Gilhaus, K., Cepok, C., Kamm, D., Surmann, B., Nedvetsky, P. I., Emich, J., Sundukova, A., Saatkamp, K., Nusse, H., Klingauf, J., Wennmann, D. O., George, B., Krahn, M. P., Pavenstadt, H. J. and Vollenbroker, B. A. (2023). Activation of Hippo Pathway Damages Slit Diaphragm by Deprivation of Ajuba Proteins. J Am Soc Nephrol. PubMed ID: 36930055

BIOLOGICAL OVERVIEW The gene warts, named for its wart-like overproliferation phenotype, is also termed lats (large tumor suppressor). Its presence was initially inferred from the observation that mitotic recombination clones homozygous for a specific deficiency produce spectacular outgrowths from the body surface (Justice, 1995). Its presence was also identified in a screen for individuals carrying clones of cells that were homozygous for either X-ray or P-element-induced mutations: a specific mutation produced an overproliferation phenotype (Xu, 1995). warts remained a relatively obscure gene until a recent study (Tao, 1999) showed that the human homolog is a negative regulator of the cyclin dependent kinase/cyclin dimer termed CDC2/cyclin A (see Drosophila CDC2 and Cyclin A). This observation places Warts in the forefront of current interest in the genes that act as central regulators of the cell cycle. Other genes related to warts, namely those more closely related to the mammalian myotonic dystrophy kinase, interact physically with the cytoskeleton regulator Rho, and it is still not clear whether the warts branch of the family functions exclusively as a cell cycle regulator or whether Warts itself also functions in regulating the cytoskeleton. This question will be dealt with more extensively below.

The loss of wts gene function leads to the cell-autonomous formation of epithelial tumors in the adult integumentary structures derived from imaginal discs. Clones have smooth edges and are round or elliptical in shape on legs and especially wings. The rounded shape of wts clones suggests that the divisions of cells lacking wts gene function are not oriented preferentially, as the clones appear to be in wild-type imaginal discs, or that there is a defect in cell adhesion that leads to abnormal cell arrangements. The presence of intact apical junctions indicates that the cellular defect in wts clones is not apical cell separation followed by cuticle deposition between the separated cells; rather, the apical ends of the cells are domed instead of flat, as in normal epithelium. Deposition of cuticle over the domed apical surface produces the altered cuticular morphology (Justice, 1995). Although mutations at wts cause many defects, an effect on cell proliferation could cause most of the phenotypes including overproliferation in mutant clones, lethality at the various stages, tissue overproliferation on the head, broadened wing blade and sterility in homozygous mutants. However, phenotypes such as extra cuticle deposits and malformed bristles and hairs are evidence of defects in differentiation (Xu, 1995).

The different behavior of the wts mutant clones and clones mutant for other previously identified Drosophila tumor suppressors points to multiple functions carried out by the different tumor suppressors. Cells mutant for discs large, lethal (2) giant larvae or hyperplastic discs seem to fail to receive growth regulation signals. They proliferate more slowly than wild-type cells during larval stages when the cells are instructed to proliferate, and they fail to terminate proliferation in late larval and pupal stages when the wild-type cells have ceased proliferation. In contrast, the warts mutant clones induced during the larval stages are overproliferated and, later, the mutant cells on the body are differentiated to form adult cuticular structures. Thus, warts could be a negative regulator that monitors the rate of proliferation (Xu, 1995).

Clues as to the function of warts comes from the cloning of the human and mouse warts homologs. Human LATS1 and fly Warts share 74% sequence identity in the kinase-containing C-terminal domain, much higher than the 45.7% identity shared by the kinase domain of Drosophila Wts and human myotonic dystrophy kinase (DM kinase), previously thought to be a potential Wts homolog. Although the overall sequence similarity between fly and human proteins in the N-terminal region is lower (22% identity and 42% similarity), stretches of highly conserved sequences are apparent. Human LATS1 was expressed in all fetal and adult tissues examined, with highest expression in the adult ovary (Tao, 1999).

Expression of human LATS1 in warts mosaic flies suppresses tumor formation; instead, transgenic warts mutant cells develop into normal adult structures. Expression of the human transgene in homozygous warts mutants rescues lethality of all warts alleles. These animal progress through all developmental stages and hatch as viable adults. The extent of phenotypic rescue correlates with the level of human LATS expression. Complete phenotype rescue requires daily induction of human LATS1, whose expression is driven by a heat shock promoter in flies. These experiments show convincingly that the human LATS1 is an authentic homolog of Drosophila warts (Tao, 1999).

LATS1 is phosphorylated, and the phosphorylation state oscillates with the cell cycle. All LATS1 protein is phosphorylated at late prophase and remains phosphorylated through metaphase. Dephosphorylated LATS can be detected when cells begin to enter anaphase, and by the start of telophase, most LATS1 is dephosphorylated. These observations suggest that LATS1 undergoes two major phosphorylation changes during the cell cycle: LATS1 is phosphorylated at the G2/M boundary or in early prophase, and becomes dephosphorylated at the metaphase/anaphase boundary (Tao, 1999).

The cyclin dependent kinase CDC2 has been shown to interact with LATS1. In immunoprecipitation experiments, the amount of co-precipitated CDC2 varies with the cell cycle. Co-precipitated CDC2 is most abundant at early mitosis, after which the amount of co-precipitated CDC2 progressively decreases as the cell cycle progresses. No co-precipitated CDC2 is detected in quiescent cells. Using the yeast two-hybrid assay, full-length LATS1 and the N-terminal region of LATS interacts with CDC2. The C-terminal kinase domain of LATS1 does not interact with CDC2. Consistent with the notion that the CDC2-associated N-terminal domain is essential for LATS function, a transgene lacking this domain does not rescue warts mutant flies. Neither full-length LATS1 nor the N-terminal region of LATS1 shows any interaction with the G1 cell-cycle kinases CDK2 and CDK4, indicating that the association between LATS1 and CDC2 is specific. The association of LATS1 and CDC2 suggests that LATS1 could act as a tumor suppressor by negatively regulating CDC2 kinase activity, although such a negative effect could not be detected in vitro (Tao, 1999).

