Note; The E3 ligase RNF43 inhibits Wnt signaling downstream of mutated beta-catenin by sequestering TCF4 to the nuclear membrane
The E3 ligase RNF43 inhibits Wnt signaling downstream of mutated beta-catenin by sequestering TCF4 to the nuclear membrane
(doi: 10.1126/scisignal.aac6757)
1.Abnormal in wnt signaling -- >90% of sporadic colon tumors
2.Mutation mostly found -- APC (adenomatous polyposis coli) or b-catenin
3.Activation of Fz causes degradation complex releases b-catenin --> b-catenin localize to nucleus
4.b-catenin bind to TCF-4 (transcription factor) --> activate the transcription corresponding to this TF
5.RNF43 involves with DNA-damage response
This paper points out three key previous findings;
1.RNF43 is tumor suppressor by reducing the abundance of Fz at plasma mb, thereby, reducing the wnt signaling.
2.Conversely, RNF43 is found at the nucleus envelope, ER and nucleoplasm --> but related to the evidence showing that RNF43 is related to DNA damage response.
3.RNF43 oncogene? or RNF43 tumor suppressor? --> numbers of research, lately, showed it might be tumor suppressor.
Questions;
RNF43 related to tumor development or prognosis in tumor patient?
The paper clarified ambiguous points step by step;
1.showing the location of RNF43 in human intestine and colon tissue -- RNF43 highly express at the "stem cell (OLFM4 -- marker for intestinal stem cell)" compartment. Even higher in adenomas and adenocarcinomas (hard to see in the fig). A; detect mRNA (in-situ hybridization) and B; detect protein (immunohistochemistry).
Picture of intestinal area and types of cells that lining along
Ref - DOI: 10.1039/C3IB40163D
2.RNF43 is "majorly" expressed in the endoderm-derived tissues and tissues having high-wnt activity
3.RNF43 is located in the nucleus; when looking in depth --> more on nuclear envelope (LaminB) and lesser nucleoplasm (PSF) and occasionally in ER (calnexin). Mutation on NLS (R437A-K655A) --> cause accumulation of RNF43 in the cytosol.
These are the images from Leica SP5 confocal -- very nice!!
4.Level of RNF43 expression depends on the colon cancer stage; stage 2 show heterogeneity (some low some high) whereas stage IV higher expression --> this stage really can't say it is the driver or the result of carcinogenesis.
5.RNF43-H292R (RING area mutation)--> no effect on localization --> meaning it works separately
6.TCF-4 is the TF binding to RNF43 promoter thereby regulation of expression (feedback loop); detech through CHIP-immunoprecipitation
7.Control of wnt activity (through the wnt receptor) requires RING domain of RNF43
8.Co-immunoprecipitation revealed RNF43 reacts with TCF-4 and the interaction occur at the C-terminal part of TCF-4 control the TF activity --> physically interact cause inhibition, no ubiquitination activity is related. (TCF-4 is reported to be ubiquitinylated by NARF, Nemo-like kinase associated RING finger protein).
endogenous testting of localization of TCF-4 once RNF43 gene is knocked down
9. Mislocation of TCF-4 caused the reduction of TCF-4 target gene expression
10.Since RNF43 physical interacts with TCF-4 via C-terminal domain of RNF43, either mislocation (truncated from TM toward the end) or lacking of C-terminal domain.
Showing the AA and protein domain in human RNF43 --> protein engineering to see the function of each domain.
Remark:
HCT116 -- chromosomal stable,activated mut-b-catenin,wt-APC,low express conductin and RNF43 mRNA expression is high --> even it is high it could not drag TCF4 to the nuclear membrane. When transfect with corresponding plasmids as show above --> RNF43 is higher enough to interact with TCF4?
Mutations of AA have been found in RNF43, pick the most candidate hit to explore (A). Proposed model for RNF43 that could play role in inhibition of wnt signaling at the downstream level of b-catenin (B).
