1 / 19

J. Biol. Chem., Vol. 278, Issue 2, 1372-1379, January 10, 2003

The Role of Calpain in the Proteolytic Cleavage of E-cadherin in Prostate and Mammary Epithelial Cells. J. Biol. Chem., Vol. 278, Issue 2, 1372-1379, January 10, 2003

herne
Download Presentation

J. Biol. Chem., Vol. 278, Issue 2, 1372-1379, January 10, 2003

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. The Role of Calpain in the Proteolytic Cleavage of E-cadherin in Prostate and Mammary Epithelial Cells • 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

  2. BACKROUND • 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

  3. 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.

  4. Calpain is involved in cell-cycle regulation, signal transduction, and apoptosis. • Activation of v-Src induces protein synthesis of calpain leading to calpain-mediated 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.

  5. 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。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。

  6. PURPOSE • 1、 Is E-cadherin a substrate for calpain？ • 2、 Is calpain-dependent proteolysis of E-cadherin associated with prostate cancer progression？

  7. 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 E-cadherin were HECD-1, E-9, 4A2 , C20820, E2, and SC-1499 . • 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.

  8. Results • 1、 Ionomycin and Calpain Inhibitor Experiments for LNCaP , MCF-7 。fig1： • Conclusion：Ionomycin Induces a Calpain-dependent Cleavage of E-cadherin to 100 kDa ，which can be significantly inhibited by calpain inhibitors.

  9. 2、 purified calpain could generate E-cad100 in vitro .fig2： • 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 caspase-3, which failed to demonstrate cleavage of E-cadherin to E-cad100 .

  10. 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 mechanismis currently unclear . qustion：E-cadherin synthesis is required for cleavage ？

  11. 4、LNCaP cells were pretreated with cycloheximide prior to TPA treatment ： • 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.

  12. 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 E-cadherin located between the C20820 and SC-1499 epitopes.

  13. 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.

  14. 7、 E-cadherin mutagenesis inhibits truncation in vivo • 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 。

  15. 8、 m-Calpain is up-regulated in localized and metastatic prostate tumors. • 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.

  16. 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% SDS-PAGE 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.

  17. Discussion

  18. Calpain induces a specific, inactivating proteolytic cleavage within the cytoplasmic domain of E-cadherin 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. • In vitro experiments also supported the hypothesis that E-cadherin is a substrate for calpain. Both µ- and m-calpain effectively cleaved full-length E-cadherin in vitro to 100 kDa. • This truncation occurred in the cytoplasmic domain of E-cadherin, and that this truncated species of E-cadherin is unable to bind to ß-catenin, Ý -catenin, and p120, which are essential for the adhesive and cell signaling functions of E-cadherin.

  19. The residues 782-787 are either critical for calpain binding or represent the actual cleavage site. • Based on these observations, we hypothesized that specific proteolytic cleavage events, such as that mediated by calpain, target and inactivate E-cadherin. 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. • Thanks！

More Related