Yeast two-hybrid experiments show that the N-terminal region of human LATS1 interacts with CDC2 more strongly than does full-length LATS1. Furthermore, the C-terminal LATS1 kinase domain does bind to the N-terminal region of LATS1 in the two-hybrid assay. These observations raise the possibility that the LATS1 kinase domain may function as a negative regulatory domain that interferes with the CDC2/LATS1 association via intramolecular binding to its N-terminal region. The association of LATS1 with CDC2 is correlated with LATS1's state of phosphorylation. This correlation could be coincidental, however, as phosphorylation is a common mechanism regulating protein activities during the cell cycle. Phosphorylation of LATS1 may be a prerequisite for its binding to DCD2 and may change its conformation, disrupting the intramolecular association between the N and C termini of LATS1 and freeing the N-terminal domain of LATS1 for CDC2 binding (Tao, 1999).

The relationship between Warts and Drosophila Cdc2 was examined in Drosophila. Although Drosophila Cdc2 remains at a constant level during the cell cycle, Cyclins A and B are degraded when Cdc2/cyclin complexes are inactivated. Thus, the levels of Cyclins A and B are sensitive indicators of Cdc2/cyclin activities. By staining eye imaginal discs containing clones of warts mutant cells with either anti-Cyclin A or B antibodies, it was found that inactivation of warts leads to abnormal accumulation of Cyclin A, but not Cyclin B. This provides further evidence that inactivation of warts deregulates Cdc2/Cyclin A activity, and suggests that the warts mutant phenotype could be suppressed by reducing Cdc2/Cyclin A activity. Indeed, both lethality and overproliferation phenotypes of various warts mutants can be suppressed by mutations in cdc2. For example, removing one copy of cdc2 is sufficient to rescue the pupal lethality and tissue overproliferation phenotypes of wts mutants. cyclin A behaves similar to cdc2 in its interaction with wts, whereas reduction of the dosage of cyclin B has no effect on the wts mutant phenotype. Furthermore, mutations in cell-cycle regulator genes such as dEF2, cyclin E and the Drosophila CDK2 homolog cdc2 do not interact with wts mutants. These observations show that the genetic interaction between wts, cdc2 and cyclin A is specific, and support the conclusion that Warts negatively modulates the activity of the CDC2/CyclinA complex (Tao, 1999).

What is the relationship between LATS/Warts and Dbf-2 related kinases and myotonic dystrophy kinase? Mammalian proteins related to myotonic dystrophy kinase interact physically with the cytoskeleton regulator Rho and Rho activates these proteins (Ishizaki, 1996; Matsui, 1996). A Caenorhabditis elegans member of the myotonic dystrophy kinase family interacts genetically with a homolog of the regulatory subunit of smooth muscle myosin phosphatase to affect cell shape (Wissmann, 1997). The 190-kDa myotonic dystrophy kinase-related Cdc42-binding kinases (MRCKs) preferentially phosphorylate nonmuscle myosin light chain at serine 19, a modification known to be crucial for activating actin-myosin contractility (Leung, 1998). Nevertheless, DBF-2 related kinases and myotonic dystrophy kinase share high levels of sequence similarity with the C-terminal kinase domain of Warts, but lack sequences corresponding to the CDC2-binding N-terminal domain (Tao, 1999). It is concluded that the myotonic dystrophy kinases serve a different function than does Warts and its mammalian homolog (Tao, 1999).

The fission yeast orb6 may be the common denominator, showing a common origin to the two kinase subfamilies. Orb6 belongs to the myotonic dystrophy kinase/cot1/warts family. The orb6 gene is required during interphase to maintain cell polarity. A decrease in Orb6 protein levels leads to loss of polarized cell shape, suggesting a role for Orb 6 in cytoskeleton dynamics. Decreased Orb6 leads to mitotic advance, while an increase in Orb6 levels maintains polarized growth and delays mitosis by affecting the p34(cdc2) mitotic kinase. Thus the Orb6 protein kinase coordinates maintenance of cell polarity during interphase. orb6 interacts genetically with orb2, which encodes the Pak1/Shk1 protein kinase, a component of the Ras1 and Cdc42-dependent signaling pathway. These results suggest that Orb6 may act downstream of Pak1/Shk1, forming part of a pathway coordinating cell morphogenesis with progression through the cell cycle. What is the role of these kinases in the regulation of the cell cycle and what is its functional significance? One possibility is that the deregulation of cell proliferation is an indirect consequence of cell shape alteration. The results with yeast suggest, however, that a decrease of Orb6 levels leads to mitotic advance before an alteration of cell shape and that Orb6 overexpression can delay onset of mitosis without altering cell morphology. This finding might point to a novel pathway in the control of mitotic onset and in the coordination of cell growth and cell proliferation (Verde, 1998).

