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Fat-regulated adaptor protein Dlish binds the growth suppressor Expanded and controls its stability and ubiquitination.

ABSTRACT: The Drosophila protocadherin Fat controls organ size through the Hippo pathway, but the biochemical links to the Hippo pathway components are still poorly defined. We previously identified Dlish, an SH3 domain protein that physically interacts with Fat and the type XX myosin Dachs, and showed that Fat's regulation of Dlish levels and activity helps limit Dachs-mediated inhibition of Hippo pathway activity. We here characterize a parallel growth control pathway downstream of Fat and Dlish. Using immunoprecipitation and mass spectrometry to search for Dlish partners, we find that Dlish binds the FERM domain growth repressor Expanded (Ex); Dlish SH3 domains directly bind sites in the Ex C terminus. We further show that, in vivo, Dlish reduces the subapical accumulation of Ex, and that loss of Dlish blocks the destabilization of Ex caused by loss of Fat. Moreover, Dlish can bind the F-box E3 ubiquitin ligase Slimb and promote Slimb-mediated ubiquitination of Expanded in vitro. Both the in vitro and in vivo effects of Dlish on Ex require Slimb, strongly suggesting that Dlish destabilizes Ex by helping recruit Slimb-containing E3 ubiquitin ligase complexes to Ex.

SUBMITTER: Wang X

PROVIDER: S-EPMC6347691 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Fat-regulated adaptor protein Dlish binds the growth suppressor Expanded and controls its stability and ubiquitination.

Wang Xing X Zhang Yifei Y Blair Seth S SS

Proceedings of the National Academy of Sciences of the United States of America 20190103 4

The <i>Drosophila</i> protocadherin Fat controls organ size through the Hippo pathway, but the biochemical links to the Hippo pathway components are still poorly defined. We previously identified Dlish, an SH3 domain protein that physically interacts with Fat and the type XX myosin Dachs, and showed that Fat's regulation of Dlish levels and activity helps limit Dachs-mediated inhibition of Hippo pathway activity. We here characterize a parallel growth control pathway downstream of Fat and Dlish. ...[more]

PMID: 30606799

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Project description:BackgroundCGRRF1 is a growth suppressor and consists of a transmembrane domain and a RING-finger domain. It functions as a RING domain E3 ubiquitin ligase involved in endoplasmic reticulum-associated degradation. The expression of CGRRF1 is decreased in cancer tissues; however, the role of CGRRF1 in breast cancer and the mechanism(s) of its growth suppressor function remain to be elucidated.MethodsTo investigate whether CGRRF1 inhibits the growth of breast cancer, we performed MTT assays and a xenograft experiment. Tumors harvested from mice were further analyzed by reverse phase protein array (RPPA) analysis to identify potential substrate(s) of CGRRF1. Co-immunoprecipitation assay was used to verify the interaction between CGRRF1 and its substrate, followed by in vivo ubiquitination assays. Western blot, subcellular fractionation, and reverse transcription quantitative polymerase chain reaction (qRT-PCR) were performed to understand the mechanism of CGRRF1 action in breast cancer. Publicly available breast cancer datasets were analyzed to examine the association between CGRRF1 and breast cancer.ResultsWe show that CGRRF1 inhibits the growth of breast cancer in vitro and in vivo, and the RING-finger domain is important for its growth-inhibitory activity. To elucidate the mechanism of CGRRF1, we identified EGFR as a new substrate of CGRRF1. CGRRF1 ubiquitinates EGFR through K48-linked ubiquitination, which leads to proteasome degradation. In addition to regulating the stability of EGFR, knockout of CGRRF1 enhances AKT phosphorylation after EGF stimulation. By analyzing the breast cancer database, we found that patients with low CGRRF1 expression have shorter survival. As compared to normal breast tissues, the mRNA levels of CGRRF1 are lower in breast carcinomas, especially in HER2-positive and basal-like breast cancers. We further noticed that CGRRF1 promoter methylation is increased in breast cancer as compared to that in normal breast tissue, suggesting that CGRRF1 is epigenetically modified in breast cancer. Treatment of 5-azactidine and panobinostat restored CGRRF1 expression, supporting that the promoter of CGRRF1 is epigenetically modified in breast cancer. Since 5-azactidine and panobinostat can increase CGRRF1 expression, they might be potential therapies for breast cancer treatment.ConclusionWe demonstrated a tumor-suppressive function of CGRRF1 in breast cancer and identified EGFR as its target.

Dataset Information

Fat-regulated adaptor protein Dlish binds the growth suppressor Expanded and controls its stability and ubiquitination.

Publications

Fat-regulated adaptor protein Dlish binds the growth suppressor Expanded and controls its stability and ubiquitination.

Similar Datasets

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