Antibodies;
OLFM4 -- marker for intestinal stem cell
LaminB receptor -- marker for inner layer of nucleus membrane
Nuclear RNA binding protein PSF -- marker for nucleoplasm
Calnexin -- marker for ER marker
Good information from selleckchem; the orange color reflects available inhibitors being seold by this company -- Ref: http://www.selleckchem.com/
Two things that I observed;
1. functional studies by using representative cell-line that is not the cancer cells, like HEK293 (lack of constitutively Wnt signaling)
2. endogenous condition -- using the corresponding cell line to test the hypothesis; either by knock down (in case of overexpression) or overexpression (in case of low expression)
(doi: 10.1126/scisignal.aac6757)
1.Abnormal in wnt signaling -- >90% of sporadic colon tumors
2.Mutation mostly found -- APC (adenomatous polyposis coli) or b-catenin
3.Activation of Fz causes degradation complex releases b-catenin --> b-catenin localize to nucleus
4.b-catenin bind to TCF-4 (transcription factor) --> activate the transcription corresponding to this TF
5.RNF43 involves with DNA-damage response
This paper points out three key previous findings;
1.RNF43 is tumor suppressor by reducing the abundance of Fz at plasma mb, thereby, reducing the wnt signaling.
2.Conversely, RNF43 is found at the nucleus envelope, ER and nucleoplasm --> but related to the evidence showing that RNF43 is related to DNA damage response.
3.RNF43 oncogene? or RNF43 tumor suppressor? --> numbers of research, lately, showed it might be tumor suppressor.
Questions;
RNF43 related to tumor development or prognosis in tumor patient?
The paper clarified ambiguous points step by step;
1.showing the location of RNF43 in human intestine and colon tissue -- RNF43 highly express at the "stem cell (OLFM4 -- marker for intestinal stem cell)" compartment. Even higher in adenomas and adenocarcinomas (hard to see in the fig). A; detect mRNA (in-situ hybridization) and B; detect protein (immunohistochemistry).
Picture of intestinal area and types of cells that lining along
Ref - DOI: 10.1039/C3IB40163D
2.RNF43 is "majorly" expressed in the endoderm-derived tissues and tissues having high-wnt activity
3.RNF43 is located in the nucleus; when looking in depth --> more on nuclear envelope (LaminB) and lesser nucleoplasm (PSF) and occasionally in ER (calnexin). Mutation on NLS (R437A-K655A) --> cause accumulation of RNF43 in the cytosol.
4.Level of RNF43 expression depends on the colon cancer stage; stage 2 show heterogeneity (some low some high) whereas stage IV higher expression --> this stage really can't say it is the driver or the result of carcinogenesis.
5.RNF43-H292R (RING area mutation)--> no effect on localization --> meaning it works separately
6.TCF-4 is the TF binding to RNF43 promoter thereby regulation of expression (feedback loop); detech through CHIP-immunoprecipitation
7.Control of wnt activity (through the wnt receptor) requires RING domain of RNF43
8.Co-immunoprecipitation revealed RNF43 reacts with TCF-4 and the interaction occur at the C-terminal part of TCF-4 control the TF activity --> physically interact cause inhibition, no ubiquitination activity is related. (TCF-4 is reported to be ubiquitinylated by NARF, Nemo-like kinase associated RING finger protein).
endogenous testting of localization of TCF-4 once RNF43 gene is knocked down
9. Mislocation of TCF-4 caused the reduction of TCF-4 target gene expression
10.Since RNF43 physical interacts with TCF-4 via C-terminal domain of RNF43, either mislocation (truncated from TM toward the end) or lacking of C-terminal domain.
Showing the AA and protein domain in human RNF43 --> protein engineering to see the function of each domain.
Remark:
HCT116 -- chromosomal stable,activated mut-b-catenin,wt-APC,low express conductin and RNF43 mRNA expression is high --> even it is high it could not drag TCF4 to the nuclear membrane. When transfect with corresponding plasmids as show above --> RNF43 is higher enough to interact with TCF4?
Mutations of AA have been found in RNF43, pick the most candidate hit to explore (A). Proposed model for RNF43 that could play role in inhibition of wnt signaling at the downstream level of b-catenin (B).
Antibodies;
OLFM4 -- marker for intestinal stem cell
LaminB receptor -- marker for inner layer of nucleus membrane
Nuclear RNA binding protein PSF -- marker for nucleoplasm
Calnexin -- marker for ER marker
Good information from selleckchem; the orange color reflects available inhibitors being seold by this company -- Ref: http://www.selleckchem.com/
Two things that I observed;
1. functional studies by using representative cell-line that is not the cancer cells, like HEK293 (lack of constitutively Wnt signaling)
2. endogenous condition -- using the corresponding cell line to test the hypothesis; either by knock down (in case of overexpression) or overexpression (in case of low expression)
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