Calcium/Calmodulin-dependent Protein Kinase IV Is
Cleaved by Caspase-3 and Calpain in SH-SY5Y Human Neuroblastoma Cells
Undergoing Apoptosis*
Kim M.
McGinnis
,
Margaret M.
Whitton§,
Margaret E.
Gnegy
, and
Kevin K. W.
Wang
¶
From the
Department of Pharmacology, University of
Michigan Medical School, Ann Arbor, Michigan 48109 and Laboratory
of Neuro-biochemistry, Departments of ¶ Neuroscience Therapeutics
and § Chemistry, Parke-Davis Pharmaceutical Research
Division of Warner-Lambert Company, Ann Arbor, Michigan 48105
 |
ABSTRACT |
We have previously demonstrated cleavage of
-spectrin by caspase-3 and calpain during apoptosis in SH-SY5Y
neuroblastoma cells (Nath, R., Raser, K. J., Stafford, D.,
Hajimohammadreza, I., Posner, A., Allen, H., Talanian, R. V.,
Yuen, P., Gilbertsen, R. B., and Wang, K. K. (1996)
Biochem. J. 319, 683-690). We demonstrate here that
calcium/calmodulin-dependent protein kinase IV (CaMK IV) is
cleaved during apoptosis by caspase-3 and calpain. We challenged SH-SY5Y cells with the pro-apoptotic agent thapsigargin. Western blot
analysis revealed major CaMK IV breakdown products of 40, 38, and 33 kDa. Digestion of control SH-SY5Y lysate with purified caspase-3
produced a 38-kDa CaMK IV fragment; digestion with purified calpain
produced a major fragment of 40 kDa. Pretreatment with carbobenzoxy-Asp-CH2OC(O)-2,6-dichlorobenzene or
Z-Val-Ala-Asp-fluoromethylketone was able to block the
caspase-3-mediated production of the 38-kDa fragment both in
situ and in vitro. Calpain inhibitor II similarly blocked formation of the calpain-mediated 40-kDa fragment both in
situ and in vitro. Digestion of recombinant CaMK IV
by other caspase family members revealed that only caspase-3 produces a fragmentation pattern consistent to that seen in situ. The
major caspase-3 and calpain cleavage sites are respectively identified as PAPD176*A and CG201*A, both within the CaMK
IV catalytic domain. Furthermore, calmodulin-stimulated protein kinase
activity decreases within 6 h in thapsigargin-treated SH-SY5Y. The
loss of activity precedes cell death.
 |
INTRODUCTION |
The identification of sequence homology between CED-3, a cysteine
protease that is absolutely required for apoptosis in the nematode
Caenorhabditis elegans, and the mammalian protein
interleukin-1-
-converting enzyme
(ICE,1 caspase-1) (1) has
focused research on the role of ICE-like cysteine proteases (caspases)
in apoptosis. To date, more than 10 caspases have been discovered and
linked to mammalian apoptosis (2-4). The overall common features of
caspases are the conservation of the active site QACXG
pentapeptide, where X is R, Q, or G, and the requirement for
an Asp residue in the P1 position (5, 6).
Among these proteases, caspase-3 (CPP32) has been proposed as a
mediator of mammalian apoptosis. Inhibition of caspase-3 activity attenuates apoptosis in osteosarcoma cells (7) and Jurkat T-cells (8,
9). Our laboratory has demonstrated that caspase-3 inhibition protects
against apoptosis in neuroblastoma cells (10, 11) and neurons in
primary culture (12). Caspase-3 knockout mice show reduced neuronal
death during brain development (13).
After apoptotic injury, the 32-kDa inactive caspase-3 proenzyme is
cleaved to 17- and 12-kDa fragments, which form the active heterodimer
(6, 14). Activated caspase-3 proteolytically cleaves important nuclear
and cytoskeletal proteins during apoptosis (7, 15, 16). Substrates
include poly(ADP-ribose) polymerase (PARP), an enzyme involved in DNA
repair (17), the 70-kDa protein component of the U1 ribonucleoprotein,
the catalytic subunit of the DNA-dependent protein kinase
(18), the cytoskeletal protein non-erythroid
-spectrin (12, 19),
huntingtin (20), and protein kinase C
(21). The functional
significance of these cleavages has not yet been determined.
Calpain is a cysteine protease with dozens of substrates; it is
activated through an increase in intracellular Ca2+ (22).
The two major calpain isoforms, µ- and m-, differ in the amount of
Ca2+ required for activation. Calpain is triggered in
necrosis (23) and in many cell types undergoing apoptosis, including
SH-SY5Y (12), probably as a consequence of a loss of Ca2+
homeostasis (24). CaM-binding proteins are particularly vulnerable to
cleavage by calpain (25, 26). We recently showed that the CaM-binding
protein
-spectrin is proteolytically cleaved in apoptotic SH-SY5Y
cells by both caspase and calpain activation (12). We suspected that
other Ca2+/CaM-binding proteins may be similarly vulnerable
to cleavage during apoptosis. The present study examines the fate of
Ca2+/CaM-dependent protein kinase IV in SH-SY5Y
cells in response to apoptotic challenge.
