Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 2 de 2
Filter
Add more filters










Database
Language
Publication year range
1.
Sci Rep ; 8(1): 671, 2018 01 12.
Article in English | MEDLINE | ID: mdl-29330435

ABSTRACT

Nuclear activation of Wnt/ß-catenin signaling is required for cell proliferation in inflammation and cancer. Studies from our group indicate that ß-catenin activation in colitis and colorectal cancer (CRC) correlates with increased nuclear levels of ß-catenin phosphorylated at serine 552 (pß-Cat552). Biochemical analysis of nuclear extracts from cancer biopsies revealed the existence of low molecular weight (LMW) pß-Cat552, increased to the exclusion of full size (FS) forms of ß-catenin. LMW ß-catenin lacks both termini, leaving residues in the armadillo repeat intact. Further experiments showed that TCF4 predominantly binds LMW pß-Cat552 in the nucleus of inflamed and cancerous cells. Nuclear chromatin bound localization of LMW pß-Cat552 was blocked in cells by inhibition of proteasomal chymotrypsin-like activity but not by other protease inhibitors. K48 polyubiquitinated FS and LMW ß-catenin were increased by treatment with bortezomib. Overexpressed in vitro double truncated ß-catenin increased transcriptional activity, cell proliferation and growth of tumor xenografts compared to FS ß-catenin. Serine 552-> alanin substitution abrogated K48 polyubiquitination,  ß-catenin nuclear translocation and tumor xenograft growth. These data suggest that a novel proteasome-dependent posttranslational modification of ß-catenin enhances transcriptional activation. Discovery of this pathway may be helpful in the development of diagnostic and therapeutic tools in colitis and cancer.


Subject(s)
Colitis/metabolism , Colorectal Neoplasms/metabolism , Transcriptional Activation , beta Catenin/genetics , beta Catenin/metabolism , Animals , Caco-2 Cells , Cell Line, Tumor , Cell Nucleus/metabolism , Cell Proliferation , Colitis/genetics , Colorectal Neoplasms/genetics , HCT116 Cells , HT29 Cells , Humans , Mice , Molecular Weight , Mutation , Neoplasm Transplantation , Proteasome Endopeptidase Complex/metabolism , Transcription Factor 4/metabolism
2.
Biochemistry ; 55(46): 6433-6444, 2016 Nov 22.
Article in English | MEDLINE | ID: mdl-27798963

ABSTRACT

IQGAP1 is a multidomain scaffold protein involved in many cellular processes. We have determined the crystal structure of an N-terminal fragment spanning residues 1-191 (CHDF hereafter) that contains the entire calponin homology domain. The structure of the CHDF is very similar to those of other type 3 calponin homology domains like those from calponin, Vav, and the yeast IQGAP1 ortholog Rng2. However, in the crystal, two CHDF molecules form a "head-to-head" or parallel dimer through mostly hydrophobic interactions. Binding experiments indicate that the CHDF binds to both F-actin and Ca2+/calmodulin, but binding is mutually exclusive. On the basis of the structure, two dimer interface substitutions were introduced. While CHDFL157D disrupts the dimer in gel filtration experiments, oxidized CHDFK161C stabilizes the dimer. These results imply that the CHDF forms the same dimer in solution that is seen in the crystal structure. The disulfide-stabilized dimer displays a reduced level of F-actin binding in sedimentation assays and shows no binding to Ca2+/calmodulin in isothermal titration calorimetry (ITC) experiments, indicating that interface residues are utilized for both binding events. The Calmodulin Target Database predicts that residues 93KK94 are important for CaM binding, and indeed, the 93EE94 double mutation displays a reduced level of binding to Ca2+/calmodulin in ITC experiments. Our results indicate that the CHDF dimer interface is used for both F-actin and Ca2+/calmodulin binding, and the 93KK94 pair, near the interface, is also used for Ca2+/calmodulin binding. These results are also consistent with full-length IQGAP1 forming a parallel homodimer.


Subject(s)
Actins/chemistry , Calmodulin/chemistry , Protein Multimerization , ras GTPase-Activating Proteins/chemistry , Actins/metabolism , Amino Acid Sequence , Calcium/metabolism , Calmodulin/metabolism , Calorimetry , Crystallization , Crystallography, X-Ray , Humans , Models, Molecular , Molecular Weight , Protein Binding , Protein Structure, Tertiary , Sequence Homology, Amino Acid , ras GTPase-Activating Proteins/genetics , ras GTPase-Activating Proteins/metabolism
SELECTION OF CITATIONS
SEARCH DETAIL
...