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).

The memory gene KIBRA is a bidirectional regulator of synaptic and structural plasticity in the adult brain.

Memory formation is associated with activity-dependent changes in synaptic plasticity. The mechanisms underlying these processes are complex and involve multiple components. Recent work has implicated the protein KIBRA in human memory, but its molecular functions in memory processes remain not fully understood. Here, we show that a selective overexpression of KIBRA in neurons increases hippocampal long-term potentiation (LTP) but prevents the induction of long-term depression (LTD), and impairs spatial long-term memory in adult mice. KIBRA overexpression increases the constitutive recycling of AMPA receptors containing GluA1 (GluA1-AMPARs), and favors their activity-dependent surface expression. It also results in dramatic dendritic rearrangements in pyramidal neurons both in vitro and in vivo. KIBRA knockdown in contrast, abolishes LTP, decreases GluA1-AMPARs recycling and reduces dendritic arborization. These results establish KIBRA as a novel bidirectional regulator of synaptic and structural plasticity in hippocampal neurons, and of long-term memory, highly relevant to cognitive processes and their pathologies.

Deletion of KIBRA, protein expressed in kidney and brain, increases filopodial-like long dendritic spines in neocortical and hippocampal neurons in vivo and in vitro.

Spines are small protrusions arising from dendrites that receive most excitatory synaptic input in the brain. Dendritic spines represent dynamic structures that undergo activity-dependent adaptations, for example, during synaptic plasticity. Alterations of spine morphology, changes of spine type ratios or density have consequently been found in paradigms of learning and memory, and accompany many neuropsychiatric disorders. Polymorphisms in the gene encoding KIBRA, a protein present in kidney and brain, are linked to memory performance and cognition in humans and mouse models. Deletion of KIBRA impairs long-term synaptic plasticity and postsynaptic receptor recycling but no information is available on the morphology of dendritic spines in null-mutant mice. Here, we directly examine the role of KIBRA in spinous synapses using knockout mice. Since KIBRA is normally highly expressed in neocortex and hippocampus at juvenile age, we analyze synapse morphology in intact tissue and in neuronal cultures from these brain regions. Quantification of different dendritic spine types in Golgi-impregnated sections and in transfected neurons coherently reveal a robust increase of filopodial-like long protrusions in the absence of KIBRA. While distribution of pre- and postsynaptic marker proteins, overall synapse ultrastructure and density of asymmetric contacts were remarkably normal, electron microscopy additionally uncovered less perforated synapses and spinules in knockout neurons. Thus, our results indicate that KIBRA is involved in the maintenance of normal ratios of spinous synapses, and may thus provide a structural correlate of altered cognitive functions when this memory-associated molecule is mutated.

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.

Topoisomerase IIß associates with Ku70 and PARP-1 during double strand break repair of DNA in neurons.

In the present study, the activity of Topoisomerase IIß (TopoIIß) is evaluated during peroxide induced double stranded DNA breaks (DSBs) repair in primary neurons. The results showed that the TopoIIß levels were enhanced during recovery from peroxide mediated damage (PED) along with Ku70, PARP-1, pol beta, and WRN helicase. Furthermore, siRNA mediated knock-down of TopoIIß in primary neurons conferred enhanced susceptibility to PED in neurons. DSBs in neurons are repaired through two pathways, one promoted by Ku70, while the other is by PARP-1 dependent manner. Participation of TopoIIß in both pathways was assessed by analysis of the interaction of TopoIIß with Ku70 and PARP-1 using co-immunoprecipitation experiments in extracts of neurons under peroxide treatment and recovery. The results of these studies showed a strong interaction of TopoIIß with Ku70 as well as PARP-1 suggesting that TopoIIß is associated both in Ku70 and PARP-dependent pathways in DSBs repair in primary neurons. The study has thus established that TopoIIß is an essential component in DSBs repair in primary neurons in both Ku70 and PARP-1 dependent pathways. We suppose that the interaction of TopoIIß may provide stabilization of the repair complex, which may assist in maintenance of tensional integrity in genomic DNA.

Regulation of AMPA receptor function by the human memory-associated gene KIBRA.

KIBRA has recently been identified as a gene associated with human memory performance. Despite the elucidation of the role of KIBRA in several diverse processes in nonneuronal cells, the molecular function of KIBRA in neurons is unknown. We found that KIBRA directly binds to the protein interacting with C-kinase 1 (PICK1) and forms a complex with α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptors (AMPARs), the major excitatory neurotransmitter receptors in the brain. KIBRA knockdown accelerates the rate of AMPAR recycling following N-methyl-D-aspartate receptor-induced internalization. Genetic deletion of KIBRA in mice impairs both long-term depression and long-term potentiation at hippocampal Schaffer collateral-CA1 synapses. Moreover, KIBRA knockout mice have severe deficits in contextual fear learning and memory. These results indicate that KIBRA regulates higher brain function by regulating AMPAR trafficking and synaptic plasticity.

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.

KIBRA: A New Gateway to Learning and Memory?

The genetic locus encoding KIBRA, a member of the WWC family of proteins, has recently been shown to be associated with human memory performance through genome-wide single nucleotide polymorphism screening. Gene expression analysis and a variety of functional studies have further indicated that such a role is biologically plausible for KIBRA. Here, we review the existing literature, illustrate connections between the different lines of evidence, and derive models based on KIBRA's function(s) in the brain that can be further tested experimentally.

Hypertension in mice lacking the CXCR3 chemokine receptor.