CaMK IV is a serine/threonine kinase that is highly homologous to the
catalytic and regulatory domains of multifunctional Ca2+/CaM-dependent kinase II (CaM kinase II)
with a calculated mass of 52 kDa (human) (27). CaMK IV expression is
mainly restricted to the brain (28, 29), thymus, and testes (30, 31).
CaMK IV is localized to the nucleus in neurons (32, 33). It has been
implicated in Ca2+-dependent transcription
regulation through activation of the cAMP response-element binding
protein (CREB) (34), activating transcription factor-1 (ATF1) (35),
activation protein-1 (AP-1) (36), and serum-response factor (SRF) (37).
Recently, Alevizopoulos et al. reported that CaMK IV
activates the transforming growth factor
-responsive transcription
factor CTF-1 (38), a hormone involved in growth regulation,
proliferation, differentiation, and apoptosis (39). The few known CaMK
IV substrates other than transcription factors include synapsin I and
the Ras-related GTP-binding protein Rap-1b (40).
CaMK IV contains a polyglutamate-rich C-terminal tail (28), which is
characteristic of chromatin-associated proteins (41). In the brain,
CaMK IV is activated by CaM kinase kinase (CaMKK) (42, 43) and
inactivated by autophosphorylation within the CaM binding domain
(44).
Several reports have demonstrated that inhibition of
Ca2+/calmodulin-dependent protein kinase
activity is associated with apoptosis. Inhibition of CaM kinase
activity with CaM kinase-specific inhibitors induces apoptosis in NIH
3T3 cells (45) and sensitizes etoposide-resistant cells to apoptotic
challenge (46). Thymic T cells from transgenic mice expressing a
catalytically inactive form of CaMK IV showed defects in survival and
activation (47).
In this study, we found that CaMK IV was proteolytically cleaved during
apoptosis. The CaMK IV fragmentation was mediated by both caspase-3 and
calpain. We also report that loss of
Ca2+/CaM-dependent kinase activity is an early
event in neuroblastoma cells undergoing apoptosis.
 |
EXPERIMENTAL PROCEDURES |
Materials--
All chemicals, unless stated otherwise, were
obtained from Sigma. N-Acetyl-Leu-Leu-Met-CHO (calpain
inhibitor II, CalpInh II), thapsigargin, staurosporine, and syntide-2
were from Calbiochem. Carbobenzoxy-Asp-CH2OC(O)-2,6-dichlorobenzene (Z-D-DCB) was
made in-house at Parke-Davis. Anti-CaMK IV (monoclonal) and anti-CaM kinase kinase were from Transduction Laboratories, anti-
-spectrin (monoclonal) was from Chemicon, and anti-PARP (monoclonal) from Biomol.
Z-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk) was obtained from Alexis
Biochemicals.
Cell Culture and Treatment--
SH-SY5Y cells were grown on
12-well plates to confluence (roughly 2 million cells/well) at
37 °C, 5% CO2 in a humidified atmosphere with
Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin. At
the beginning of the experiment, cultures were washed three times with
serum-free Dulbecco's modified Eagle's medium. As indicated, cells
were pretreated for 1 h with protease inhibitors. The cultures were then challenged with 0.5 µM staurosporine or 2 µM thapsigargin and maintained for indicated time, when
protein was extracted. Cerebellar granule neurons and mixed cortical
cells were isolated as described previously (12).
Protein Extraction and Western Blotting--
Total protein was
extracted by lysing cells with 2% SDS/Tris buffer, precipitating
proteins with trichloroacetic acid and solubilizing with Tris base as
described previously (48). Protein concentration was determined with a
modified Lowry (Bio-Rad D-C protein assay kit). Equal
amounts of protein were loaded on each lane and subjected to sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 4-20%
acrylamide gradient gel; Novex) with a Tris/glycine running buffer. The
separated proteins were transferred to a polyvinylidene difluoride
(PVDF) membrane (0.2 µm) by semidry electrotransfer (Bio-Rad semidry
transfer unit) for 2 h at 20 V. The blots were probed with primary
antibody, a biotinylated secondary antibody, and avidin-conjugated
alkaline phosphatase (Amersham Pharmacia Biotech). The immunoblots were developed with nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate.
Cell Extract and Purified CaMK IV Digestion in Vitro with
Caspases and Calpain--
Total protein was extracted from untreated
confluent SH-SY5Y cells by the Triton X-100 method (12). SH-SY5Y cell
extract (30 µg of protein) was digested with 2.5 µg of mature
purified recombinant caspases (supplied by Dr. Robert Talanian, BASF)
or 1 µg of purified µ- or m-calpain (Calbiochem) in 100 mM Hepes buffer (pH 7.4 at room temperature), 10 mM dithiothreitol, 10% (v, v) glycerol, 1 mM
EGTA (for caspases), or 1 mM Ca2+ (for calpain)
for indicated times. The digestion was halted by the addition of
SDS-containing sample buffer for PAGE. Alternatively, 1.5 µg of
purified, recombinant CaMK IV was subject to digestion under identical
conditions. Samples were subjected to SDS-PAGE, transferred to PVDF,
and probed with indicated antibody.
