45
60 uL
I or II.
Ei&!.L3...-i.rlininhibitorvQfnfnative inducedtim.
‘land
LI
Calpastatin I (dashed lines) or calpastatin I1 (solid lines) was incubated in the absence (closed symbols) or in the presence (open symbols) of 90 units of alkaline phosphatase as described in Methods. Calpastatin activity was assayed with 4 units of )I-calpain (A) or m-calpain (B) as reported in Methods.
calpastatin
II undergoes conversion
p-calpain
and much leas active
3A) into
on m-calpain (Fig.
a new form highly 3B).
NO
active
on
changes in
the
I were observed following treatment with alkaline 32 phosphatase (Fig.3). Furthermore, P-labelled calpastatin, produced by Peak II 32 protein kinase in the presence of [y- PIATP (see Fig. 1) when treated with 32 alkaline phosphatase progressively looses almost all the P label in a linear properties
of
(Fig.
calpastatin
TIME,
min
Ei&LCorrelationQftiSzP-labelled~~~* W ef ti native inhibitorv ProPerties. 32P-labelled calpastatin I (50 units),produced as reported in the legend to Fig. lA, was incubated with alkaline phosphatase (90 units). At the times indicated, 0.1 ml aliquots were removed to evaluate the extent of dephosphorylation (closed symbols) as described in the legend to Fig. 1A. At the same times, aliquots of 0.1 ml were removed and assayed for inhibitory efficiency againstp-calpain (open symbols) , as described in Methods. 756
Vol.
187,
No.
2, 1992
BIOCHEMICAL
01”““‘1
20
30
40
50
60
70
FRACTION
.
80
AND
90
BIOPHYSICAL
01”““’
100
20
NUMBER
30
40
RESEARCH
50
60
70
FRACTION
80
COMMUNICATIONS
90 1 0
NUMBER
c2lculpiIl~~QfefD
folloulne dephosphor7llatlon. (A) Native calpastatin E (% incubated in the absence (closed symbols) or in the presence (open symbols) of 700 units of alkaline phosphataseas described in Methods. The two samples were separately loaded on DE-32columns (0.5 x 8 cm) previously equilibrated in 50 mMacetate, pH 6.7,containing 0.1 mMEIYTAand 0.5 mM Z-mercaptoethanol. The columnswere washedwith 5 columnvolume of the samebuffer and the absorbedproteins eluted with a linear gradient of sodiumchloride from 0 to 0.3 M (40+40 ml). The flow rate was 0.2 ml/min and fractions of 0.6 ml were collected. The calpastatin activity was assayed as described in Methods. (B) Purified calpastatin I (800 units) was phosphorylated with 80 units of Peak II protein kinase as described in Methods. The phospho-calpastatin (350 units) was than incubated in the absence (closed symbols) or in the presence (open symbols) of alkaline phosphatase(700 units). The sampleswere than submitted to DE-32 chromatography and assayed for calpastatin activity as reported in Fig. 5A. -calDastatln was
units)
correlation Effect and
properties
(Fig.
4).
Qf~Q.nf;hechromatoE!raD~ProDertiesQfcalPastatin IL
results
The
dephosphorylation,
properties
ween the
two inhibitor
phorylation
was
calpastatin
II
of calpastatin
effective in those
I (Fig.
5B),
peak of activity
alkaline
phosphatase,
identical
suggest
that
phosphatase calpastatin
II
pattern,
in converting
the
characteristics
acquires the
of
chromatographic calpastatin
I. by
to that of native
0.185 M NaCl.
profile
calpastatin
Calpastatin
757
of II,
phosphorylation
Following
of both forms is shifted I (approximately
if dephos-
properties
when submitted to DE-32 chromatography at approximately
bet-
are also characterized
we have investigated
5A) or produced "in vitro"
the elution
following
I thus suggesting an interconversion
forms. Since the two calpastatins
from rat muscle (Fig.
calpastatin single
by alkaline
chromatographic elution
by a different
isolated
;1.
presented the previous section,
catalyzed
inhibitory
tion
inhibitory
with the reapparance of the native
emerges as exposure
of a to
to a posi-
0.125 M NaCl).
Vol.
