Download The Role of Calpain in the Proteolytic Cleavage of E

The Role of Calpain in the
Proteolytic Cleavage of Ecadherin in Prostate and
Mammary Epithelial Cells
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J. Biol. Chem., Vol. 278, Issue 2, 1372-1379, January 10, 2003
 Jonathan Rios-Doria §, Kathleen C. Day ¶, Rainer Kuefer , Michael G. Rashid ,
Arul M. Chinnaiyan **¶, Mark A. Rubin **¶, and Mark L. Day §¶
 From the Department of Urology and the § Program in Cellular and Molecular
Biology, University of Michigan, the Department of Urology, University of Ulm,
Prittwitz-Strasse 43, 89075 Ulm, Germany, and the ** Department of Pathology,
and the ¶ University of Michigan Comprehensive Cancer Center, University of
Michigan, Ann Arbor, Michigan 48109
BACKROUND
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E-cadherinThe cadherin structure consists of an extracellular
domain with five tandem repeats, a transmembrane domain,
and a cytoplasmic domain that interacts with the catenin family
binding proteins.
 Association of E-cadherin with the catenin family of proteins is
critical for the maintenance of a functional adhesive complex.
 Inactivation of E-cadherin through gene mutation,
transcriptional inactivation, or promoter methylation has been
demonstrated in many adenocarcinomas
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Calpain’s structure
The 80 kDa catalytic subunit :
domain I is a short pro-domain
region; domain II represents a
distinct papain-like cysteine
protease domain; domain III has
no significant homology to any
other protein, may regulate
protease activity; domain IV is a
calcium binding domain that has
been proposed to play a role in
substrate recognition.
The small 30 kDa: domain IV of
the catalytic subunit and domain
VI of the regulatory subunit, each
possess five sets of EF hand
calcium binding motifs that have
been proposed to confer calcium
dependency upon the activity of
calpain.
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Calpain is involved in cell-cycle
regulation, signal transduction, and
apoptosis.
Activation of v-Src induces protein
synthesis of calpain leading to calpainmediated degradation of its own
inhibitor calpastatin thereby, further
enhancing calpain activity.
promotes proteolytic cleavage of FAK
accelerates the progression of
transformed cells through the G1 stage
of the cell-cycle and contributes to
anchorage-independent growth.
EGFR induced phosphorylation of
calpain and increased calpain activity
that subsequently increased motility.
Proteins that have been identified as
substrates of calpain include the
cytoskeletal proteins spectrin and
members of the integrin family.
 E-cadherin和calpain均是钙依赖的，也都
与细胞粘附关系密切，但以前的研究并没有
将二者联系起来。
 文章作者在研究钙离子在上皮钙粘素分解中
作用的时候，发现了一种新的100kda的钙
粘素水解片段，即E-cad100。
 而E-cad100产生可以被calpain抑制剂所阻
止。所以E-cadherin很可能也是calpain的
底物。
PURPOSE
 1、
Is E-cadherin a substrate for calpain？
 2、 Is calpain-dependent proteolysis of Ecadherin associated with prostate cancer
progression？
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Cell Lines ：LNCaP , MCF-7, and SKBR3(no ecad)
Ionomycin：A divalent calcium ionophore that is widely
used as a tool to investigate the role of intracellular
calcium in cellular processes. ↑Ca++
TPA：12-o-tetradecanoylphorbol-13-acetate. ↑PKC
Antibodies-- Antibodies used for the detection of Ecadherin were HECD-1, E-9, 4A2 , C20820, E2, and SC1499 .
Tissue Procurement : fresh radical prostatectomy
specimens were frozen in liquid nitrogen within 30 min
after surgical excision. A Rapid Autopsy Protocol has been
previously described. The metastases were confirmed as
prostate cancer in origin by a genitourinary pathologist at
the University of Michigan.
Results
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1、 Ionomycin and Calpain Inhibitor Experiments for LNCaP , MCF-7 。
fig1：
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Conclusion：Ionomycin Induces a Calpain-dependent
Cleavage of E-cadherin to 100 kDa ，which can be
significantly inhibited by calpain inhibitors.
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2、 purified calpain could generate E-cad100 in vitro .fig2：
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Conclusion ： both µ- and m-calpain can specifically cleave
mature E-cadherin to the 100-kDa fragment in vitro.
A similar in vitro assay was performed using purified caspase3, which failed to demonstrate cleavage of E-cadherin to Ecad100 .
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3、Expression and truncation of exogenous E-cadherin in SKBR3 cells.