N-terminal Sequencing of Digested Recombinant CaMK
IV--
Purified, recombinant mouse CaMK IV fragments produced by
digestion with purified caspase-3 or µ-calpain were subjected to Edman degradation to obtain N-terminal sequences (in-house at Parke-Davis). Mouse CaMK IV (20 µg, a gift from Dr. Thomas Soderling) was incubated for time indicated at room temperature with purified recombinant caspase-3 (5 µg) or purified µ-calpain (3 µg). The digestion was halted with the addition of SDS-PAGE sample buffer. Samples were subjected to SDS-PAGE and transferred in a CAPS/methanol buffer to PVDF membrane using the method of Matsudaira (49). The
membranes were stained with 0.1% Coomassie in 50% methanol until
bands appeared. The stained bands were excised and subjected to
N-terminal sequencing.
Assay for CaM-stimulated Protein Kinase Activity--
SH-SY5Y
cells were treated as described, and cells were collected by scraping
and centrifugation. Cells washed once with cold Tris-buffered saline
plus 1 mM EDTA. Cell lysates were made by resuspending the
cell pellet in homogenization buffer (20 mM Tris-HCl, pH
7.5, 0.5 mM EGTA, 1 mM EDTA, 10 mM
sodium pyrophosphate, 0.4 mM sodium molybdate, 2 mM dithiothreitol, 0.5% Triton X-100, and protease
inhibitors) (50). Cells were kept on ice 10 min with vortexing and then
sonicated for 15 s. Total CaM kinase activity in 15 µg of sample
was assayed by phosphorylation of the CaM kinase-selective substrate
syntide-2 (Pro-Leu-Ala-Arg-Thr-Leu-Ser-Val-Ala-Gly-Leu-Pro-Gly-Lys-Lys, 40 µM), in the presence of Ca2+ (0.5 mM) and calmodulin (1.5 µg/assay tube) with 5 µCi/assay tube [
-32P]ATP (ICN). Kinase reactions were carried
out for 5 min at 30 °C and halted by spotting onto P-81
phosphocellulose paper (Whatman) and washing in 75 mM
phosphoric acid (3 × 10 min). The extent of phosphorylation was
quantified by a Beckman scintillation counter.
Cell Death Measurement--
SH-SY5Y cell viability was assessed
by measuring release of the cytosolic enzyme, lactate dehydrogenase
(LDH) into the culture medium (25 µl samples). Quantification of LDH
release was done using the Cytotox 96 colorimetric LDH assay kit
(Promega), following the manufacturer's directions.
 |
RESULTS |
CaMK IV, but Not CaMK II (
or
), Is Expressed in SH-SY5Y
Human Neuroblastoma Cells--
To determine whether CaMK II or CaMK IV
are expressed in SH-SY5Y cells, we subjected control lysate from
SH-SY5Y cells to Western blotting with antibodies to CaMK IV (Fig.
1A, top), and to
the major neuronal isoforms of CaMK II:
(Fig. 1A,
bottom) and
(Fig. 1A, middle). Rat
cerebral cortical and cerebellar granule cell extracts were used as
positive controls for the presence of CaMK II
and
. In our
hands, human CaMK IV appeared as a roughly 55-kDa doublet (probably
and
CaMK IV isoforms) with rat CaMK IV running slightly higher. The
predicted molecular mass for human CaMK IV is 52 kDa (27). Although
neither
nor
isoform of CaMK II is detectable in SH-SY5Y cells,
the presence of other CaMK II isoforms, such as
or
, or CaM
kinase I cannot be disregarded.

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Fig. 1.
CaMK IV, but not CaMK II ( or ), is
expressed in SH-SY5Y human neuroblastoma cells. A,
total cellular protein was extracted from untreated cells, separated on
SDS-PAGE, 4-20% Tris-glycine gradient (15 µg of protein/lane),
electrotransferred, and probed with antibodies to CaMK IV
(top), CaMK II (middle), or CaMK II
(bottom). As positive CaMK controls, lysate from rat mixed
cortical cultures and rat cerebellar granule neurons (CGC)
were run in parallel with SH-SY5Y lysate. B, purified,
recombinant CaMK II and CaMK IV (1 µg) were immunoblotted as
above. Each was probed with antibodies to CaMK II and CaMK IV.
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To validate our antibodies, purified recombinant CaMK II
and CaMK
IV were each Western blotted with antibodies to CaMK II
and CaMK IV
(Fig. 1B). The CaMK IV antibody reacted with only a 55-kDa
protein, whereas the CaMK II
antibody reacted with only a 50-kDa
protein. These molecular masses are consistent with masses for these
proteins observed in situ (Fig. 1A). No
cross-reactivity occurred, demonstrating the specificity of the
antibodies used.