187,
No.
BIOCHEMICAL
2, 1992
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
DISCUSSION In
the understanding
neutral the
proteinase,
natural
isozymes
and of a single
understand the
how calpain
Infact,
to the proteinase
and to
calpastatin
of this
it
form,
can be activated
understanding
open question
calpastatin.
protein,
with respect
mechanisms of the soluble a still
termed calpain,
inhibitory
the inhibitor
to
of the regulatory
the existence
in rat skeletal
nism
for the interconversion
of the single
calpastatin
rent
forms each characterized
phosphatase
calpain
reaction
identified
muscle.
nant calpastatin
type
specificity
less represented an homologous
forms. regulated reactions
kinase,
properties by alkaline
can
provides
efficiently
The precise and
understanding
the nature
is at present
calpastatin
the activity of how this
of the signal
that
of
rat
the predomi-
to those
inhiof
the
"in vitro"
I following
of this
by
inhibitory
cyclic
of how a single two different
activates
in
acquires
acquire
interconversion
here
phosphorylation
calpastatin
explanation
the and a
are
Conversely
II
The demonstration
the first
regulate
following kinase).
to those of
phosphatase.
that
identical
II)
(Peak II protein
identical
derivative
demonstrate
properties
II or "native"
by a kinase
from rat muscle,
type (calpastatin
calpastatin
interconversion
isozyme
chromatographic
calpastatin
chromatographic sphorylation
and
I) isolated
diffe-
versus
found to be present
the data presented
(calpastatin
protein
phosphorylated
of
Specifically,
in
mecha-
two
specificity
is operated
forms previously
presented
muscle of a
and thus a phospho- and a dephospho-
to
A contribution
isozyme into
inhibitory
Interconversion
as the two calpastatin
skeletal
bitory
isozymes.
so far
by the results
paper which demonstrate
homologous
difficult
in a cell.
this
two
of
the presence of two calpain
has been
is provided
by a different
concerns the role
due to the large excess of
and regulated
problem
intracellular
and depho-
mechanism calpastatin
calpain of calpastatin
phospho- or
isois
dephospho-
under investigation.
ACKNCWLEDCMENTS This work was supported in part by grants from National Research Council (CNR). Target Projects: Prevention and Control Disease Factors, SP 8. Control of cardiovascular diseases, contratto n"91.00237.PF 41., PP. Ingegneria Cenetica and Biotecnologia and Biostrumentazione and from Associazione Italiana per la Ricerca sul Cancro. 758
Vol.
187,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
Murachi, T. (1983). Trends Biochem. Sci. 0, 167-169 Pontremoli, S. and Melloni, E. (1986) Ann. Rev. Biochem. 55, 455-481 Mellgren, R.L. (1987). FASEB J. 1, 110-115 Mellgren, R.L. (1980). FBBS Lett. 109, 129-133 Suzuki, K. (1987). Trends Biochem. Sci. 12, 103-105 Takano, E., Maki, M., Hatanaka, M., Marti, T., Mori, H., Titani, K., Kannagi, R., Ooi, T., and Murachi, T. (1988). Biochemistry 27, 1964-1972 Kawasaki, H., Emori, Y., Imajoh-Ohmi, S., Minami, Y.,and Suzuki, K. (1989) J. Biochem. 106, 274,281 Pontremoli, S., Melloni, E., Viotti, P-L., Michetti, M., Salamino, P., and Horecker, B.L. (1991) Arch. Biochem. Biophys. 288, 644-652 Murachi, T. (1989). Biochem. Intern. 18, 263-294 Mellgren, R.L. and Carr, T.C. (1983) Arch. Biochem. Biophys. 255, 779-786 Pontremoli, S., Melloni, E., Viotti, P-L., Michetti, M., Di Lisa, P., and Siliprandi, N. (1990). Res. Commun. 167, 373-380 Pontremoli, S., Viotti, P.L., Michetti, M., Sparatore, B., Salamino, F., and Melloni, E. (1990). Biochem. Biophys. Res. Commun.171, 569-574 Pontremoli, S., Melloni, E., Sparatore, B., Michetti, M., Salamino, F., and Horecker, B.L. (1986) F'roc. Natl. Acad. Sci., U.S.A., 83, 1685-1689 Toshima, Y., and Yoshimura, N. (1975) J. Biochem. 78, 1161-1169 Pontremoli, S., Melloni, E., Salamino, F., Sparatore, B., Michetti, M., Sacco, O., and Horecker B.L. (1986) Arch. Biochem. Biophys. 250, 23-29 Melloni, E., and Pontremoli, S., (1991) J. Nutr. Biochem. 2, 467-476 Kapprell, H.P. and Goll, D.E. (1989) J. Biol. Chem. 264, 17888-17896 Pontremoli, S., Melloni, E., Sparatore, B., Michetti, M., Salamino, F., and Horecker B.L. (1990) J. Biol. Chem. 265, 706-712
759
187,
No.
