Abstract
Cell-cell contacts play a vital role in intracellular signaling, although the molecular mechanisms of these signaling pathways are not fully understood. E-cadherin, an important mediator of cell-cell adhesions, has been shown to be cleaved by γ-secretase. This cleavage releases a fragment of E-cadherin, E-cadherin C-terminal fragment 2 (E-cad/CTF2), into the cytosol. Here, we study the fate and function of this fragment. First, we show that coexpression of the cadherin-binding protein, p120 catenin (p120), enhances the nuclear translocation of E-cad/CTF2. By knocking down p120 with short interfering RNA, we also demonstrate that p120 is necessary for the nuclear localization of E-cad/CTF2. Furthermore, p120 enhances and is required for the specific binding of E-cad/CTF2 to DNA. Finally, we show that E-cad/CTF2 can regulate the p120-Kaiso-mediated signaling pathway in the nucleus. These data indicate a novel role for cleaved E-cadherin in the nucleus.
Highlights
In multicellular organisms, individual cells are often connected with each other via cell-cell adhesions to form threedimensionally structured tissues or organs
Cadherins are a family of transmembrane cell-cell adhesion proteins that can be subdivided into several groups including classical cadherins and protocadherins [3]
Cadherins are proteolytically cleaved by a number of proteases including extracellular metalloproteases, ␥-secretase, and caspase (24 –27). ␥-Secretase cleavage of several transmembrane molecules following initial cleavage by metalloproteases has emerged as a common mechanism for signaling to the nucleus [28]
Summary
Antibodies, Plasmids, and Materials—Antibodies against E-cadherin, p120, N-cadherin, GM130, BiP/GRP78, and Lamp-1 were purchased from BD Biosciences. To construct pCMV-E-cad/ CTF2-NLS-myc and pCMV-E-cad/CTF2(AAA)-NLS-myc, the cytoplasmic domain of E-cadherin was amplified by PCR from pBAT-E-cadherin and pLK-E-cadherin(762AAA764), respectively, using the following primers: 5Ј-GAATTCGCGGCCGCAATGCGGAGGAGAACGGTGGTC and 3Ј-GGAATTCGCGGCCGCGTCGTCCTCGCCACCGCCG. These were cloned into a NotI site of the pCMV-myc-nuc pShooter vector (Invitrogen). To construct pcDNA/TO/GFP-E-cad/CTF2NLS, the cDNA of E-cad/CTF2-NLS was amplified by PCR from pCMV-E-cad/CTF2-NLS-myc using the following primers: 5Ј-CGGCGCCTCGAGGCGGAGGAGAACGGTGGTC and 3Ј-CGGCGCCGAATTCCTATGCGGCCCCATTCAGATCC This was cloned into the EcoRI and XhoI sites of pcDNA/TO/GFP. Purified GST-E-cad/CTF2 and GST proteins were produced, and GST pull-down assays were performed as previously described [40]. For reporter assays using the matrilysin promoter, HEK293 cells were transfected with pGL3-Basic-2.3kb HMat together with FLAG-p120, E-cad/CTF2-NLS-myc, or E-cad/ CTF2(AAA)-NLS-myc. Statistical Analysis—Student’s t tests assuming equal or unequal variance and a two-tailed distribution were performed for statistical analysis
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