title: Regulation of calcium influx and signaling pathway in cancer cells via TRPV6-Numb1 interaction.
abstract: Ca(2+) is a critical factor in the regulation of signal transduction and Ca(2+) homeostasis is altered in different human diseases. The level of Ca(2+) in cells is highly regulated through a diverse class of regulators. Among them is the transient receptor potential vanilloid 6 (TRPV6), which is a Ca(2+) selective channel that absorbs Ca(2+) in the small intestine. TRPV6 is overexpressed in some cancers and exhibits oncogenic potential, but its exact mechanism is still poorly understood. The Numb protein is a cell fate determinant that functions in endocytosis and as a tumor suppressor via the stabilization of p53. Numb protein consisted of four isoforms. Here, we showed a novel function of Numb1, which negatively regulates TRPV6 activity. The expression of Numb1 decreased cytosolic Ca(2+) concentrations in TRPV6-transfected HEK293 cells. When all the isoforms of Numb were depleted using siRNA in a TRPV6 stable cell line, the levels of cytosolic Ca(2+) increased. We observed an interaction between Numb1 and TRPV6 using co-immunoprecipitation. We confirmed this interaction using Fluorescence Resolution Energy Transfer (FRET). We identified the TRPV6 and Numb1 binding site using TRPV6 C-terminal truncation mutants and Numb1 deletion mutants. The binding site in TRPV6 was an aspartic acid at amino acid residue 716, and that binding site in Numb1 was arginine at amino acid residue 434. A Numb1 mutant, lacking TRPV6 binding activity, failed to inhibit TRPV6 activity. Every isoform of Numb knockdown, using an siRNA-based approach in MCF-7 breast cancer cells, not only showed enhanced TRPV6 expression but also both the cytosolic Ca(2+) concentration and cell proliferation were increased. The down-regulated expression of TRPV6 using siRNA increased Numb protein expression; however, the cytosolic influx of Ca(2+) and proliferation of the cell were decreased. To examine downstream signaling during Ca(2+) influx, we performed Western blotting analysis on TRPV6 upregulated cancer cells (MCF-7, PC-3). Taken together, these results demonstrated that Numb1 interacts with TRPV6 through charged residues and inhibits its activity via the regulation of protein expression. Moreover, we provided evidence for a Ca(2+)-regulated cancer cell signaling pathway and that the Ca(2+) channel is a target of cancer cells.
authors.affiliation: Department of Physiology, Seoul National University College of Medicine, Seoul 110-799, Republic of Korea.
authors.forename: Sung-Young
authors.initials: SY
authors.lastname: Kim
authors.forename: Dongki
authors.initials: D
authors.lastname: Yang
authors.forename: Jongyoun
authors.initials: J
authors.lastname: Myeong
authors.forename: Kotdaji
authors.initials: K
authors.lastname: Ha
authors.forename: Su-Hwa
authors.initials: SH
authors.lastname: Kim
authors.forename: Eun-Jung
authors.initials: EJ
authors.lastname: Park
authors.forename: In-Gyu
authors.initials: IG
authors.lastname: Kim
authors.forename: Nam-Hyuk
authors.initials: NH
authors.lastname: Cho
authors.forename: Kyu Pil
authors.initials: KP
authors.lastname: Lee
authors.forename: Ju-Hong
authors.initials: JH
authors.lastname: Jeon
authors.forename: Insuk
authors.initials: I
authors.lastname: So
mesh_terms: D001665:Binding Sites; D001943:Breast Neoplasms / Q000378:metabolism / Q000473:pathology; D002118:Calcium / Q000378:metabolism*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D049109:Cell Proliferation; D005260:Female; D057809:HEK293 Cells; D006801:Humans; D061986:MCF-7 Cells; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D020033:Protein Isoforms / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D015398:Signal Transduction; D050916:TRPV Cation Channels / Q000235:genetics / Q000378:metabolism*
chemical_list: D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; C113026:NUMB protein, human; D020033:Protein Isoforms; D034741:RNA, Small Interfering; D050916:TRPV Cation Channels; C120776:TRPV6 channel; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002118:Calcium