September
2, 1992
BIOCHEMICAL
Pontremoli,
Institute Received
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
MODULATION OF INHIBITORY
S.
AND
16, 1992
EFFICIENCY OF RAT SKELRTAL MUSCLR CALPASTATIN BY PHOSPHORYLATION
P.L.
Viotti,
of Biological
July
15,
751-759
M. Michetti, and E. Melloni
Chemistry
F. Salamino,
, University
B. Sparatore
of Genoa, Genoa, Italy
1992
SUMMARY. Rat skeletal muscle calpastatin form is markedly modified in its inhibitory properties by means of a reverse reaction which involves both phosphorylation and dephosphorylation. Dephospho-calpastatin shows greater inhibitory efficiency versus p-calpain, whereas phospho-calpastatin shows maximal inhibition versus m-calpain. Both forma are present in fresh rat muscle. Phosphorylation has been reproduced "in vitro" using a homologous C&Z'+ independent protein kinase and found to result in the incorporation of approximately one mole of 32P per mole of protein. Dephosphorylation was induced by treatment with alkaline phosphatase and 32P release shown found to correlate with modifications of the inhibitory properties. This reversible covalent modification of calpastatin is considered en important advancement in the understanding of how different calpain isoforms can be more efficiently controlled by a single inhibitor isozyme form. 0 1992AcademicPress, 1°C.
The
INTRODUCTION. present the
in all
animal
calpain
of the metal
ion.
protein,
compartment
(l-3,
16,
in a single
form,
containing
for
interaction
activity
predominant
(8).
17).
calpain
from skeletal
chromatography inhibitory
with
the
form
localized
At difference
with calpain,
in
(6,7).
We have recently into
calpastatin
is on
molecular
form (termed calpastatin
by a cell
is present
consensus
shown that
two peaks following
to the two homologous
751
is regulated
the same soluble
four domains with an identical
The two forms show similar
calpastatin
activity
calpastatin,
with respect
calpain,
of two isozymes distinguishable
2+ to Ca , the proteinase
muscle separates
efficiency
proteinase,
and accordingly definedp-calpain and m2+ at yM Ca the latter at mM concentration
is active
In addition
inhibitor
in
dependent neutral
requirement,
The former
(l-5).
natural
cells 2+ Ca
basis of their
2+ Ca
soluble
sequence
calpastatin ion
exchange
mass, but a different calpain
isoforms.
I) shows maximum
The
efficiency
0006-291X/92 $4.00 Copyright 0 1992 by Academic Press. Inc. All rights of reproduction in any form reserved.
Vol.
187,
versus tin
No.
2, 1992
p-calpain,
II)
on
suggested
the basis
rat
skeletal
single
calpastatin
mental
evidences
phosphorylation decrease inhibition calpastatin line
versus
evidence muscle
indicating catalyzed
in
form.
of two
calpastatins
was
modifications, phosphorylation
of the inhibitor
was In this
protein.
the two calpastatin
the dephospho-
calpasta-
species
present
and the phospho-form
is also
supported
of a
by experi-
that
the predominant calpastatin I form undergoes 2+ by a Ca resulting in a independent protein kinase, efficiency
m-calpain.