Time Course of CaMK IV, Poly(ADP-ribose) Polymerase, and
-Spectrin Fragmentation in Apoptotic SH-SY5Y Cells--
To
investigate the fate of CaMK IV in neuronal cells undergoing apoptosis,
we challenged SH-SY5Y cell with 2 µM thapsigargin, a well
established pro-apoptotic agent (51-53). Thapsigargin increases intracellular Ca2+ concentration through inhibition of the
endoplasmic reticulum Ca2+/Mg2+-ATPase (54).
Whole cell lysate was prepared and subjected to Western blot analysis.
Treatment with thapsigargin resulted in the appearance of two major
CaMK IV fragments, 40 kDa and 38 kDa, and two minor fragments, 50 kDa
and 33 kDa (Fig. 2A, top
panel). The 40-kDa fragment is seen faintly in control lysate, but
increases by 3 h of thapsigargin treatment. The 38-kDa band is
readily apparent 3 h after start of thapsigargin treatment.

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Fig. 2.
CaMK IV breakdown in apoptotic SH-SY5Y
cells. A, SH-SY5Y cells were untreated or treated for
various times with 2 µM thapsigargin (thapsi).
Total protein lysate was separated by SDS-PAGE (4-20% acrylamide, 15 µg/lane), electrotransferred to PVDF membrane and probed with
antibody to CaMK IV (top panel), PARP (middle),
and -spectrin (bottom). Intact proteins are indicated as
well as the major breakdown product(s). B, densitometric
quantification from data in A of major breakdown products of
CaMK IV (38 and 40 kDa, open columns), PARP (85 kDa,
solid columns), and -spectrin (120 kDa, striped
columns). Data are representative of two separate
experiments.
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We compared the time course for CaMK IV breakdown with those of PARP
(Fig. 2A, middle panel) and
-spectrin (Fig.
2A, bottom panel) in thapsigargin-treated SH-SY5Y
cells.
-Spectrin (19, 55, 56) and PARP (57, 58) are caspase
substrates cleaved in many cell types undergoing apoptosis, including
SH-SY5Y (11, 12). Caspase-3 activity mediates the formation of the
120-kDa
-spectrin breakdown product (BDP) and the 85-kDa PARP BDP.
Densitometric analysis was performed on the fragments (Fig.
2B). CaMK IV, PARP, and
-spectrin fragments were apparent
beginning at 3 h post-treatment. The early appearance of a greater
amount the 120-kDa
-spectrin BDP is consistent with reports that
-spectrin cleavage is one of the early events of apoptosis (56). The
increase of the CaMK IV fragments occurs in a time frame similar to
that of PARP and
-spectrin.
Caspase and Calpain Inhibitors Alter the CaMK IV Breakdown Pattern
in SH-SY5Y Cells Undergoing Staurosporine- and Thapsigargin-mediated
Apoptosis--
We wanted to confirm that CaMK IV would be cleaved
under a different apoptotic paradigm. SH-SY5Y cells were challenged
with staurosporine, a nonspecific kinase inhibitor and well established pro-apoptotic agent (10, 11, 57, 59). Staurosporine treatment produced
the same breakdown pattern as thapsigargin (Fig.
3A, second lane
versus fifth lane).

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Fig. 3.
Effect of caspase and calpain inhibitors on
CaMK IV breakdown in apoptotic cells. A, SH-SY5Y
protein extracts were subjected to Western blotting with anti-CaMK IV.
Intact CaMK IV is indicated (55 kDa). Cells were either untreated
(Con) or treated for 24 h with 0.5 µM
staurosporine or 2 µM thapsigargin in the presence or
absence of 50 µM Z-D-DCB (Z-D), 20 µM CalpInh II (CI), or 2 mM EGTA.
B, CaMK IV Western blot of SH-SY5Y treated with thapsigargin
for 24 h in the presence or absence of 50 µM
Z-VAD-fmk (Z-V).
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We have previously demonstrated that pretreatment with the pan-caspase
inhibitor Z-D-DCB blocks formation of the 120-kDa
-spectrin fragment
(12). Here, we examine its ability to prevent CaMK IV cleavage during
apoptosis. Z-D-DCB prevented formation of the 38-kDa CaMK IV fragment
in both staurosporine- and thapsigargin-treated SH-SY5Y cells (Fig.
3A). Ability of Z-D-DCB to block formation of a
subset of CaMK IV fragments suggests that caspase is in part, but not
fully, mediating CaMK IV proteolysis.
In staurosporine-treated, but not thapsigargin-treated, cells,
formation of the 50- and 33-kDa CaMK IV fragments was attenuated. This
discrepancy may be a result of the different mechanisms that lead to
apoptosis in staurosporine- versus thapsigargin-treated cells or differences in time to onset for apoptosis. Staurosporine activates caspase-3 much more rapidly than thapsigargin in these cells
(data not shown). Furthermore, because staurosporine is a protein
kinase inhibitor, it may directly interact with CaMK IV and alter
susceptibility to cleavage.
