KRAS-induced actin-interacting protein regulates inositol 1,4,5-trisphosphate-receptor-mediated calcium release.

Abstract

KRAS-induced actin-interacting protein (KRAP) was originally characterized as a filamentous- actin-interacting protein. We have recently found that KRAP is an associated molecule with inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R) and is responsible for the proper subcellular localization of IP(3)R. Since it remains unknown whether KRAP regulates the IP(3)R-mediated Ca(2+) signaling, we herein examined the effects of KRAP on the IP(3)R-mediated Ca(2+) release by Ca(2+) imagings in the cultured HEK293 or MCF7 cells. Reduction of KRAP protein by KRAP-specific siRNA diminishes ATP-induced Ca(2+) release and the ATP-induced Ca(2+) release is completely quenched by the pretreatment with the IP(3)R inhibitor but not with the ryanodine receptor inhibitor, indicating that KRAP regulates IP(3)R-mediated Ca(2+) release. To further reveal mechanistic insights into the regulation of IP(3)R-mediated Ca(2+) release by KRAP, we examined the effects of the KRAP-knockdown on the releasable Ca(2+) content of intracellular Ca(2+) stores. Consequently, reduction of KRAP does not affect the amount of ionophore- or Ca(2+)-ATPase inhibitor-induced Ca(2+) release in the HEK293 cells, indicating that releasable Ca(2+) content of intracellular Ca(2+) stores is not altered by KRAP. Thus, KRAP is involved in the proper regulation of IP(3)R-mediated Ca(2+) release.

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KRAS-induced actin-interacting protein regulates inositol
1,4,5-trisphosphate-receptor-mediated calcium release
Takahiro Fujimotoa,b, Takashi Machidaa, Toshiyuki Tsunodaa,b, Keiko Doia,
Takeharu Otaa, Masahide Kurokib, Senji Shirasawaa,b,⇑
aDepartment of Cell Biology, Faculty of Medicine, and Fukuoka University, 7-45-1 Nanakuma, Jonan-ku, Fukuoka 814-0180, Japan
bCentral Research Institute for Advanced Molecular Medicine, Fukuoka University, 7-45-1 Nanakuma, Jonan-ku, Fukuoka 814-0180, Japan
a r t i c l ei n f o
Article history:
Received 24 March 2011
Available online 30 March 2011
Keywords:
Inositol 1,4,5-trisphosphate receptor
KRAS-induced actin-interacting protein
Calcium release
Calcium signaling
a b s t r a c t
KRAS-induced actin-interacting protein (KRAP) was originally characterized as a filamentous- actin-inter-
acting protein. We have recently found that KRAP is an associated molecule with inositol 1,4,5-trisphos-
phate (IP3) receptor (IP3R) and is responsible for the proper subcellular localization of IP3R. Since it
remains unknown whether KRAP regulates the IP3R-mediated Ca2+signaling, we herein examined the
effects of KRAP on the IP3R-mediated Ca2+release by Ca2+imagings in the cultured HEK293 or MCF7 cells.
Reduction of KRAP protein by KRAP-specific siRNA diminishes ATP-induced Ca2+release and the ATP-
induced Ca2+release is completely quenched by the pretreatment with the IP3R inhibitor but not with
the ryanodine receptor inhibitor, indicating that KRAP regulates IP3R-mediated Ca2+release. To further
reveal mechanistic insights into the regulation of IP3R-mediated Ca2+release by KRAP, we examined
the effects of the KRAP-knockdown on the releasable Ca2+content of intracellular Ca2+stores. Conse-
quently, reduction of KRAP does not affect the amount of ionophore- or Ca2+-ATPase inhibitor-induced
Ca2+release in the HEK293 cells, indicating that releasable Ca2+content of intracellular Ca2+stores is
not altered by KRAP. Thus, KRAP is involved in the proper regulation of IP3R-mediated Ca2+release.
? 2011 Elsevier Inc. All rights reserved.
1. Introduction
Regulation of intracellular Ca2+concentration is critical in
numerous biological processes [1]. Three inositol 1,4,5-trisphos-
phate receptor (IP3R) subtypes are differentially expressed among
tissues [2–5] and function as the Ca2+release channel on special-
ized endoplasmic reticulum (ER) membranes [6–9]. The proper
subcellular localization of IP3R is crucial for its proper function
[10–13].
KRAP (KRAS-induced actin-interacting protein) was originally
identified as one of the deregulated expression genes in colorectal
cancer [14]. KRAP encodes a cytoplasmic protein associated with
filamentous-actin (F-actin), and this protein is localized around
the apical pole of hepatocytes and pancreatic acinar cells [14,15].
