WW and C2 domain-containing proteins regulate hepatic cell differentiation and tumorigenesis through the hippo signaling pathway.

The Hippo pathway regulates cell differentiation, proliferation, and apoptosis. Upon activation, it inhibits the import of the transcriptional coactivator yes-associated protein (YAP) into the nucleus, thus suppressing transcription of pro-proliferative genes. Hence, dynamic and precise control of the Hippo pathway is crucial for organ size control and the prevention of tumor formation. Hippo signaling is controlled by a growing number of upstream regulators, including WW and C2 domain-containing (WWC) proteins, which trigger a serine/threonine kinase pathway. One component of this is the large tumor suppressor (LATS) kinase, which phosphorylates YAP, trapping it in the cytoplasm. WWC proteins have been shown to interact with LATS in vitro and stimulate its kinase activity, thus directly promoting cytoplasmic accumulation of phosphorylated YAP. However, the function of the WWC proteins in the regulation of cell proliferation, organ size control, and tumor prevention in vivo has not yet been determined. Here, we show that loss of hepatic WWC expression in mice leads to tissue overgrowth, inflammation, fibrosis, and formation of liver carcinoma. WWC-deficient mouse livers display reduced LATS activity, increased YAP-mediated gene transcription, and enhanced proliferation of hepatic progenitor cells. In addition, loss of WWC expression in the liver accelerates the turnover of angiomotin proteins, which act as negative regulators of YAP activity. CONCLUSION: Our data define an essential in vivo function for WWC proteins as regulators of canonical and noncanonical Hippo signaling in hepatic cell growth and liver tumorigenesis. Thus, expression of WWC proteins may serve as novel prognostic factors in human liver carcinoma. (Hepatology 2018;67:1546-1559).

Evolutionary and molecular facts link the WWC protein family to Hippo signaling.

The scaffolding protein KIBRA (also called WWC1) is involved in the regulation of important intracellular transport processes and the establishment of cell polarity. Furthermore, KIBRA/WWC1 is an upstream regulator of the Hippo signaling pathway that controls cell proliferation and organ size in animals. KIBRA/WWC1 represents only one member of the WWC protein family that also includes the highly similar proteins WWC2 and WWC3. Although the function of KIBRA/WWC1 was studied intensively in cells and animal models, the importance of WWC2 and WWC3 was not yet elucidated. Here, we describe evolutionary, molecular, and functional aspects of the WWC family. We show that the WWC genes arose in the ancestor of bilateral animals (clades such as insects and vertebrates) from a single founder gene most similar to the present KIBRA/WWC1-like sequence of Drosophila. This situation was still maintained until the common ancestor of lancelet and vertebrates. In fish, a progenitor-like sequence of mammalian KIBRA/WWC1 and WWC2 is expressed together with WWC3. Finally, in all tetrapods, the three family members, KIBRA/WWC1, WWC2, and WWC3, are found, except for a large genomic deletion including WWC3 in Mus musculus. At the molecular level, the highly conserved WWC proteins share a similar primary structure, the ability to form homo- and heterodimers and the interaction with a common set of binding proteins. Furthermore, all WWC proteins negatively regulate cell proliferation and organ growth due to a suppression of the transcriptional activity of YAP, the major effector of the Hippo pathway.

Tissue-specific differences in the regulation of KIBRA gene expression involve transcription factor TCF7L2 and a complex alternative promoter system.

UNLABELLED: KIBRA has been described as a key regulator of the Hippo signaling pathway, regulating organ size control, cell contact inhibition, cell growth, as well as tumorigenesis and cystogenesis. Since there is scarce information on KIBRA gene expression regulation, we analyzed the molecular basis of tissue-specific KIBRA expression in human kidney epithelial (IHKE, HPCT) and neuroblastoma (SH-SY5Y, SK-SN-SH) cells. We detected four novel and differentially used transcription start sites, two of which positioned in the first intron, generating two novel alternative exons. We identified one constitutively active core promoter (P1a) and three alternative promoters (P1b, P2, and P3), which were exclusively active in kidney cells. Transcription factor 7-like 2 (TCF7L2) selectively activated KIBRA at P1a, P2, and P3 in kidney cells. The two genetic variants -580C>T (p < 0.05) and -1691C>T (p < 0.01) significantly affected the transcriptional activity of the KIBRA core promoter. We propose a novel functional structure of the KIBRA gene and provide detailed insight into molecular cell type-specific KIBRA transcriptional regulation by TCF7L2, the Yes-associated protein 1 and TEA domain family member. Our findings provide a potential basis for future studies on malfunctioning KIBRA regulation in pathophysiological conditions such as cancer development. KEY MESSAGE: KIBRA expression is regulated by three independent, cell type-specific promoters Two novel TSS were located within intron one resulting in two alternative exons TSS utilization is cell type-specific TCF7L2, YAP1, and TEAD are involved in the differential KIBRA expression regulation.