Because calpain is also activated in SH-SY5Y cells undergoing apoptosis
(12), we investigated its role in CaMK IV fragmentation. We inhibited
calpain in two ways: by incubation with 2 mM EGTA and by
treatment with CalpInh II before the onset of apoptosis. Pretreatment
with EGTA inhibits formation of the 40-kDa fragment in
thapsigargin-treated SH-SY5Y (Fig. 3A). In thapsigargin- and staurosporine-induced apoptosis, pretreatment with CalpInh II completely blocked formation of the 40-kDa fragment but did not affect
the 38-kDa fragment. Blockade of the 33-kDa fragment was more variable;
in some experiments it was attenuated by Z-D-DCB, in others
by CalpInh II.
To further confirm the role of caspases in the proteolysis of CaMK IV
in SH-SY5Y cells undergoing apoptosis, we pretreated with another
commonly used caspase inhibitor, Z-VAD-fmk (60), and then challenged
for 24 h with thapsigargin. In thapsigargin-treated cells,
Z-VAD-fmk acted identically to Z-D-DCB in its ability to block formation of the 38-kDa CaMK IV BDP (Fig. 3B).
Characterization of CaMK IV Breakdown in Vitro--
To examine the
effect of specific caspases or calpain on CaMK IV fragmentation, cell
lysate (30 µg) from control SH-SY5Y cells was digested with purified
recombinant caspase-3 (
form) or µ-calpain. Digestion with
caspase-3 or caspase-1 produced a 38-kDa CaMK IV breakdown product
(Fig. 4). Digestion with caspases in the
presence of Z-D-DCB eliminated the 38-kDa fragment, whereas
CalpInh II had no effect on it. µ-Calpain produced CaMK IV fragments
of 40 kDa and several fragments of 30-33 kDa (Fig. 4, last
lane). The presence of 40- and 33-kDa fragments in the control
lysate probably reflects basal activation of endogenous calpain.

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Fig. 4.
In vitro digestion of SH-SY5Y cell
lysate by purified caspase-3 and calpain. Protein was extracted
from untreated SH-SY5Y by incubation with 1% Triton-Tris buffer as
described under "Experimental Procedures." Lysate were untreated or
incubated in a volume of 100 µl with 2.5 µg of caspase-3 or
caspase-1 (5 h) or 1 µg of µ-calpain (10 min) in the presence or
absence of 20 µM CalpInh II or 50 µM
Z-D-DCB. The incubation was halted with the addition of 20 µl of SDS sample buffer. The lysates were subjected to SDS-PAGE and
analyzed by Western blotting with CaMK IV (top) or
-spectrin (bottom) antibodies. Intact protein and major
breakdown products are indicated.
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We also probed the digested lysate with
-spectrin antibody as a
positive control for caspase and calpain activity. The appearance of
150- and 120-kDa
-spectrin fragments is consistent with caspase-3 activation as shown previously. Digestion with µ-calpain produced only a 150-kDa fragment. The calpain-mediated 150-kDa fragment is
generated at a cleavage site, which is distinct from the
caspase-mediated 150-kDa fragment (12).
Purified, Recombinant CaMK IV Is Cleaved by Calpain and Caspase-3
in Pattern Consistent with in Situ Results--
Purified recombinant
CaMK IV was incubated with purified caspase-3 or m-calpain as described
under "Experimental Procedures" (Fig.
5A). Caspase-3 digestion
produced fragments of 50 and 38 kDa, whereas calpain produced a major
40-kDa fragment in addition to minor fragments. A number of different
caspases can co-exist in mammalian cells as part of a caspase cascade
(61). We wanted to examine the CaMK IV cleavage pattern produced by
various members of the caspase family. We used purified recombinant
caspase-1 (ICE), caspase-2 (ICH-1), caspase-3 (CPP32), caspase-4
(ICH-2), and caspase-6 (Mch-2) to digest purified CaMK IV for 1 or
4 h (Fig. 5B). Only caspase-3 and caspase-1 produced
the 38-kDa fragment that appears in situ during apoptosis.
However, caspase-1 produced additional fragments of 47 and 45 kDa,
which were not observed in situ.

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Fig. 5.
Purified recombinant CaMK IV digested with
caspase-3 and m-calpain produces cleavage patterns consistent with
in situ breakdown. A, purified, recombinant
CaMK IV (5 µg) was either untreated (Con) or digested for
1 h with 1.5 µg of caspase-3 or for 4 min with 1 µg of
m-calpain. B, purified recombinant CaMK IV was untreated
(Con) or digested with 1.5 µg of caspase-3, caspase-1,
caspase-4, or caspase-6 for 1 or 4 h. Digests were subjected to
SDS-PAGE and probed with antibody against CaMK IV.
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Cleavage Sites of Caspase-3- and Calpain-mediated CaMK IV
Fragments--
To determine the caspase-3- and calpain-mediated CaMK
IV cleavage sites, we performed N-terminal sequencing on the major
fragments using Edman degradation as described under "Experimental
Procedures." Table I summarizes the
N-terminal sequence data.