A coordinated phosphorylation by Lats and CK1 regulates YAP stability through SCF^β-TRCP

The Yes-associated protein (YAP) transcription coactivator (a homolog of Drosophila Yorkie) is a key regulator of organ size and a candidate human oncogene. YAP is inhibited by the Hippo pathway kinase cascade, at least in part via phosphorylation of Ser 127, which results in YAP 14-3-3 binding and cytoplasmic retention. This study reports that YAP is phosphorylated by Lats on all of the five consensus HXRXXS motifs. Phosphorylation of Ser 381 in one of them primes YAP for subsequent phosphorylation by CK1delta/epsilon (Drosophila homolog: Discs overgrown) in a phosphodegron. The phosphorylated phosphodegron then recruits the SCF^β-TRCP E3 ubiquitin ligase (see Drosophila Slmb), which catalyzes YAP ubiquitination, ultimately leading to YAP degradation. The phosphodegron-mediated degradation and the Ser 127 phosphorylation-dependent translocation coordinately suppress YAP oncogenic activity. This study identified CK1delta/epsilon as new regulators of YAP and uncovered an intricate mechanism of YAP regulation by the Hippo pathway via both S127 phosphorylation-mediated spatial regulation (nuclear-cytoplasmic shuttling) and the phosphodegron-mediated temporal regulation (degradation) (Zhao, 2010).

Accumulating evidence supports the role of YAP as a key controller of organ size and as a human oncogene. Elucidating the mechanisms regulating YAP activity will have implications in the normal physiology of organ size regulation and pathogenesis of human cancer. The Hippo pathway is the only inhibitor of YAP known to date. It has been shown to play a key role in limiting organ size in Drosophila, and deregulation of several components of this pathway, such as NF2 mutation, has been implicated in human cancer. It has been shown that the Hippo pathway inhibits YAP by S127 phosphorylation-mediated 14-3-3 binding and cytoplasmic retention, therefore providing a mechanism of spatial separation of YAP from its nuclear target transcription factors, such as TEAD (Zhao, 2010).

YAP has been shown to be ubiquitinated, although the mechanism was unknown. The data presented in this study elucidated another layer of YAP regulation. By phosphorylation on S381, the Hippo pathway primes YAP for phosphorylation by CK1delta/epsilon, and subsequent ubiquitination and degradation. This provides a mechanism of temporal regulation of YAP protein levels upon activation of the Hippo pathway. Under physiological conditions like high cell density, the S381 phosphorylation-mediated degradation might be the major cause for YAP degradation. Relative S381 phosphorylation dropped dramatically when cell density increased, although relative S127 phosphorylation of YAP was increased, indicating that the S381-phosphorylated YAP could not be accumulated, possibly due to degradation. However, it is possible that there exists a S127 phosphorylation-dependent fail-safe mechanism for YAP destabilization when S381-mediated degradation is not working properly. Such a mechanism may explain why both S127 and S381 mutations are required for YAP stabilization. This study reveals that inhibition of YAP by the Hippo pathway is more complex than expected, with both spatial and temporal mechanisms. It is speculated that the spatial regulation could provide a reversible short-term inhibition of YAP, while the temporal regulation through YAP degradation may provide an irreversible long-term inhibition. Dysregulation of both mechanisms could lead to oncogenic transformation (Zhao, 2010).

It is worth noting that the S381-initiated degradation of YAP is not conserved in Drosophila Yki, because this phosphorylation site and the phosphodegron are not present in Yki, although they are conserved through vertebrates. However, this does not exclude the possibility that Yki protein stability is controlled by the Hippo pathway through other mechanisms. The phosphodegron is conserved in TAZ, a YAP paralog, and also modulates TAZ stability in a similar manner (Zhao, 2010).

Are there additional mechanisms of YAP regulation by the Hippo pathway? The possibility exists. The current studies confirmed three other Lats phosphorylation sites in YAP, but their functions are unknown. Although these sites do not seem to play an obvious role in controlling the oncogenic activity of YAP, as indicated by NIH-3T3 cell transformation assay, they may function in other contexts (Zhao, 2010).

The similarity between YAP and β-catenin is quite interesting. β-catenin is also a transcription coactivator implicated in malignant transformation. Without Wnt signaling, β-catenin is constantly degraded through SCF^β-TRCP-mediated ubiquitination. Similar to YAP, β-catenin binding with SCF^β-TRCP depends strictly on multistep phosphorylation of the phosphodegron involving CK1α and GSK-3. Perturbation of this process leads to β-catenin accumulation in colorectal cancer, HCCs, and malignant melanomas. There are similarities between YAP and β-catenin in many aspects, including their function as transcription coactivators with growth-promoting activity and as latent oncogenes. They are both subject to multistep phosphorylation and phosphodegron-dependent ubiquitination by SCF^β-TRCP, and deregulation of the degradation leads to oncogenic transformation (Zhao, 2010).

Extensive studies have been done to analyze mutations leading to β-catenin stabilization, which should shed light on future studies of YAP. In the case of β-catenin, its stabilization in cancer is frequently due to failure to recruit GSK3 as a result of inactivating mutations of adenomatous polyposis coli (APC) or axin. In some cases, stabilization of β-catenin also results from mutation in the phosphodegron and its priming phosphorylation sites. Interestingly, elevated YAP protein levels have been observed in some cancers. It will be interesting to survey possible YAP mutations in cancer samples and identify proteins regulating YAP phosphodegron phosphorylation. It will also be important to examine deregulation of YAP protein levels as a result of Hippo pathway component mutations in cancer (Zhao, 2010).

CK1 is a family of multifunctional kinases with unique substrate specificity as pS/T-X_1-2-S/T. Phosphorylation by CK1 requires preceding phosphorylation of residue at the -2 or -3 position of the target residue. This requirement of a priming phosphorylation by another kinase provides a possible mechanism of signal integration in complex biological processes. For example, in the case of YAP destabilization, the requirement of CK1delta/epsilon phosphorylation following Lats phosphorylation may integrate other signals besides the Hippo pathway to regulate YAP. CK1 is often referred to as constitutively active kinase. However, it has also been reported that CK1 is regulated by subcellular localization and inhibitory autophosphorylation by stimuli such as γ irradiation and Wnt signaling. At high cell density, a clear drop of relative YAP-S381 phosphorylation and an increase of relative YAP-S127 phosphorylation are observed. The fact that both sites are phosphorylated by Lats kinase suggests that phosphorylation of S384 might induce YAP degradation. It will be interesting to investigate if cell density increases CK1 activity (Zhao, 2010).