KIBRA (KIdney/BRAin protein) regulates learning and memory and stabilizes Protein kinase Mζ.

The WWC1 gene has been genetically associated with human episodic memory performance, and its product KIdney/BRAin protein (KIBRA) has been shown to interact with the atypical protein kinase protein kinase M ζ (PKMζ). Although recently challenged, PKMζ remains a candidate postsynaptic regulator of memory maintenance. Here, we show that PKMζ is subject to rapid proteasomal degradation and that KIBRA is both necessary and sufficient to counteract this process, thus stabilizing the kinase and maintaining its function for a prolonged time. We define the binding sequence on KIBRA, a short amino acid motif near the C-terminus. Both hippocampal knock-down of KIBRA in rats and KIBRA knock-out in mice result in decreased learning and memory performance in spatial memory tasks supporting the notion that KIBRA is a player in episodic memory. Interestingly, decreased memory performance is accompanied by decreased PKMζ protein levels. We speculate that the stabilization of synaptic PKMζ protein levels by KIBRA may be one mechanism by which KIBRA acts in memory maintenance. KIBRA/WWC1 has been genetically associated with human episodic memory. KIBRA has been shown to be post-synaptically localized, but its function remained obscure. Here, we show that KIBRA shields PKMζ, a kinase previously linked to memory maintenance, from proteasomal degradation via direct interaction. KIBRA levels in the rodent hippocampus correlate closely both to spatial memory performance in rodents and to PKMζ levels. Our findings support a role for KIBRA in memory, and unveil a novel function for this protein.

Polycystin-2 activity is controlled by transcriptional coactivator with PDZ binding motif and PALS1-associated tight junction protein.

Autosomal dominant polycystic kidney disease (ADPKD) is the most frequent monogenic cause of kidney failure, characterized by the development of renal cysts. ADPKD is caused by mutations of the polycystin-1 (PC1) or polycystin-2 (PC2) genes. PC2 encodes a Ca(2+)-permeable cation channel, and its dysfunction has been implicated in cyst development. The transcriptional coactivator with PDZ binding motif (TAZ) is required for the integrity of renal cilia. Its absence results in the development of renal cysts in a knock-out mouse model. TAZ directly interacts with PC2, and it has been suggested that another yet unidentified PDZ domain protein may be involved in the TAZ/PC2 interaction. Here we describe a novel interaction of TAZ with the multi-PDZ-containing PALS1-associated tight junction protein (PATJ). TAZ interacts with both the N-terminal PDZ domains 1-3 and the C-terminal PDZ domains 8-10 of PATJ, suggesting two distinct TAZ binding domains. We also show that the C terminus of PC2 strongly interacts with PDZ domains 8-10 and to a weaker extent with PDZ domains 1-3 of PATJ. Finally, we demonstrate that both TAZ and PATJ impair PC2 channel activity when co-expressed with PC2 in oocytes of Xenopus laevis. These results implicate TAZ and PATJ as novel regulatory elements of the PC2 channel and might thus be involved in ADPKD pathology.

Poly(A)-binding protein is associated with neuronal BC1 and BC200 ribonucleoprotein particles.

BC1 RNA and BC200 RNA are two non-homologous, small non-messenger RNAs (snmRNAs) that were generated, evolutionarily, quite recently by retroposition. This process endowed the RNA polymerase III transcripts with central adenosine-rich regions. Both RNAs are expressed almost exclusively in neurons, where they are transported into dendritic processes as ribonucleoprotein particles (RNPs). Here, we demonstrate with a variety of experimental approaches that poly(A)-binding protein (PABP1), a regulator of translation initiation, binds to both RNAs in vitro and in vivo. We identified the association of PABP with BC200 RNA in a tri-hybrid screen and confirmed this binding in electrophoretic mobility-shift assays and via anti-PABP immunoprecipitation of BC1 and BC200 RNAs from crude extracts, immunodepleted extracts, partially purified RNPs and cells transfected with naked RNA. Furthermore, PABP immunoreactivity was localized to neuronal dendrites. Competition experiments using variants of BC1 and BC200 RNAs demonstrated that the central adenosine-rich region of both RNAs mediates binding to PABP. These findings lend support to the hypothesis that the BC1 and BC200 RNPs are involved in protein translation in neuronal dendrites.