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Table I
N-terminal sequences of major CaM kinase IV fragments
Mouse recombinant CaM kinase IV digested by caspase-3 or m-calpain as
described under "Experimental Procedures."
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Multiple sequences were detected within the major Coomassie-stained
fragments. Intact 55-kDa CaMK IV has an N-terminal sequence of
V4TVPSXPSS, suggesting that the recombinant
protein is truncated upon expression in Escherichia coli
(Table I). In the case of both caspase-3 and m-calpain digestion, the
38-40-kDa bands also contained this truncated N terminus
(V4TVPSXPSS, Table I). Because the CaMK IV
antibody was made to residues 1-271, the fragments seen in Western
blots (Figs. 2-5) were likely this N terminus.
In the case of calpain, we detected a 38-kDa fragment with an
N-terminal sequence of TPGYXAPEIL, corresponding to a
cleavage of CG201T202PGYCAPEIL,
which co-migrated with the 40-kDa N-terminal fragment. A smaller
fragment of about 33 kDa with an N terminus of LVPDYXIDGS was also sequenced. This fragment indicates a calpain cleavage site at
EN23L24VPDYWIDGS.
On the other hand, caspase-3 produced a major fragment with an
N-terminal sequence of APLKIADFXL, corresponding to cleavage at PAPD176A177PLKIADFGL of CaMK IV.
The N-terminal fragment of the caspase digested CaMK IV is therefore
slightly smaller than the calpain-mediated fragment, reflecting what we
observed in situ (Figs. 2 and 3); a 38-kDa fragment produced
by caspase and a 40-kDa fragment by calpain. A second caspase cleavage
site was found in the 33-kDa fragment with an N terminus of GSNRDPLGDF,
corresponding to a cleavage at
YWID31G32SNRDPLGDF. Fig.
6 is a schematic of the CaMK IV molecule
with the caspase-3 and m-calpain cleavage sites marked and fragment sizes indicated.

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Fig. 6.
Schematic for CaMK IV fragmentation by
caspase-3 and calpain. The intact 53-kDa CaMK IV is illustrated as
a linear molecule with its major regions indicated. One pair of caspase
and calpain cleavage sites is located within the catalytic domain, and
an additional pair of cleavage sites is located in the N-terminal
domain.
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Ca2+/CaM-stimulated Protein Kinase Activity Reduction
Is an Early Event of Thapsigargin-mediated Apoptosis in SH-SY5Y
Cells--
We investigated whether CaM kinase IV proteolysis could be
correlated with physiological changes in cells undergoing apoptosis. We
treated SH-SY5Y cells for various times with thapsigargin and monitored
CaM-stimulated protein kinase activity in the cell homogenate using the
CaM kinase-selective substrate syntide-2. This peptide has been used to
measure CaMK IV activity in vitro (43, 62, 63). Syntide-2 is
homologous to phosphorylation site 2 of glycogen synthase (64). In
order to prevent changes in the phosphorylation state and further
degradation of the CaM kinase, the homogenization buffer contained
protein phosphatase inhibitors and protease inhibitors. Because
syntide-2 phosphorylation may reflect nonspecific kinase activity, we
determined Ca2+/CaM-stimulated protein kinase activity by
calculating the difference between phosphorylation in the presence or
absence of Ca2+/CaM. Additionally, although we were unable
to detect CaMK II
or
in SH-SY5Y cells (Fig. 1), the influence
on syntide-2 phosphorylation of other CaM kinases that may be expressed
in these cells, such as CaMK II
, could not be discounted.
A 30% loss in CaMK activity in SH-SY5Y cells treated with thapsigargin
was apparent at 6 h and increased through 24 h, when 55% of
activity was lost (Fig. 7A).
Importantly, the loss in CaMK activity preceded loss of cell viability
as measured by LDH leaked into the culture medium (Fig.
7A).

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Fig. 7.
Ca2+/CaM-stimulated protein
kinase activity decreases over time in SH-SY5Y cells challenged with
thapsigargin. A, SH-SY5Y cells were treated for 0, 1, 3, 6, 12, or 24 h with 2 µM thapsigargin. Cell
lysate was incubated at 30 °C for 5 min with either 1 mM
CaCl2 and 10 µM CaM or 1 mM EGTA
and 40 µM syntide-2. The reaction was stopped by spotting
onto phosphocellulose. Data are expressed as percent loss of maximal
Ca2+/CaM-stimulated protein kinase activity
(circles). The time course for thapsigargin-mediated LDH
release (squares) is shown on the right-hand
y-axis. Data are mean ± S.E. values. (**, p < 0.01, compared with time zero, Student's t test,
n = 6.) B, cells were treated in the
presence or absence of 50 µM Z-D-DCB or 20 µM CalpInh II. Cells were either unchallenged (open
bars) or treated for 6 h with 2 µM thapsigargin
(solid bars). Data are mean ± S.E. Owing to
variability among experiments, results are shown as a percentage of
control. (**, p < 0.0002; *, p = 0.007, compared with control treatment, Student's t test,
n = 6-8.)