In Drosophila, the CK1delta/epsilon homolog discs overgrown (dco) has been positioned in the Hippo pathway upstream of dachs by its regulation of the Hippo pathway downstream target genes and by genetic epistasis experiments. Recently, dco has further been shown to phosphorylate Fat, although it has not been determined if this phosphorylation directly affects Fat function and the Hippo pathway activity. However, the function of CK1delta/epsilon in regulating YAP-β-TRCP interaction is not due to inhibition of the Hippo pathway, as both YAP-4SA/S381 and YAP-S381D mutants are still inhibited by IC261. Conversely, the mechanism of CK1delta/epsilon in regulating YAP stability is unlikely to be conserved in dco, as the phosphodegron is not conserved in Yki. Nevertheless, the function of dco/CK1delta/epsilon in inhibiting Yki/YAP is conserved between Drosophila and mammals, although different mechanisms may be employed (Zhao, 2010).

YAP contains a phosphodegron, DSGXS, that is highly similar to but does not exactly match the canonical DSGXXS phosphodegron. However, the requirement of the second serine residue for β-TRCP binding is less stringent compared with the first one. In the reported phosphodegron variants, some of them require the second serine to be further away from the DSG, and, in certain cases like CDC25A, the second S is not even required. In the case of YAP, the second serine (S387) is not absolutely required, but contributes to YAP interaction with β-TRCP and YAP ubiquitination. This was shown by the residual binding between β-TRCP and the phosphorylation-deficient S387A, and the largely normal binding between β-TRCP and the phosphomimetic S387D (Zhao, 2010).

The exact YAP sequence S_(-3)TDS₍₀₎G, where S_(-3) (S381) serves as a priming phosphorylation site for S₍₀₎ (S384), is conserved in some other β-TRCP substrates like CDC25A, which contains S_(-6)XXS_(-3)TDS₍₀₎G. In this case, the -6 position serine phosphorylation by Chk1 is shown to be required for CDC25A binding with β-TRCP and subsequent degradation in vivo. However, in an in vitro binding assay, a peptide with phosphorylation on the S₍₀₎ but not S_(-3) showed a strong binding to β-TRCP, which was not further enhanced by phosphorylation on S_(-3). This in vitro binding assay using peptides sharing similar phosphodegron structure with YAP helps to exclude the function of YAP-S381 as an integral part of the phosphodegron directly involved in β-TRCP binding, but rather supports S381 as a priming phosphorylation site for S384 phosphorylation by CK1delta/epsilon. Compared with YAP, it is speculated that the main function of the S_(-3) in the CDC25A phosphodegron might be a phosphorylation-relaying residue passing the signal from the -6 position to the 0 position instead of being directly involved in β-TRCP binding. Phosphodegron with a phosphorylated -3 position serine also exists in other known SCF^β-TRCP substrates, such as RE-1 silencing transcription factor (REST). Together with YAP, they may represent a class of SCF^β-TRCP substrates containing a SXDSG phosphodegron, in which the first serine serves as a priming phosphorylation site. In the case of CDC25A and REST, the kinase responsible for phosphorylating the second serine residue is unknown. The CK1 family kinases are attractive candidates for this function because of their pS/T-X_1-2-S/T target consensus. It is speculated that there may be a broader role for the CK1 family in SCF^β-TRCP-mediated protein ubiquitination and degradation (Zhao, 2010).

In close proximity with the YAP phosphodegron, there is a tyrosine residue (Y391) reported to be phosphorylated by c-Abl in response to DNA damage, which results in YAP stabilization. Future studies are needed to test if the Y391 phosphorylation modulates SCF^β-TRCP-mediated YAP ubiquitination and degradation (Zhao, 2010).

Identification of Happyhour/MAP4K as alternative Hpo/Mst-like kinases in the Hippo kinase cascade

In Drosophila and mammals, the canonical Hippo kinase cascade is mediated by Hpo/Mst acting through the intermediary kinase Wts/Lats to phosphorylate the transcriptional coactivator Yki/YAP/TAZ. Despite recent reports linking Yki/YAP/TAZ activity to the actin cytoskeleton, the underlying mechanisms are poorly understood and/or controversial. Using Drosophila imaginal discs as an in vivo model, this study shows that Wts, but not Hpo, is genetically indispensable for cytoskeleton-mediated subcellular localization of Yki. Through a systematic screen, the Ste-20 kinase Happyhour (Hppy) and its mammalian counterpart MAP4K1/2/3/5 were identified as an alternative kinase that phosphorylates the hydrophobic motif of Wts/Lats in a similar manner as Hpo/Mst. Consistent with their redundant function as activating kinases of Wts/Lats, combined loss of Hpo/Mst and Hppy/MAP4K abolishes cytoskeleton-mediated regulation of Yki/YAP subcellular localization, as well as YAP cytoplasmic translocation induced by contact inhibition. These Hpo/Mst-like kinases provide an expanded view of the Hippo kinase cascade in development and physiology (Zheng, 2015).