Conversely
is restored
form (termed
in which
This observation
the inhibitory of
(9,lO)
that
COMMUNICATIONS
post-translational
modification
are presumably
isozyme
calpastatin
of
reports
indicating
RESEARCH
The presence
as the result
covalent
BIOPHYSICAL
abundant
m-calpain.
of preliminary
as a possible
AND
the less
interpreted
we present
paper, in
whereas
is more active
temptatively also
BIOCHEMICAL
following
versus
p-calpain
the original
dephosphorylation
and
in
inhibitory of
calpastatin
an
increased
specificity
of
II by alka-
phosphatase.
MATERIAL, AND METHODS
a. . p-C&pain and m-calpain were purified from CalDaind-Durlflcatlon rat skeletal muscle as previously reported (11). The specific activities of the purified proteinases were 3950 units/mg and 4000 units& for )1-calpain and mcalpain, respectively. The two calpastatin forms were isolated by ion exchange chromatography as previously described (8). For a better separation, calpastatin II was resubmitted the to same chromatographic step. The specific activities of purified calpastatin I and II were 5600 and 4400 units/mg, respectively. . . p-Calpain, m-calpain and calpastatin BssaYnfcalDaindcalPastatinactlvltv activities were routinely assayed as pieviously reported (12). One unit of calpain activity is defined as the amount that causes release of 1 nmole of free aminogroups per minute in the specified conditions. One unit of calpastatin is defined as the amount that inhibits a unit of calpain activity. a. . Purlflcatlon Qfalkaline~frnmtiDlasmamembraneaf~ ner&rophi&. Neutrophils, prepared from freshly human blood as described in (13), were suspended in 6 ml of 0.25 M sucrose (25 x lo4 /ml) and distrupted with 50 strokes in a glass-teflon homogenizer. The crude lisate was then loaded on a 55% Percoll solution and centrifuged for 20 min at 100,000 x g as described in (15). The membrane fraction was collected and than suspended in 4 ml of 2 mg/ml sodium deoxicolate. The insoluble material was discarded by centrifugation for 10 min at 100,000 x g and the supernatant was loaded on a DE-52 column (1xlOcm) previously equilibrated in 50 mM borate pH 7.5 containing 0.1 mM EDNA. The column was washed with 10 column-volume of the same buffer, and the proteins were eluted with a linear gradient of sodium chloride from 0 to 0.3 M (lOO+lOO ml). The phosphatase activity emerges in a single peak eluted at O-12-0.15 M NaCl. The fractions under the peak were collected and concentrated to 4 ml by ultrafiltration on an Amicon YM-10 membrane. The alkaline phosphatase was then submitted to hydrophobic chromatography on a butyl-agarose column in the same condition as reported in (8). The peak containing the phosphatase activity was collected, concentrated and dialyzed against 50mM borate, pH 7.5, containing 0.1 mM EDTA. The alkaline phosphatase activity was assayed in 0.5 ml of 5OmM sodium borate, pH 8.3, containing 2mM 2-glycerol-phosphate, and an appropiate amount of phos752
Vol. 187, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
phatase. The mixture was incubated for 15 min at 37°C and the inorganic phosphate liberated was determinated by procedure reported in (14). One unit is defined as the amount of enzyme which catalyzes the release of 1 nmol/min of Pi from 2-glycerol-phosphate under the assay conditions. The specific activity for the purified alkaline phosphatase was 4300 (units/mg protein). Alternatively, the alkaline phosphatase was assayed in the conditions reported above, following the dephosphorylation of 32P-histone, previously phosphorylated by Peak II protein kinase. ** . PurlflcatlonQfPeaksIdIILLmkinasefrPmm muscle. Freshly collected rat skeletal muscle (120 g) was minced and suspended in 600 ml of cold 10 mM HEPES, pH 7.5, containing 0.25 M sucrose, 10 mM 2-mercaptoethanol, 5 mM EDTA, 0.1 mM leupeptin, and 2 mM PMSF. The tissues was homogenized on Waring Blendor homogenizer set up at maximum speed and centrifuged at 40,000 x g for 15 and loaded on a DE-52 column (3.0 x 15 min. The