A, Protein extracts from SKBR3 parental cells (lanes 1-2) and SK-Ecad
cells (lanes 3-6), treated with TPA at the indicated times, were
immunoblotted with the HECD-1 antibody, Fig. 3
1、 Activation of PKC in these E-cad-expressing SKBR3 cells
(termed SK-Ecad) revealed the rapid accumulation of E-cad100
over a course of 12 h ；
2、PKC activation by TPA treatment could induce expression of any
endogenous E-cadherin protein in transfected SKBR3 cells. the
mechanism is currently unclear .
qustion：E-cadherin synthesis is required for cleavage ？
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4、LNCaP cells were pretreated with cycloheximide prior
to TPA treatment ：
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Fig. 4. LNCaP cells were pretreated with 50 µM
cycloheximide 30 min prior to TPA treatment. 50 µg of
protein extracts from cells that were harvested at the
indicated times were resolved by 6% SDS-PAGE and
immunoblotted with HECD-1 antibody.
Conclusion ： E-cadherin synthesis is not required for
cleavage.
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5、Epitope and Functional Mapping of E-cad100.
Fig. 5. A schematic of full-length E-cadherin and a table of the antibodies and
epitopes to which they are mapped are shown. TPA-treated SK-Ecad protein
extracts were resolved by SDS-PAGE and immunoblotted with E-9 (site A), 4A2
(site B), C20820 (site C), and SC-1499 (site D) antibodies. Lysates were
resolved by 6% SDS-PAGE
Conclusion ：the cleavage site was in the cytoplasmic domain of Ecadherin located between the C20820 and SC-1499 epitopes.
6、E-cad100 does not associate with the catenin complex.
The ß -catenin and Ý -catenin binding domains : 815-839 of
the cytoplasmic domain. The p120 binding site: 758-773.
Fig. 6.TPA-treated LNCaP protein extracts were
immunoprecipitated with E-cadherin antibody 4A2, -catenin,
p120, and -catenin antibodies. A control
immunoprecipitation was also performed using a normal
goat IgG antibody and an immunoprecipation using protein
A Sepharose beads alone.
Conclusion :E-cad100 has lost the ß - and Ý -catenin binding
domains.
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7、 E-cadherin mutagenesis inhibits truncation in vivo
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Fig. 7. A, E-cadherin amino acid sequence, from 771-810, is
shown with the targeted mutation sites. B, the corresponding
mutant constructs containing in-frame internal deletions were
stably transfected into SKBR3 cells and protein extracts harvested
at the indicated times following TPA treatment.SKBR3-Ecad wild
type cells are also shown (lane 10).
Conclusion ：1、Mutation of the E-cadherin Cytoplasmic
Domain Inhibits Truncation of E-cadherin in Vivo ;2、amino acid
residues 782-787 are essential for calpain cleavage 。
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8、 m-Calpain is up-regulated in localized and metastatic
prostate tumors.
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Fig. 8. cDNA microarray analysis was performed using
26 benign prostate, 49 localized prostate cancer, and
19 metastatic prostate cancer samples. m-Calpain transcript
levels were found to be significantly up-regulated in localized
prostate and metastatic cancer samples compared with benign
prostate.
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9、E-cad100 is detected in localized and metastatic prostate cancer ：
Fig 9. A, protein extracts (30 µg) of prostate tissue from two different
patients were analyzed for E-cadherin expression. Extracts from both the
normal and tumor aspect of the prostate gland were run on a 6% SDSPAGE gel and immunoblotted with HECD-1 antibody. B, protein extracts
were prepared from metastatic lesions removed from the following sites: 1,
liver; 2, diaphragm; 3, soft tissue; 4, peritoneum; 5, adrenal gland; 6,
paratracheal lymph node .
 Conclusion :increased expression of calpain is associated with the
generation of the 100-kDa fragment in localized and metastatic disease.
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Discussion
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Calpain induces a specific, inactivating proteolytic
cleavage within the cytoplasmic domain of Ecadherin in prostate and mammary epithelial cells.
Pretreatment of LNCaP and MCF-7 cells with
calpain inhibitors blocked the generation of
ionomycin-induced E-cad100, strongly implicating
calpain in this process.
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In vitro experiments also supported the hypothesis
that E-cadherin is a substrate for calpain. Both µand m-calpain effectively cleaved full-length Ecadherin in vitro to 100 kDa.
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This truncation occurred in the cytoplasmic domain
of E-cadherin, and that this truncated species of Ecadherin is unable to bind to ß -catenin, Ý -catenin,
and p120, which are essential for the adhesive and
cell signaling functions of E-cadherin.
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The residues 782-787 are either critical for calpain
binding or represent the actual cleavage site.
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Based on these observations, we hypothesized that
specific proteolytic cleavage events, such as that
mediated by calpain, target and inactivate Ecadherin. Such a loss of E-cadherin function may
result in the net reduction of interepithelial adhesion
and promote the malignant transformation of
prostate epithelial cells.
 Over.
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Thanks！