Understanding of the core kinase cascade of the Hippo pathway has been aided by multiple lines of investigation. First, genetic screens for tumor suppressors using mosaic flies have identified Hpo, Sav, Wts, and Mats as main constituents of the core kinase cassette. Second, biochemical studies of the activation mechanism of NDR family kinases, which include Lats1/2 and NDR1/2, demonstrate the importance of regulatory phosphorylation sites on the activation loop and the hydrophobic motif. The realization that the hydrophobic motif of Wts is phosphorylated by Hpo provides a fitting molecular explanation for the linear genetic pathway uncovered by in vivo studies. The simplicity of this linear pathway begun to be challenged based on the observation that Mst1/2 null cells still showed high levels of Lats phosphorylation on the hydrophobic motif. Indeed, in many subsequent reports, various signals have been reported to still regulate YAP/TAZ activity in Mst1/2 null cells. While semantically these observations were implied to support the existence of 'Mst1/2-independent' mechanisms, the molecular underpinning of this phenomenon has been elusive. It was also unclear whether this represents a mammalian-specific phenomenon as there has been no evidence to date that a similar mechanism operates in Drosophila (Zheng, 2015).

The current study addresses these issues in several significant ways. Definitive evidence is provided supporting an alternative Hpo-independent mechanism of Hippo pathway activation in Drosophila by demonstrating the genetic requirement of Wts, but not Hpo, in LatB-induced nuclear exclusion of Yki. Through a systematic screen, the Ste-20 family kinase Hppy/MAP4K was identified as a plausible molecular explanation for Mst1/2-independent regulation of Hippo signaling. Not only does Hppy/MAP4K directly phosphorylate the hydrophobic motif of Wts/Lats in vitro and in cell cultures, but loss of Hppy/MAP4K also abolishes LatB-induced Yki/YAP cytoplasmic translocation in Hpo/Mst null cells in both Drosophila tissues and mammalian cell cultures. These findings support the view that Hpo/Mst and Hppy/MAP4K act as redundant kinases targeting the hydrophobic motif of Wts/Lats. It is also noted that analysis of Hpo/Mst and Hppy/MAP4K in F-actin-mediated Hippo signaling was largely based on LatB treatment. Thus, it remains to be determined how these kinases cooperate with each other in a more physiological setting of cytoskeleton modulation. Nevertheless, the fact that MAP4K mediates Mst-independent regulation of YAP target gene expression and contact inhibition of YAP nuclear localization suggests that these kinases co-regulate Wts/Lats in multiple contexts beyond LatB-induced F-actin disruption. Since the hydrophobic motif of NDR1/2 can be phosphorylated by both Mst1 and Mst3, it is suggested that phosphorylation of hydrophobic motif by multiple Ste-20 kinases may be a common feature of the NDR family kinases. It is noted that two other kinases, CK2 and MSN/MAP4K4, were recently reported to promote Wts/Lats activity toward Yki. However, neither kinase was shown to directly phosphorylate the hydrophobic motif of Wts/Lats. Furthermore, although MSN was shown to promote Yki phosphorylation when Wts was co-expressed in S2 cells, MSN alone did not affect Yki phosphorylation, as was observed in this study. Thus, the mechanisms by which these kinases promote Hippo signaling remain to be determined (Zheng, 2015).

Recent studies have implicated cellular mechanical force as a regulator of Yki/YAP/TAZ activity. Reorganization of F-actin cytoskeleton has been suggested as the common mediator of mechanical forces arising from cell-cell and cell-matrix interactions. However, the underlying mechanism by which F-actin controls Yki/YAP/TAZ activity remains poorly understood and/or controversial. While some studies suggested that cytoskeleton-mediated regulation of YAP/TAZ is independent of the Hippo kinase cascade, others suggested that it requires the Hippo kinase cascade. The observation that LatB-induced Yki cytoplasmic localization is Wts dependent is more consistent with a Hippo signaling-dependent mechanism. An important modification brought by the current study is that the canonical Hippo kinase cascade should be expanded to include Hppy/MAP4K at the level of Hpo. This expanded Hippo kinase cascade may also include NDR1/2 at the level of Lats1/2, given the recent report of NDR1/2 as Lats1/2-like kinases capable of phosphorylating YAP (Zheng, 2015).

Finally, it is suggested that the Hippo-signaling-dependent and -independent regulation of YAP/TAZ by F-actin may be potentially reconciled with each other. A major discrepancy between the two models came from the analysis of mutant YAP/TAZ that lacks all the Lats phosphorylation sites (YAP5SA or TAZ4SA). It is noted, however, that a different readout was used to assay the regulation of these YAP/TAZ mutants in the different studies. A luciferase reporter assay was used to show that the transcriptional activity of YAP5SA or TAZ4SA still responded to F-actin reorganization, whereas subcellular localization was used to show that YAP5SA no longer responded to cytoplasmic localization of YAP induced by F-actin disruption. These results may reflect the functionality of different subcellular pools of F-actin ; inasmuch as YAP/TAZ localization is regulated by F-actin through the Hippo pathway, F-actin may also play a separate role in regulating the transcriptional activity of YAP/TAZ in the nucleus, especially given the increasing appreciation of a more direct role of nuclear F-actin in transcriptional regulation (Zheng, 2015).

GENE STRUCTURE

No effort was made to isolate cDNA clones corresponding to the 4.7 kb transcript; thus the exact sequence of this short transcript is not known. However, a polyadenylation signal consensus sequence was found at nucleotide position 4655-4660 in the 5.7 kb transcript and in the corresponding genomic DNA and a 0.51 kb probe from the 3' end of the 5.7 kb transcript does not hybridize to the 4.7 kb transcript, while a 1 kb probe from the 5' untranslated region of the 5.7 kb transcript hybridizes to both the 5.7 kb and 4.7 kb transcripts. This suggests that the 4.7 kb transcript may be a truncated version of the 5.7 kb transcript. The genomic and cDNA sequence corresponding to the 5.7 kb transcript was determined. The entire 5720 bp cDNA sequence is interrupted by seven introns (Xu, 1995).