clear supernatant was collected cm) previously equilibrated in 10 mM HEPES, pH 7.5, containing 10 mM P-mercaptoethanol and 1 mM PD'A. The column was washed with the same buffer, until the absorbance at 280 nm was below 0.02. The absorbed proteins were then eluted with a linear gredient of sodium chloride from 0 to 0.3 M (200+200 ml) dissolved in the equilibrating buffer. The flow rate was 0.4 ml/min and fractions of 2.5 ml were collected. The histone-phosphorylating activity emerged in two separate pAC3: a first one eluted at a NaCl concentration of 0.08-0.11 M and named Peak I protein kinase, contains a protein kinase activity requiring C&Z+, phosphatidylserine and diacylglycerol for catalytic activity ; the second one named Peak II protein kinase, was eluted at approximately 0.22 M NaCl, and found to contain a protein kinase activity independent by the presence or the absence of any effecters. The two peaks were separately colleted (Peak I proteine kinase, fractions 34-65; Peak II protein kinase, fractions 95-130), concentrated and further purified by affinity chromatography on threonine-sepharose columns equilibrated in 10 mM borate pH 7.5 containing 0.1 mM EDTA, 10 mM 2-mercaptoethanol, and 10% glycerol (13). This procedure increases the specific activity of Peak I protein kinase approximately 250 fold and that of Peak II protein kinase more than 500 fold. The final specific activity of Peak I and II protein kinase was 108 units/mg and 280 units/mg respectively. Protein kinase activity was assayed pH 7.5, containing 0.4 uCi of (J'32-P) ATP, 10 in 0.2 ml of 50 mM sodium borate, and 1 mM PDTA. For Peak I ).JM ATP, 5mM MgC12, 0.1 mg of type III-S histone, protein kinase EIYIA was replaced with 10 )lg of phosphatidylserine, 0.2pg of The mixture was incubated for 10 min at 30°C diacylglycerol and 0.5 mM CaC12. and stopped by addition of 1 ml of 10% (W:V) trichloroacetic acid and 1 mg bovine serum albumin. One unit of protein kinase activity is defined as the amount of enzyme which catalyzes the incorporation of 1 nmol of 32P/min into histone under the assay conditions. v nf c&pa&&& 1. Purified calpastatin I(50 units) was incubated at 30°C for 10 min in 0.4 ml of 50 mM sodium borate, containing 5 mM PH 7.5, Peak II protin kinase. The samples MgC12, 10 JJM ATP with 5 units of purified were then heated for 2 min at 90°C and assayed for their inhibitory efficiency against the calpain isoforms. m af c,&astatj.ns. Purified calpastatins (50 units) were incubated at 37°C for 30 min in 0.8 ml of 50 mM sodium borate pH 8.3 with alkaline phosphatase (90 units). The mixtures were than heated for 2 min at 90°C and assayed for their inhibitory efficiency against calpain isoforms.
RESULTS Muscle
cells
protein
kinase
contain
a protein
(see Methods),
homologous
calpaatatin
dependent
and
(Fig.
is completed
kinase that
activity,
herewith
defined
catalyzes
the phosphorylation 32 P-groups 1A). The incorporation of when one equivalent 753
of phosphate
as
Peak
of
purified
results
is bound
II
doseto
the
Vol.
187,
No.
2, 1992
BIOCHEMICAL
Eik. L m
AND
ef -
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
I. hu rsak I.I Protein kinase. (A) Calpa-
statin I (50 units) was phosphorylated with 5 units of Peak II protein kinase aa described in Methods, in the presence of 0.5pCi of ATP 732P. At the time indicated aliquots of 0.1 ml of the incubation mixture were collected and the reaction stopped by addition of 1 ml of 10% (W:V) trichloroacetic acid together with 1 mg of bovine serum albumin. The radiolabelled proteins were recovered and washed with 5% trichloroacetic acid on glass microfibre filters and then counted. (B) Calpastatin I (50 units),phosphorylated as described in Fig. lA, was loaded on twoseparate lanes of an 8% SDS-PAGE,gel. The first lane was stained with blue Coamassie, dried and than submitted to autoradiography. The second one aas cut in 0.4 cm slices and the position of calpastatin activity was identified as reported in (8) and in Methods.