KRAP-deficient (KO) mice display decreased adiposity and im-
proved glucose metabolism [16], suggesting that KRAP physiologi-
cally participates in the regulation of systemic energy homeostasis.
Furthermore, the pancreatic acinar cells in KRAP-KO mice contain
an increased amount of zymogen granules, indicating the critical
roles for KRAP in the exocrine system of the pancreas [17]. We
have recently demonstrated that KRAP is relevant to the proper
localization of IP3R through physical association in the epithelial
tissues [18]. However, it remains unknown whether KRAP regu-
lates the IP3R-mediated Ca2+signaling. Thus, we herein examined
the effects of KRAP on the IP3R-mediated Ca2+release in the cul-
tured cells.
2. Materials and methods
2.1. Antibodies
The antibodies used were: anti-actin (A2066) from Sigma,
anti-IP3R1 (ab5840) from Abcam, anti-IP3R3 (610313) from BD
Transduction Laboratories, anti-PLCc (E-12) from Santa Cruz, and
anti-KRAP was prepared as described previously [15].
2.2. Animals
All animals used in this study were treated in accordance with
the rules of Fukuoka University. KRAP-knockout mice were gener-
ated as described in a previous publication [16].
2.3. Cell culture and transfection
HEK293 and MCF7 cells were maintained at 37 ?C, with 5% CO2
in DMEM containing 10% FCS and penicillin–streptomycin–glutamine.
0006-291X/$ - see front matter ? 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2011.03.112
⇑Corresponding author at: Department of Cell Biology, Faculty of Medicine, and
Fukuoka University, 7-45-1 Nanakuma, Jonan-ku, Fukuoka 814-0180, Japan. Fax:
+81 92 864 3865.
E-mail address: sshirasa@fukuoka-u.ac.jp (S. Shirasawa).
Biochemical and Biophysical Research Communications 408 (2011) 214–217
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HEK293 or MCF7 cells were transfected with a small inhibitory
RNA (siRNA) using a MicroPorator MP-100 (Digital Bio) as
described [15].
2.4. Ca2+imaging
For the Ca2+imaging, transfected cells were plated at a density
of 13,000 cells in 200 ll of medium per well in collagen-coated 96-
well multiwell plates. Two days after transfection, the HEK293 or
MCF7 cells were incubated with 4 lM Fluo-4 acetoxymethyl ester
(Invitrogen) and 5 lM Hoechst 33342 (Invitrogen) in the growth
medium for 50 min at 37 ?C. The Fluo-4-loaded cells were washed
once with calcium-free HBSS supplemented with 0.5 mM EGTA,
and then mounted on the stage of the In Cell Analyzer 1000
equipped with a 20 ? objective (GE Healthcare). To detect the fluo-
rescence images of Ca2+signals, Fluo-4 was excited using a xenon
lamp and a HQ480/40 nm excitation filter, and the emission fluo-
rescence signals were collected through a HQ535/50 nm band-pass
filter. To detect the fluorescence images of nuclear signals, Hoechst
33342 was excited using a D360/40 nm excitation filter, and the
emission fluorescence signals were collected through a HQ460/
40 nm band-pass filter. Images were acquired every 2.5 s after
administration of ATP as an agonist, and were analyzed using the
region-of-interest function of the Work station Ver3.2 software
package (GE Healthcare). The mean fluorescence intensity of cal-
cium signals within Hoechst-positive regions of interest (nuclear
region) of all the cells in each image was calculated.
2.5. [3H] IP3-binding assay
We have used the protocol described by Chadwick [19] with a
slight modification. Microsomes (300 lg of protein) of the liver
or kidneys were incubated with [3H] IP3(PerkinElmer Life Sci-
ences) at 2.2 nM in the assay solution containing 50 mM Tris–
HCl, pH8.3, 1 mM EDTA, 100 mM KCl for 15 min on ice, centrifuged,
and the microsomal pellets were washed twice with the assay
solution. The incorporated radioactivity was determined by liquid
scintillation counting.
2.6. Statistical analysis
Data are presented as the means ± standard errors of the means
(SEM) and statistical analysis was performed using an unpaired
Student’s t-test when comparing the means of two groups. Differ-
ences of P values of less than 0.05 were considered to be statisti-
cally significant.