|
|
Next, we investigated the effects of Z-D-DCB and CalpInh II
on CaM kinase activity in thapsigargin-treated SH-SY5Y cells. We chose
6 h of thapsigargin treatment because at that time there is a
significant decrease in CaM kinase activity with little increase in LDH
release. Loss of CaM-stimulated protein kinase activity at 6 h is
not attributable to loss in cell viability. Additionally, by 6 h,
although CaMK IV fragmentation is evident, there is very little PARP
breakdown. Pretreatment with the pan-caspase inhibitor Z-D-DCB for 1 h prevented the thapsigargin-mediated
loss of CaM-stimulated protein kinase activity at 6 h (Fig.
7B). However, pretreatment with CalpInh II did not reverse
the loss of activity. Neither Z-D-DCB nor CalpInh II alone
affected CaM kinase activity.
CaM Kinase II
and CaM Kinase Kinase Are Also Caspase Substrates
in Neuronal Cells Undergoing Apoptosis--
Because the percent loss
in CaM kinase activity is greater than the loss of intact CaMK IV, we
suspected that other components of the CaM kinase signal transduction
pathway are affected by apoptosis. We examined the fate of the upstream
activator of CaMK IV, CaM kinase kinase (CaMKK), in SH-SY5Y cells
undergoing apoptosis (Fig.
8A). Western blot analysis of
CaMKK from control and staurosporine-treated SH-SY5Y cells revealed a
caspase-dependent digestion of CaMKK to a 58-kDa fragment.
The fragmentation was blocked by pretreatment with Z-D-DCB,
but not CalpInh II.

View larger version (43K):
[in this window]
[in a new window]
|
Fig. 8.
Caspase-mediated breakdown of CaMKK in
SH-SY5Y cells and CaMK II in rat cortical neurons.
A, SH-SY5Y cells were untreated or treated for 8 h with
0.5 µM staurosporine in the presence or absence of
Z-D-DCB (50 µM) or CalpInh II (20 µM). Whole cell lysate was subjected to Western blot
analysis with anti-CaMKK. B, mixed cortical cells were
treated as in A and lysate was probed with anti-CaMK
II .
|
|
Because we could not discount the possibility that CaM kinases that we
were unable to detect may be expressed in SH-SY5Y, we examined the
vulnerability of CaMK II
to fragmentation in neurons undergoing
apoptosis (Fig. 8B). Rat mixed cortical neurons were treated
for 24 h with staurosporine in the presence or absence of
Z-D-DCB or CalpInh II. A 35-kDa CaMK II
BDP was observed
that was inhibited by Z-D-DCB but not CalpInh II.
 |
DISCUSSION |
This is the first report describing caspase-3- and
calpain-mediated CaMK IV degradation in apoptotic cells. We identify
both caspase- and calpain-mediated CaMK IV cleavage sites. This is also
the first report demonstrating a loss in
Ca2+/calmodulin-dependent kinase activity in
cells undergoing apoptosis.
Mounting evidence has clearly shown that caspase-3 plays a key role in
mammalian apoptosis. Caspase-3 is activated by a wide range of
apoptotic challenges in a variety of cell types (11, 18, 65). We have
previously demonstrated its activation in apoptotic SH-SY5Y (11, 12).
An increasing number of caspase-3 substrates have now been identified.
These substrates all share the DXXD consensus site preferred
by caspase-3. However, Talanian et al. (66) recently
reported that caspase-3 and the closely related caspase-7 (Mch-3) would
also accept other less preferred residues in the P4
position in peptidic substrates. The sites of caspase-3-mediated CaMK
IV cleavage have an Asp in the P1 but not in the
P4 position. CaMK IV cleavage is not as efficient as PARP
and
-spectrin cleavage (Fig. 2A), which may be a result of the absence of Asp in the P4 position of the caspase-3
cleavage sites. Caspase-3-mediated fragmentation of CaMK IV is an early event in SH-SY5Y cells undergoing apoptosis. In thapsigargin-treated SH-SY5Y cells, the 38-kDa CaMK IV BDP (cleavage site
PAPD176APLK) appears in a time frame consistent
with caspase-3-mediated PARP and
-spectrin fragmentation (Fig.
2).
We found that CaM-stimulated protein kinase activity decreased over
time in thapsigargin-treated SH-SY5Y cells (Fig. 7A). CaMK
II activity has recently been shown to decrease in cultured neurons
exposed to excitotoxic insult (67). In addition, inhibition of CaM
kinase activity with CaM kinase-specific inhibitors induces apoptosis
in fibroblasts (45) and sensitizes etoposide-resistant cells to
apoptotic challenge (46). To address whether fragmentation of CaMK IV
by caspase-3 and calpain has physiological consequences in the
apoptosis cascade, we considered expressing a caspase/calpain cleavage-resistant CaMK IV mutant. Owing to the presence of multiple cleavages by both proteases, this strategy would not be feasible.