Genomic size - over 17 kb

Bases in 5' UTR - 1136

Exons - 8

Bases in 3' UTR - 927

PROTEIN STRUCTURE

Amino Acids - 1099

Structural Domains

The warts gene is located in a complex region. The 5' end of the WTS 5.7 kb transcript (cDNA) is only about 550 bp away from the T2 transcript and its 3' end is about 1.5 kb away from the zfh-1 transcript. Furthermore, all three of these closely located transcripts are located in an intron of the T1 transcription unit. An interesting feature of the 5.7 kb transcript is the existence of a 141 bp segment located in the 3' untranslated region, which is identical to the first 141 bp of the 5' untranslated region of the class I transcript from the Drosophila phospholipase C gene, plc-21 (Shortridge, 1991). The functional significance of this sequence motif is unknown. It could be a regulatory target sequence that is shared by both genes. The C-terminal half of Warts shares extensive sequence similarity with a group of six proteins, including the Dbf20 and Dbf2 cell cycle protein-ser/thr kinases from Saccharomyces cerevisiae and the COT-1 putative protein kinase from Neurospora crassa. The sequence similarity between the kinase domains of Warts and these proteins (39%-49% identity) is much higher than the sequence similarity observed between the different subgroups of protein-ser/thr kinases (20%-25% identity). However, there is an insertion of about 40 amino acid residues within the kinase domains of these proteins that shares little sequence similarity with Warts. The human myotonic dystrophy protein kinases (MDPK) also have significant similarity with the C-terminal region of LATS, but their kinase domains do not contain this ~40 amino acid insertion. In addition, LATS and these proteins also share significant levels of sequence similarity in the two regions (each contains ~100-150 a.a.) flanking the kinase domain (20%- 28% identity). In the case of Dbf20, its entire sequence except for the 20 C-terminal most residues can be aligned with Warts, indicating Warts is a close relative of Dbf20. A polyglutamine opa repeat is located near the middle of the protein. The N-terminal half of Wts contains many short homopolymeric runs including polyproline which makes up about 15% of the residues. At least one of the proline-rich stretches closely matches the consensus of SH3-binding sites, raising the possibility that it may interact with SH3-containing proteins (Xu, 1995 and references).

EVOLUTIONARY HOMOLOGS

Warts homologs in yeast and fungi

The yeast Saccharomyces cerevisiae typically divides asymmetrically to give a large mother cell and a smaller daughter cell. Mothers and daughters have distinct fates. Cbk1 kinase and its interacting protein Mob2 (see Drosophila Mats) regulate this asymmetry by inducing daughter-specific genetic programs. Daughter-specific expression is due to Cbk1/Mob2-dependent activation and localization of the Ace2 transcription factor to the daughter nucleus. Ectopic localization of active Ace2 to mother nuclei is sufficient to activate daughter-specific genes in mothers. Eight genes are daughter-specific under the tested conditions, while two are daughter-specific only in saturated cultures. Some daughter-specific gene products contribute to cell separation by degrading the cell wall. These experiments define programs of gene expression specific to daughters and describe how those programs are controlled (Colman-Lerner, 2001).

Protein kinases in the Cot-1/Orb6/Ndr/Warts family are important regulators of cell morphogenesis and proliferation. Cbk1p, a member of this family in Saccharomyces cerevisiae, has been shown to be required for normal morphogenesis in vegetatively growing cells and in haploid cells responding to mating pheromone. A mutant of PAG1, a novel gene in S. cerevisiae, displays defects similar to those of cbk1 mutants. pag1 and cbk1 mutants share a common set of suppressors, including the disruption of SSD1, a gene encoding an RNA binding protein, and the overexpression of Sim1p, an extracellular protein. These genetic results suggest that PAG1 and CBK1 act in the same pathway. Furthermore, Pag1p and Cbk1p localize to the same polarized peripheral sites and they coimmunoprecipitate with each other. Pag1p is a conserved protein. The homologs of Pag1p in other organisms are likely to form complexes with the Cbk1p-related kinases and function with those kinases in the same biological processes (Du, 2001).

The opportunistic fungal pathogen, Candida albicans, is reported to have several potential virulence factors. A potentially significant factor is the ability to undergo morphological transition from yeast to hypha. This alteration of form is accompanied by many changes within the cell, including alterations in gene expression and cell wall composition. A gene has been isolated that encodes a highly conserved serine/threonine kinase that appears to be involved in the regulation of proteins associated with the cell wall. The designation CBK1 (cell wall biosynthesis kinase 1) has been assigned to this gene. Mutants lacking CBK1 form large aggregates of round cells under all growth conditions and lack the ability to undergo morphological differentiation. Additionally, these mutants show an altered pattern of expression of several transcripts encoding proteins associated with the cell wall. The results suggest that the kinase encoded by CBK1 plays a general role in the maintenance and alteration of the cell wall of C. albicans in all morphologies (McNemar, 2002).

The Saccharomyces cerevisiae mitotic exit network (MEN) is a conserved signaling network that coordinates events associated with the M to G1 transition. The function was investigated of two S. cerevisiae proteins related to the MEN proteins Mob1p and Dbf2p kinase. Previous work indicates that cells lacking the Dbf2p-related protein Cbk1p fail to sustain polarized growth during early bud morphogenesis and mating projection formation. Cbk1p is also required for Ace2p-dependent transcription of genes involved in mother/daughter separation after cytokinesis. The Mob1p-related protein Mob2p physically associates with Cbk1p kinase throughout the cell cycle and is required for full Cbk1p kinase activity, which is periodically activated during polarized growth and mitosis. Both Mob2p and Cbk1p localize interdependently to the bud cortex during polarized growth and to the bud neck and daughter cell nucleus during late mitosis. Ace2p is restricted to daughter cell nuclei via a novel mechanism requiring Mob2p, Cbk1p, and a functional nuclear export pathway. Furthermore, nuclear localization of Mob2p and Ace2p does not occur in mob1-77 or cdc14-1 mutants, which are defective in MEN signaling, even when cell cycle arrest is bypassed. Collectively, these data indicate that Mob2p-Cbk1p functions to (1) maintain polarized cell growth, (2) prevent the nuclear export of Ace2p from the daughter cell nucleus after mitotic exit, and (3) coordinate Ace2p-dependent transcription with MEN activation. These findings may implicate related proteins in linking the regulation of cell morphology and cell cycle transitions with cell fate determination and development (Weiss, 2002).