calpastatin tor
protein
tion
each
identify to
was incubated
mixture
and
characterize for
one separately
radioactive
for
the migration
at
the radiolabelled
experiments
is
agents
and its
(Peek
I protein
gels.
insensitive nature
activity.
kinase,
see Methods)
the incuba-
in two was
aliquots
utilized
migrate kinase ions,
lB,
the
in the same
posi-
utilized
these
lipids
A second protein isolated
to
and the second one
As shown in Fig.
by metal
was also
divided
bands,
activity
to be elucidated.
the inhibi-
II kinase,
sample
The Peak II protein
to activation
remains
One
protein
band and the peak of calpastatin
on the electrophoretic
90 "C,
to SDS-PAGE.
of calpastatin
process,
Peek protein
2 minutes
submitted
by autoradiography
the phosphorylation
10 min with
was then heated
localize
tion
To
molecule.
in
or
chelating
kinase
from muscle
activity cells
and
2+
found
to be activated
sulted
however
by Ca
to be very
;
its
modest (data
capacity
to phosphorylate
not shown). 754
calpastatin
re-
Vol.
187,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
0’ 0
10 20 30 CALPASTATIN I. UL
* . .
EliLLcJllmw3iIlinhibitorvsPeclflcltv
RESEARCH
COMMUNICATIONS
10 20 30 CALPASTATIN I, uL
QfcalPastatinIinducedbY
m. Calpastatin I (50 units) was incubated in the presence (open symbols) and in the absence (closed symbols) of 5 units of Peak II protein kinase as Calpastatin activity was assayed using 4 units of pdescribed in Methods. calpain (A) or m-calpain (B) as reported in Methods.
Effect
nf
w
previously
skeletal
following
its
muscle is more active
calpastatin
II
efficiency
ef
calDastatin.
(calpastatin
on m-calpain.
skeletal
nf m
order to
calpastatin
acquires properties
muscle calpastatin
the existence
isolated
for
30 min at 37°C with a sample of alkaline
established
to inactivate
then assayed onp-calpain,
to those charac-
II.
II could be converted
reaction.
Purified muscle,
phosphatase.
at 90°C for 2 min,
and m-calpain isoforms (Fig.
The
a condition
phosphatase but not calpastatin
755
its
versus ,u-calpain.
nf w
described from rat skeletal
by heating the mixture
in
In
mechanism of the two calpa-
calpastatin
I by means of a dephosphorylating
in
2,
II.
of an interconversion
II,
terminated
Pig.
increase
similar
monerties
we have explored if native
as previously
present
As shown in
by a remarkable
in j,& m
verify
forms,
I
I)
I undergoes a profound modification
characterized
calpastatin
We have
and that the less represen-
versus m-calpain and a decrease in the inhibition
of native
Bffecf
statin
calpastatin
specificity,
Thus, phosphorylated teristic
onp-calpain,
form is mostly active
phosphorylation
inhibitory
pmpstia
that the major form of calpastatin
reported
in rat ted
nn the inhU2hx
3).
activity,
to
calpastatin was incubated reaction
was
previously which
was
In these conditions
Vol.
187,
No.
BIOCHEMICAL
2, 1992
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
001
50.
15 30 CALPASTATIN
45 I or
O0
60 UL
II.
15 30 CALPASTATIN
45
60 uL
I or II.
Ei&!.L3...-i.rlininhibitorvQfnfnative inducedtim.
‘land
LI
Calpastatin I (dashed lines) or calpastatin I1 (solid lines) was incubated in the absence (closed symbols) or in the presence (open symbols) of 90 units of alkaline phosphatase as described in Methods. Calpastatin activity was assayed with 4 units of )I-calpain (A) or m-calpain (B) as reported in Methods.
calpastatin
II undergoes conversion
p-calpain
and much leas active
3A) into
on m-calpain (Fig.
a new form highly 3B).