The CXC chemokine receptor 3 (CXCR3) has been linked to autoimmune and inflammatory disease, allograft rejection, and ischemic nephropathy. CXCR3 is expressed on endothelial and smooth muscle cells. Although a recent study posited that antagonizing of CXCR3 function may reduce atherosclerosis, the role of CXCR3 in controlling physiological vascular functions remains unclear. This study demonstrates that disruption of CXCR3 leads to elevated mean arterial pressures in anesthetized and conscious mice, respectively. Stimulation of isolated resistance vessels with various vasoconstrictors showed increased contractibility in CXCR3-/- mice in response to angiotensin II (ANG II) and a decreased vasodilatation in response to acetylcholine (ACh). The increased contractibility was related to higher ANG II type 1 receptor (AT1R) expression, whereas the decreased vasodilatation was related to lower M3-ACh receptor expression in the mesenteric arteries of CXCR3-/- mice compared with wild-type mice. The vasodilatatory response to ACh could be antagonized by the nonselective ACh receptor antagonist atropine and the selective M3 receptor antagonist 4-DAMP, but not by M1, M2, and M4 receptor antagonists. Additionally, EMSA studies revealed that transcription factors SP-1 and EGR-1 interact as a complex with the murine AT1R promoter region. Furthermore, we could show increased expression of SP-1 in CXCR3-/- mice indicating an imbalanced SP-1 and EGR-1 complex formation which causes increased AT1R expression and hypertension. The data indicate that CXCR3 receptor is important in vascular contractility and hypertension, possibly through upregulated AT1R expression.

KIBRA modulates directional migration of podocytes.

Asymmetric delivery and distribution of macromolecules are essential for cell polarity and for cellular functions such as differentiation, division, and signaling. Injury of podocytes, which are polarized epithelial cells, changes the dynamics of the actin meshwork, resulting in foot process retraction and proteinuria. Although the spatiotemporal control of specific protein-protein interactions is crucial for the establishment of cell polarity, the mechanisms controlling polarity-dependent differentiation and division are incompletely understood. In this study, yeast two-hybrid screens were performed using a podocyte cDNA library and the polarity protein PATJ as bait. The protein KIBRA was identified as an interaction partner of PATJ and was localized to podocytes, tubular structures, and collecting ducts. The last four amino acids of KIBRA mediated binding to the eighth PDZ domain of PATJ. In addition, KIBRA directly bound to synaptopodin, an essential organizer of the podocyte cytoskeleton. Stable knockdown of KIBRA in immortalized podocytes impaired directed cell migration, suggesting that KIBRA modulates the motility of podocytes by linking polarity proteins and cytoskeleton-associated protein complexes.

SYNCRIP, a component of dendritically localized mRNPs, binds to the translation regulator BC200 RNA.

Dendritic transport of (m)RNA molecules and localized translation at post-synaptic sites is connected to synaptic plasticity and memory formation. Brain cytoplasmic RNA, 200nt (BC200 RNA) is a brain-specific, small non-messenger RNA with a somatodendritic distribution in primate neurons. The transcript is a component of a ribonucleoprotein particle that is thought to act as a regulator of decentralized translation in dendrites. To elucidate the cellular function of the BC200 ribonucleoprotein particle, we purified BC200 RNA-binding proteins from human brain. Here, we describe the interaction of human Synaptotagmin-binding cytoplasmic RNA interacting protein (SYNCRIP) with BC200 RNA. SYNCRIP was recently characterized as a component of large mRNA transport granules in neurons and is probably involved in local protein synthesis at post-synaptic sites. Our in vitro binding studies demonstrate that SYNCRIP interacts specifically with BC200 RNA and that binding is mediated through its N-terminal RNA recognition motifs and the internal A-rich region of BC200 RNA, respectively. Furthermore, immunoprecipitation experiments indicate an in vivo association of SYNCRIP and BC200 RNA in human brain. Thus, SYNCRIP may recruit BC200 RNA into mRNA transport complexes involved in the regulation of localized translation in dendrites.

Characterization of KIAA0513, a novel signaling molecule that interacts with modulators of neuroplasticity, apoptosis, and the cytoskeleton.

KIAA0513 was previously identified as upregulated in the dorsolateral prefrontal cortex of subjects with schizophrenia by microarray analysis. In the present study, the differential expression in the schizophrenic subjects was confirmed by quantitative RT-PCR. The limited homology to proteins of known function and lack of functional domains in the encoded protein have made it difficult to predict a function for KIAA0513. We used in situ hybridization, RNA blots, western blots, and immunocytochemistry to examine KIAA0513 expression in normal brain and peripheral tissues. The gene is ubiquitously expressed but is enriched in the brain, particularly in the cerebellum. Finally, interacting proteins were identified using a yeast two-hybrid screen to functionally characterize the protein. KIAA0513 interacts with KIBRA, HAX-1, and INTS4, which also interact with proteins involved in neuroplasticity, apoptosis, and cytoskeletal regulation. Therefore, KIAA0513 is likely to be involved in signaling pathways related to these processes.

Interaction of rat poly(A)-binding protein with poly(A)- and non-poly(A) sequences is preferentially mediated by RNA recognition motifs 3+4.

Vasopressin (VP) mRNA and the non-coding BC200 RNA are sorted to neuronal dendrites. Among proteins interacting specifically with both RNAs is the multifunctional poly(A)-binding protein (PABP) consisting of four RNA recognition motifs (RRMs) and a C-terminal auxiliary domain. The protein/RNA interaction studies presented here reveal that PABPs association with VP- and BC200 RNA is exclusively mediated by RRMs 3+4. Quantitative binding studies with PABP deletion mutants demonstrate preferential binding of RRMs 3+4 even to poly(A)-homopolymers, while RRMs 1+2 exhibit a lower affinity for those sequences. An optimal interaction with both poly(A)- and non-poly(A) sequences is only achieved by full-size PABP.

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.