In Saccharomyces cerevisiae, Ras proteins connect nutrient availability to cell growth through regulation of protein kinase A (PKA) activity. Ras proteins also have PKA-independent functions in mitosis and actin repolarization. Mutations in MOB2 or CBK1 confer a slow-growth phenotype in a ras2Delta background. The slow-growth phenotype of mob2Delta ras2Delta cells results from a G1 delay that is accompanied by an increase in size, suggesting a G1/S role for Ras not previously described. In addition, mob2Delta strains have imprecise bud site selection, a defect exacerbated by deletion of RAS2. Mob2 and Cbk1 act to properly localize Ace2, a transcription factor that directs daughter cell-specific transcription of several genes. The growth and budding phenotypes of the double-deletion strains are Ace2 independent but are suppressed by overexpression of the PKA catalytic subunit, Tpk1. From these observations, it is concluded that the PKA pathway and Mob2/Cbk1 act in parallel to determine bud site selection and promote cell cycle progression (Schnaper, 2004).

Mammalian Warts homologs

Although many tumour suppressors have been identified in Drosophila melanogaster, it is not clear whether these fly genes are directly relevant to tumorigenesis in mammals. Mammalian homologues of Drosophila lats have been identified. Human LATS1 suppresses tumour growth and rescues all developmental defects, including embryonic lethality in flies. In mammalian cells, LATS1 is phosphorylated in a cell-cycle-dependent manner and complexes with CDC2 in early mitosis. LATS1-associated CDC2 has no mitotic cyclin partner and no kinase activity for histone H1. Furthermore, lats mutant cells in Drosophila abnormally accumulate cyclin A. These biochemical observations indicate that LATS is a novel negative regulator of CDC2/cyclin A, a finding supported by genetic data in Drosophila demonstrating that lats specifically interacts with cdc2 and cyclin A (Tao, 1999).

To elucidate the function of mammalian LATS1, Lats1-/- mice were generated. Lats1-/- animals exhibit a lack of mammary gland development, infertility and growth retardation. Accompanying these defects are hyperplastic changes in the pituitary and decreased serum hormone levels. The reproductive hormone defects of Lats1-/- mice are reminiscent of isolated LH-hypogonadotropic hypogonadism and corpus luteum insufficiency in humans. Furthermore, Lats1-/- mice develop soft-tissue sarcomas and ovarian stromal cell tumours and are highly sensitive to carcinogenic treatments. These data demonstrate a role for Lats1 in mammalian tumorigenesis and specific endocrine dysfunction (St John, 1999).

A novel human protein kinase, designated kpm, was identified and molecularly cloned. The isolated cDNA clone had an open reading frame consisting of 1088 amino acid residues with a putative kinase domain located near the carboxy-terminus. A homology search revealed that kpm belongs to a subfamily of serine/threonine protein kinases including warts/lats, a Drosophila tumor suppressor. Among these, kpm is most homologous to, but distinct from, recently reported LATS1, a human homolog of Drosophila warts/lats. Northern blot analysis has disclosed that kpm is expressed as a 6.0 kb transcript in most of the tissues examined and also as an additional shorter 4.0 kb transcript in testis. Western blotting detected kpm protein as a band with an apparent Mr of 150 kD. Immune complex kinase assay of HA-tagged kpm shows that kpm has kinase activity and phosphorylates itself in vitro. Studies with cultured cells show that kpm protein is expressed relatively constantly throughout the cell cycle and undergoes significant phosphorylation at mitotic phase. These results suggest that kpm plays a role in cell cycle progression during mitosis and its deletion or dysfunction might be involved in certain types of human cancers (Hori, 2000).

Protein interaction of mammalian Warts homologs

NDR (nuclear Dbf2-related) kinase belongs to a family of kinases that is highly conserved throughout the eukaryotic world. NDR is regulated by phosphorylation and by the Ca(2+)-binding protein, S100B. The budding yeast relatives of Homo sapiens NDR, Cbk1, and Dbf2, have been shown to interact with Mob2 (Mps one binder 2) and Mob1, respectively. This interaction is required for the activity and biological function of these kinases. In this study, it is shown that hMOB1, the closest relative of yeast Mob1 and Mob2, stimulates NDR kinase activity and interacts with NDR both in vivo and in vitro. The point mutations of highly conserved residues within the N-terminal domain of NDR reduced NDR kinase activity as well as human MOB1 binding. A novel feature of NDR kinases is an insert within the catalytic domain between subdomains VII and VIII. The amino acid sequence within this insert shows a high basic amino acid content in all of the kinases of the NDR family known to interact with MOB proteins. This sequence is autoinhibitory, and the data indicate that the binding of human MOB1 to the N-terminal domain of NDR induces the release of this autoinhibition (Bichsel, 2004).