NO
active
on
changes in
the
I were observed following treatment with alkaline 32 phosphatase (Fig.3). Furthermore, P-labelled calpastatin, produced by Peak II 32 protein kinase in the presence of [y- PIATP (see Fig. 1) when treated with 32 alkaline phosphatase progressively looses almost all the P label in a linear properties
of
(Fig.
calpastatin
TIME,
min
Ei&LCorrelationQftiSzP-labelled~~~* W ef ti native inhibitorv ProPerties. 32P-labelled calpastatin I (50 units),produced as reported in the legend to Fig. lA, was incubated with alkaline phosphatase (90 units). At the times indicated, 0.1 ml aliquots were removed to evaluate the extent of dephosphorylation (closed symbols) as described in the legend to Fig. 1A. At the same times, aliquots of 0.1 ml were removed and assayed for inhibitory efficiency againstp-calpain (open symbols) , as described in Methods. 756
Vol.
187,
No.
2, 1992
BIOCHEMICAL
01”““‘1
20
30
40
50
60
70
FRACTION
.
80
AND
90
BIOPHYSICAL
01”““’
100
20
NUMBER
30
40
RESEARCH
50
60
70
FRACTION
80
COMMUNICATIONS
90 1 0
NUMBER
c2lculpiIl~~QfefD
folloulne dephosphor7llatlon. (A) Native calpastatin E (% incubated in the absence (closed symbols) or in the presence (open symbols) of 700 units of alkaline phosphataseas described in Methods. The two samples were separately loaded on DE-32columns (0.5 x 8 cm) previously equilibrated in 50 mMacetate, pH 6.7,containing 0.1 mMEIYTAand 0.5 mM Z-mercaptoethanol. The columnswere washedwith 5 columnvolume of the samebuffer and the absorbedproteins eluted with a linear gradient of sodiumchloride from 0 to 0.3 M (40+40 ml). The flow rate was 0.2 ml/min and fractions of 0.6 ml were collected. The calpastatin activity was assayed as described in Methods. (B) Purified calpastatin I (800 units) was phosphorylated with 80 units of Peak II protein kinase as described in Methods. The phospho-calpastatin (350 units) was than incubated in the absence (closed symbols) or in the presence (open symbols) of alkaline phosphatase(700 units). The sampleswere than submitted to DE-32 chromatography and assayed for calpastatin activity as reported in Fig. 5A. -calDastatln was
units)
correlation Effect and
properties
(Fig.
4).
Qf~Q.nf;hechromatoE!raD~ProDertiesQfcalPastatin IL
results
The
dephosphorylation,
properties
ween the
two inhibitor
phorylation
was
calpastatin
II
of calpastatin
effective in those
I (Fig.
5B),
peak of activity
alkaline
phosphatase,
identical
suggest
that
phosphatase calpastatin
II
pattern,
in converting
the
characteristics
acquires the
of
chromatographic calpastatin
I. by
to that of native
0.185 M NaCl.
profile
calpastatin
Calpastatin
757
of II,
phosphorylation
Following
of both forms is shifted I (approximately
if dephos-
properties
when submitted to DE-32 chromatography at approximately
bet-
are also characterized
we have investigated
5A) or produced "in vitro"
the elution
following
I thus suggesting an interconversion
forms. Since the two calpastatins
from rat muscle (Fig.
calpastatin single
by alkaline
chromatographic elution
by a different
isolated
;1.
presented the previous section,
catalyzed
inhibitory
tion
inhibitory
with the reapparance of the native
emerges as exposure
of a to
to a posi-
0.125 M NaCl).
Vol.
187,
No.
BIOCHEMICAL
2, 1992
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
DISCUSSION In
the understanding
neutral the
proteinase,
natural
isozymes
and of a single
understand the
how calpain
Infact,
to the proteinase
and to
calpastatin
of this
it
form,
can be activated
understanding
open question
calpastatin.
protein,
with respect
mechanisms of the soluble a still
termed calpain,
inhibitory
the inhibitor
to
of the regulatory
the existence
in rat skeletal
nism
for the interconversion
of the single
calpastatin
rent
forms each characterized
phosphatase
calpain
reaction
identified
muscle.
