The calpain system is involved in the constitutive regulation of beta-catenin signaling functions.
Beta-catenin is a multifunctional protein serving both as a structural element in cell adhesion and as a signaling component in the Wnt pathway, regulating embryogenesis and tumorigenesis. The signaling fraction of beta-catenin is tightly controlled by the adenomatous polyposis coli-axin-glycogen synthase kinase 3beta complex, which targets it for proteasomal degradation. It has been recently shown that Ca(2+) release from internal stores results in nuclear export and calpain-mediated degradation of beta-catenin in the cytoplasm. Here we have highlighted the critical relevance of constitutive calpain pathway in the control of beta-catenin levels and functions, showing that small interference RNA knock down of endogenous calpain per se (i.e. in the absence of external stimuli) induces an increase in the free transcriptional competent pool of endogenous beta-catenin. We further characterized the role of the known calpain inhibitors, Gas2 and Calpastatin, demonstrating that they can also control levels, function, and localization of beta-catenin through endogenous calpain regulation. Finally we present Gas2 dominant negative (Gas2DN) as a new tool for regulating calpain activity, providing evidence that it counteracts the described effects of both Gas2 and Calpastatin on beta-catenin and that it works via calpain independently of the classical glycogen synthase kinase 3beta and proteasome pathway. Moreover, we provide in vitro biochemical evidence showing that Gas2DN can increase the activity of calpain and that in vivo it can induce degradation of stabilized/mutated beta-catenin. In fact, in a context where the classical proteasome pathway is impaired, as in colon cancer cells, Gas2DN biological effects accounted for a significant reduction in proliferation and anchorage-independent growth of colon cancer.
Benetti
Roberta
R
Laboratorio Nazionale del Consorzio Interuniversitario per le Biotecnologie, Area Science Park, Padriciano 99, 34012 Trieste, Italy.
Copetti
Tamara
T
Dell'Orso
Stefania
S
Melloni
Edon
E
Brancolini
Claudio
C
Monte
Martin
M
Schneider
Claudio
C
D025601:Adenomatous Polyposis Coli Protein / Q000378:metabolism; D000818:Animals; D060466:Axin Protein; D015153:Blotting, Western; D002135:Calcium-Binding Proteins / Q000737:chemistry; D002154:Calpain / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D002448:Cell Adhesion; D002460:Cell Line; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D003110:Colonic Neoplasms / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D017930:Genes, Reporter; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008840:Microfilament Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism; D015398:Signal Transduction; D013347:Subcellular Fractions; D013997:Time Factors; D015534:Trans-Activators / Q000378:metabolism*; D014162:Transfection; D051153:Wnt Proteins; D051176:beta Catenin
D025601:Adenomatous Polyposis Coli Protein; D060466:Axin Protein; C495270:CTNNB1 protein, human; C495267:CTNNB1 protein, mouse; D002135:Calcium-Binding Proteins; D003598:Cytoskeletal Proteins; C099165:Gas2 protein, mouse; D036341:Intercellular Signaling Peptides and Proteins; D008840:Microfilament Proteins; D034741:RNA, Small Interfering; D012097:Repressor Proteins; D015534:Trans-Activators; D051153:Wnt Proteins; D051176:beta Catenin; D049452:Green Fluorescent Proteins; C033668:calpastatin; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; C000605507:Gsk3b protein, mouse; D038362:Glycogen Synthase Kinase 3; D002154:Calpain; C449000:m-calpain; D046988:Proteasome Endopeptidase Complex