One reason for the loss of CaM-stimulated protein kinase activity
in apoptotic SH-SY5Y cells may be proteolytic fragmentation. CaMK IV
was cleaved within the catalytic domain (Fig. 6). The possibility also
exists that other CaM kinases are expressed in SH-SY5Y cells. Tombes
et al. (45) have presented data that CaMK II
may be
involved in apoptosis in NIH 3T3 cells. We propose that multiple CaM
kinases are susceptible to proteolytic fragmentation in neurons
undergoing apoptosis. In this report, we show that CaM kinase II
,
which has significant sequence homology to CaMK IV within the catalytic
domain, is also cleaved by caspases in apoptotic cortical neurons (Fig.
8A). Thus, the loss of activity of other CaM kinases may
contribute to the loss of total CaM-stimulated protein kinase
activity.
Because most of the parent CaMK IV remained intact, even after 24 h of thapsigargin treatment, the 60% loss in CaM-stimulated protein
kinase activity (Fig. 7A) is not likely to be caused by CaMK
IV fragmentation alone. The activity decrease could also be attributed
to events occurring upstream from CaMK IV in the CaM kinase cascade.
CaMK IV activity is enhanced by the
Ca2+/calmodulin-dependent CaM kinase kinase
(CaMKK) (68). We report here that CaMKK is cleaved by caspase(s) in
apoptotic SH-SY5Y cells (Fig. 8B). The caspase-mediated
CaMKK cleavage may further contribute to the loss of CaMK IV activity.
This is a subject for future investigation. CaMK IV activity is also
altered by autophosphorylation on Ser12-Ser13
at the N terminus (69). Changes in phosphorylation state as a result of
apoptotic challenge may also lead to decreases in CaMK activity.
Caspase inhibition eliminated the loss of CaM-stimulated protein
kinase activity, whereas calpain inhibition had no effect (Fig.
7B). Thus, the caspase-mediated fragmentation appears to be
predominant over the calpain pathway in neuronal cells undergoing apoptosis, as we described previously with staurosporine-mediated apoptosis in SH-SY5Y and low-potassium-mediated apoptosis in rat cerebellar granule neurons (12). The attenuation of CaMKK cleavage by
caspase inhibition, but not calpain inhibition (Fig. 8A),
may explain why caspase inhibition alone is sufficient to prevent loss
of CaM-stimulated protein kinase activity (Fig. 7B). Further investigation of the activity changes in purified CaMK IV and CaMKK in
response to digestion by caspases and calpain are planned.
CaMK IV is involved in gene transcription factor phosphorylation (34,
35, 63) leading to the expression of immediate early genes. In
addition, CaMK IV has recently been reported to inhibit type I adenylyl
cyclase, a Ca2+/CaM-dependent neurospecific
enzyme (70) and activate transforming growth factor
, which is
involved in cell cycle regulation (38). The degradation of CaMK IV and
loss of activity in apoptotic neurons may be functionally related to
the morphological and nuclear changes accompanying apoptosis. Although
its physiological role is not well defined, CaMK IV expression is
restricted primarily to the brain and thymus. CaMK IV may regulate
important neuronal processes that are altered as the neuron undergoes
apoptosis.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Robert Talanian (BASF) for the
gift of recombinant caspases. We are also grateful to Drs. Tom
Soderling and Debra Brickey (Vollum Institute, Oregon Health Sciences
University, OR) for generously supplying recombinant CaMK IV.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grant MH36044, National Institutes of Health Training Grant
5T32GM07767, a National Science Foundation graduate research fellowship
(to K. M. M.), and an advanced predoctoral fellowship in
pharmacology from the Pharmaceutical Research and Manufacturers of
America Foundation (to K. M. M.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Laboratory of
Neuro-biochemistry, Department of Neuroscience Therapeutics,
Parke-Davis Pharmaceutical Research Div., Warner-Lambert Co., 2800 Plymouth Rd., Ann Arbor, MI 48105. Tel.: 734-622-7132; Fax:
734-622-7178; E-mail: kevin.wang{at}wl.com.
The abbreviations used are:
ICE, interleukin-1-
-converting enzyme; BDP, breakdown product; CaM, calmodulin; CaMK II, Ca2+/calmodulin-dependent
protein kinase IICaMK IV, Ca2+/calmodulin-dependent protein kinase IVCaMKK, CaM kinase kinaseCalpInh II, calpain inhibitor IIZ-D-DCB, carbobenzoxy-Asp-CH2OC(O)-2,6-dichlorobenzenePARP, poly(ADP-ribose) polymerasePVDF, polyvinyldifluorideZ-VAD-fmk, Z-Val-Ala-Asp-fluoromethylketonePAGE, polyacrylamide gel
electrophoresisLDH, lactate dehydrogenaseCAPS, 3-(cyclohexylamino)propanesulfonic acid.
 |
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