3. Results and discussion
3.1. Reduction of KRAP expression inhibits IP3R-mediated Ca2+release
in the HEK293 or MCF7 cells
We examined the knockdown effect of KRAP on ATP-induced
Ca2+release via IP3R by Ca2+imaging with the fluorescent Ca2+
indicator in the HEK293 cells. KRAP-siRNAs specifically suppressed
the endogenous KRAP expression without interfering with the
expression of other proteins including IP3R1, IP3R3, PLCc and actin
in the HEK293 cells (Fig. 1A). When cells were stimulated with the
IP3-generating agonist ATP, both the average peak amplitude of
Ca2+released and the total amount of Ca2+released after the stim-
ulations were decreased in the KRAP-knockdown cells compared
with those of the control scramble siRNA-treated cells at all ATP
concentrations examined (Fig. 1B–D). These results indicated that
KRAP is required for the normal ATP-induced Ca2+signaling in
the HEK293 cells. To further examine the relevance of KRAP to
the ATP-induced Ca2+release, the IP3R inhibitor (xestospongin C)
was applied to the Ca2+imaging assay. Pretreatment of the
HEK293 cells by xestospongin C completely abolished the ATP-
induced Ca2+release (Fig. 1E, left), indicating that Ca2+release
detected in our assays is totally mediated by IP3R. Indeed, the
ATP-induced Ca2+release was still observed in the presence of
the ryanodine receptor inhibitor (ruthenium red) and the peak
amplitude of the Ca2+response was attenuated by the KRAP-
knockdown (Fig. 1E, right). These results indicate that KRAP
regulates IP3R-mediated Ca2+release in the HEK293 cells.
To check the generality of the functional relevance of KRAP in
the regulation of IP3R-mediated Ca2+release, the same experiment
was carried out using the MCF7 cells. As obtained from the assays
in the HEK293 cells, both the average peak amplitude of Ca2+re-
leased and the total amount of Ca2+released after the stimulations
were decreased in the KRAP-knockdown cells compared with those
of the control scramble siRNA-treated cells at all ATP concentra-
tions examined (Fig. 2A-D). These results show that the functional
relevance to the regulation of IP3R-mediated Ca2+release of KRAP
is also conserved in the MCF7 cells.
3.2. KRAP affects neither the intracellular Ca2+content nor the IP3-
binding itself to IP3R
Since we previously demonstrated that KRAP is critical for the
proper subcellular localization of IP3R [18], the mislocalization of
IP3R upon KRAP-knockdown seems to be the primary mechanistic
explanation for the impairment of the IP3R-mediated Ca2+release
in the KRAP-knockdown cells. However, we examined the effects
of the KRAP on the releasable Ca2+content of intracellular Ca2+
stores and on the IP3binding to IP3R, because critical region of
IP3R for the association with KRAP is the amino-terminal amino-
acid residues 1-610, in which IP3binding domain exists [18].
When the HEK293 cells were treated with either an ionophore
(A23187) or the Ca2+-ATPase inhibitor (thapsigargin), both the
average peak amplitude of Ca2+released and the total amount of
Ca2+released after the stimulations were comparable between
the KRAP-knockdown and control cells (Fig. 3A–D), indicating that
KRAP-knockdown does not affect the amount of the releasable Ca2+
content.
Furthermore, analysis of the tritium-labeled IP3-binding to IP3R
by using microsomes prepared from liver or kidneys showed that
comparable amounts of the IP3-binding were detected between
the KRAP-deficient and the wild-type control microsomes (Fig. 4).
This result indicates that the IP3-binding to IP3R normally occurs
in the absence of KRAP in vitro, suggesting that KRAP seems not
to be required for the IP3-binding itself to the receptor in vitro.
How about the in vivo status of the IP3-binding to IP3R in the ab-
sence of KRAP? Considering the critical relevance of KRAP in the
regulation of the localization of IP3R in vivo [18], KRAP may affect
the IP3-binding to the receptor possibly through the spatial regula-
tion of the receptor in vivo. Although a full understanding of the ex-
act molecular mechanisms of the IP3R-mediated Ca2+regulation by
KRAP and the precise functional relevance in biological processes
should await further studies, we provide evidence to implicate a
critical role of KRAP in the regulation of IP3-mediated Ca2+signal-
ing in the cultured cells.
As proper subcellular localization of the IP3R subtype is essen-
tial for the regulation of well-organized Ca2+dynamics [20–27],
the regulatory mechanisms of the IP3R-mediated Ca2+release by
KRAP possibly through the spatial regulation of IP3R will partici-
pate in diverse biological events. Actually, the pancreatic acinar
cells in KRAP-KO mice, in which IP3R localization is impaired,
showed an increased amount of zymogen granules [17,18], sug-
gesting the implications of KRAP-mediated IP3R functions in the
exocrine system. As KRAP is also a cancer-related gene [14,15]
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and is involved in the regulation of whole-body energy homeosta-
sis [16], it is likely that the protein plays a critical role in numerous
biological processes through distinct molecular mechanisms.