nant calpastatin
type
specificity
less represented an homologous
forms. regulated reactions
kinase,
properties by alkaline
can
provides
efficiently
The precise and
understanding
the nature
is at present
calpastatin
the activity of how this
of the signal
that
of
rat
the predomi-
to those
inhiof
the
"in vitro"
I following
of this
by
inhibitory
cyclic
of how a single two different
activates
in
acquires
acquire
interconversion
here
phosphorylation
calpastatin
explanation
the and a
are
Conversely
II
The demonstration
the first
regulate
following kinase).
to those of
phosphatase.
that
identical
II)
(Peak II protein
identical
derivative
demonstrate
properties
II or "native"
by a kinase
from rat muscle,
type (calpastatin
calpastatin
interconversion
isozyme
chromatographic
calpastatin
chromatographic sphorylation
and
I) isolated
diffe-
versus
found to be present
the data presented
(calpastatin
protein
phosphorylated
of
Specifically,
in
mecha-
two
specificity
is operated
forms previously
presented
muscle of a
and thus a phospho- and a dephospho-
to
A contribution
isozyme into
inhibitory
Interconversion
as the two calpastatin
skeletal
bitory
isozymes.
so far
by the results
paper which demonstrate
homologous
difficult
in a cell.
this
two
of
the presence of two calpain
has been
is provided
by a different
concerns the role
due to the large excess of
and regulated
problem
intracellular
and depho-
mechanism calpastatin
calpain of calpastatin
phospho- or
isois
dephospho-
under investigation.
ACKNCWLEDCMENTS This work was supported in part by grants from National Research Council (CNR). Target Projects: Prevention and Control Disease Factors, SP 8. Control of cardiovascular diseases, contratto n"91.00237.PF 41., PP. Ingegneria Cenetica and Biotecnologia and Biostrumentazione and from Associazione Italiana per la Ricerca sul Cancro. 758
Vol.
187,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
Murachi, T. (1983). Trends Biochem. Sci. 0, 167-169 Pontremoli, S. and Melloni, E. (1986) Ann. Rev. Biochem. 55, 455-481 Mellgren, R.L. (1987). FASEB J. 1, 110-115 Mellgren, R.L. (1980). FBBS Lett. 109, 129-133 Suzuki, K. (1987). Trends Biochem. Sci. 12, 103-105 Takano, E., Maki, M., Hatanaka, M., Marti, T., Mori, H., Titani, K., Kannagi, R., Ooi, T., and Murachi, T. (1988). Biochemistry 27, 1964-1972 Kawasaki, H., Emori, Y., Imajoh-Ohmi, S., Minami, Y.,and Suzuki, K. (1989) J. Biochem. 106, 274,281 Pontremoli, S., Melloni, E., Viotti, P-L., Michetti, M., Salamino, P., and Horecker, B.L. (1991) Arch. Biochem. Biophys. 288, 644-652 Murachi, T. (1989). Biochem. Intern. 18, 263-294 Mellgren, R.L. and Carr, T.C. (1983) Arch. Biochem. Biophys. 255, 779-786 Pontremoli, S., Melloni, E., Viotti, P-L., Michetti, M., Di Lisa, P., and Siliprandi, N. (1990). Res. Commun. 167, 373-380 Pontremoli, S., Viotti, P.L., Michetti, M., Sparatore, B., Salamino, F., and Melloni, E. (1990). Biochem. Biophys. Res. Commun.171, 569-574 Pontremoli, S., Melloni, E., Sparatore, B., Michetti, M., Salamino, F., and Horecker, B.L. (1986) F'roc. Natl. Acad. Sci., U.S.A., 83, 1685-1689 Toshima, Y., and Yoshimura, N. (1975) J. Biochem. 78, 1161-1169 Pontremoli, S., Melloni, E., Salamino, F., Sparatore, B., Michetti, M., Sacco, O., and Horecker B.L. (1986) Arch. Biochem. Biophys. 250, 23-29 Melloni, E., and Pontremoli, S., (1991) J. Nutr. Biochem. 2, 467-476 Kapprell, H.P. and Goll, D.E. (1989) J. Biol. Chem. 264, 17888-17896 Pontremoli, S., Melloni, E., Sparatore, B., Michetti, M., Salamino, F., and Horecker B.L. (1990) J. Biol. Chem. 265, 706-712
759