Elucidation of the full spectrum of the activities of KRAP will lead
to a better understanding of cellular programs, cancer develop-
ment and metabolism-related diseases.
Fig. 1. Knockdown of KRAP inhibits IP3R-mediated Ca2+release in the HEK293 cells. (A) The western blot analysis of KRAP, IP3R1, IP3R3, PLCc and actin expression levels in
HEK293 cells 48 h after the transfection of siRNAs (specific KRAP-siRNA: KRAP1, KRAP2). Scramble-siRNAs were used as controls. (B) Representative Ca2+responses in siRNA-
treated HEK293 cells stimulated with the ATP concentrations indicated. All traces are averaged from multiple (87–140) cells. (C) Quantification of the Ca2+peak amplitude in
(B). The amplitude of the response was defined as the ratio of the relative fluorescence of the maximum fluorescence intensity post-ATP stimulation (fmax) to the baseline
fluorescence intensity at time zero (f0). (D) Quantification of the total fluorescence intensity in (B). (E) Quantification of the peak amplitude of Ca2+responses in siRNA-treated
HEK293 cells stimulated with 1 lM ATP in the presence of 2 lM xestospongin C (XeC) or 10 lM ruthenium red (RR). Data were presented as the means ± SEM, n = 4.
Independent t-tests were used for statistical comparisons.⁄P < 0.05;⁄⁄P < 0.01 relative to the scramble siRNA-treated control; N.S., not significant.
Fig. 2. Knockdown of KRAP inhibits IP3R-mediated Ca2+release in the MCF7 cells. (A) Representative Ca2+responses in siRNA-treated MCF7 cells stimulated with the
indicated concentrations of ATP. All traces are averaged from multiple (146–200) cells. (B) Quantification of the Ca2+peak amplitude in (A). The amplitude of the response was
defined as the ratio of the relative fluorescence of the maximum fluorescence intensity post-ATP stimulation (fmax) to the baseline fluorescence intensity at time zero (f0). (C)
Quantification of the total fluorescence intensity in (A). (D) The western blot analysis of KRAP, IP3R1, IP3R3, PLCc and actin expression levels in MCF7 cells 48 h after the
transfection of siRNAs. Data are presented as the means ± SEM, n = 4. Independent t-tests were used for statistical comparisons.⁄P < 0.05;⁄⁄P < 0.01 relative to the scramble
siRNA-treated control.
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Conflict of interest statement
None declared.
Acknowledgments
This work was supported in part by a Grant-in-Aid for the FCAM
from the Ministry of Education, Culture, Sports, Science, and Tech-
nology, a Grant-in-Aid for Scientific Research from the Japan Soci-
ety for the Promotion of the Science, a Grant-in-Aid from the
Clinical Research Foundation, and a Grant-in-Aid from the Sumito-
mo Foundation. We thank Takami Danno for technical assistance.
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relevanceandmolecular
Fig. 3. Knockdown of KRAP does not affect the intracellular Ca2+content. (A) Quantification of the peak amplitude of Ca2+responses in siRNA-treated HEK293 cells stimulated
with 2 lM A23187. The amplitude of the response was defined as the ratio of the relative fluorescence of the maximum fluorescence intensity post-ATP stimulation (fmax) to
the baseline fluorescence intensity at time zero (f0). (B) Quantification of the total fluorescence intensity of Ca2+responses in siRNA-treated HEK293 cells stimulated with
2 lM A23187. (C) Quantification of the peak amplitude of Ca2+responses in siRNA-treated HEK293 cells stimulated with 5 lM thapsigargin. (D) Quantification of the total
fluorescence intensity of Ca2+responses in siRNA-treated HEK293 cells stimulated with 5 lM thapsigargin. Data are presented as the means ± SEM, n = 4. Independent t-tests
were used for statistical comparisons. N.S., not significant.
Fig. 4. No difference in the amount of IP3binding to IP3R in the absence or presence
of KRAP. Data were presented as the means ± SEM, n = 4. Independent t-tests were
used for statistical comparisons. N.S., not significant.
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KRAP
IP3R1
IP3R3
Actin
Scramble1 Scramble2
KRAP1 KRAP2
HeLa
ATP1 mM
ATP3 mM
ATP10 mM
Scramble1 KRAP1
Scramble2 KRAP2
Fluorescence intensity
Time after stimulation (sec)
0
100
200
300
400
0
100
200
300
400
A
B
Supplementary Fig. 1