Nuclear Dbf2-related (NDR) protein kinases are a family of AGC group kinases that are involved in the regulation of cell division and cell morphology. The human and mouse NDR2, a second mammalian isoform of NDR protein kinase, have been cloned and characterized. NDR1 and NDR2 share 86% amino acid identity and are highly conserved between human and mouse. However, they differ in expression pattern: mouse Ndr1 is expressed mainly in spleen, lung and thymus, whereas mouse Ndr2 shows highest expression in the gastrointestinal tract. NDR2 is potently activated in cells following treatment with the protein phosphatase 2A inhibitor okadaic acid, which also results in phosphorylation on the activation segment residue Ser-282 and the hydrophobic motif residue Thr-442. Ser-282 becomes autophosphorylated in vivo, whereas Thr-442 is targeted by an upstream kinase. This phosphorylation can be mimicked by replacing the hydrophobic motif of NDR2 with a PRK2-derived sequence, resulting in a constitutively active kinase. Similar to NDR1, the autophosphorylation of NDR2 protein kinase is stimulated in vitro by S100B, an EF-hand Ca(2+)-binding protein of the S100 family, suggesting that the two isoforms are regulated by the same mechanisms. Further a predominant cytoplasmic localization of ectopically expressed NDR2 is demonstrated (Stegert, 2004).

Originally identified in Drosophila, the Warts(Wts)/Lats protein kinase has been proposed to function with two other Drosophila proteins, Hippo (Hpo) and Salvador (Sav), in the regulation of cell cycle exit and apoptosis. In mammals, two candidate Warts/Lats homologs, termed Lats1 and Lats2, have been described, and the targeted disruption of LATS1 in mice increases tumor formation. Little, however, is known about the function and regulation of human Lats kinases. Human Mst2, a STE20-family member and purported Hpo ortholog, phosphorylates and activates both Lats1 and Lats2. Deletion analysis reveals that regulation of Lats1 occurs through the C-terminal, catalytic domain. Within this domain, two regulatory phosphorylation sites were identified by mass spectrometry. These sites, S909 in the activation loop and T1079 within a hydrophobic motif, have been highly conserved during evolution. Moreover, a direct interaction is observed between Mst2 and hWW45, a putative ortholog of Drosophila Sav. These results indicate that Mst2-like kinases regulate Lats kinase activities in an evolutionarily conserved regulatory pathway. Although the function of this pathway remains poorly understood in mammals, it is intriguing that, in Drosophila, it has been linked to development and tissue homeostasis (Chan, 2005).

The conserved Hippo signaling pathway regulates organ size in Drosophila melanogaster and mammals and has an essential role in tumor suppression and the control of cell proliferation. Recent studies identified activators of Hippo signaling, but antagonists of the pathway have remained largely elusive. This paper shows that NPHP4, a known cilia-associated protein that is mutated in the severe degenerative renal disease nephronophthisis, acts as a potent negative regulator of mammalian Hippo signaling. NPHP4 directly interacts with the kinase Lats1 and inhibits Lats1-mediated phosphorylation of the Yes-associated protein (YAP) and TAZ (transcriptional coactivator with PDZ-binding domain), leading to derepression of these protooncogenic transcriptional regulators. Moreover, NPHP4 induces release from 14-3-3 binding and nuclear translocation of YAP and TAZ, promoting TEA domain (TEAD)/TAZ/YAP-dependent transcriptional activity. Consistent with these data, knockdown of NPHP4 negatively affects cellular proliferation and TEAD/TAZ activity, essentially phenocopying loss of TAZ function. These data identify NPHP4 as a negative regulator of the Hippo pathway and suggest that NPHP4 regulates cell proliferation through its effects on Hippo signaling (Habbig, 2011).

Mammalian Warts homologs and the cell cycle

The mitotic apparatus plays a pivotal role in dividing cells to ensure each daughter cell receives a full set of chromosomes and complement of cytoplasm during mitosis. A human homolog of the Drosophila warts tumor suppressor, h-warts/LATS1, is an evolutionarily conserved serine/threonine kinase and a dynamic component of the mitotic apparatus. An interaction of h-warts/LATS1 with zyxin, a regulator of actin filament assembly, has been identified. Zyxin is a component of focal adhesion: however, during mitosis a fraction of cytoplasmic-dispersed zyxin becomes associated with h-warts/LATS1 on the mitotic apparatus. Zyxin is phosphorylated specifically during mitosis, most likely by Cdc2 kinase, and the phosphorylation regulates association with h-warts/LATS1. Furthermore, microinjection of truncated h-warts/LATS1 protein, including the zyxin-binding portion, interfers with localization of zyxin to mitotic apparatus, and the duration of mitosis of these injected cells is significantly longer than that of control cells. These findings suggest that h-warts/LATS1 and zyxin play a crucial role in controlling mitosis progression by forming a regulatory complex on mitotic apparatus (Hirota, 2000).

LATS (large tumour suppressor) is a family of conserved tumour suppressors identified in Drosophila and mammals. Human LATS1 binds to LIMK1 (see Drosophila LIM-kinase1) in vitro and in vivo and colocalizes with LIMK1 at the actomyosin contractile ring during cytokinesis. LATS1 inhibits both the phosphorylation of cofilin by LIMK1 and LIMK1-induced cytokinesis defects. Inactivation of LATS1 by antibody microinjection or RNA-mediated interference in cells, or gene knockout in mice, abrogates cytokinesis and increases the percentage of multinucleate cells. These findings indicate that LATS1 is a novel cytoskeleton regulator that affects cytokinesis by regulating actin polymerization through negative modulation of LIMK1 (Yang, 2004).

Two forms of the large tumor suppressor gene (Lats1) protein expressed in the vertebrate retina

The large tumor suppressor gene (Lats1) encodes a protein kinase that is highly conserved from fly to human, and plays a crucial role in the prevention of tumor formation by controlling mitosis progression. In addition to the previously isolated 7.5 kb long form of Lats1 (Lats1L) mRNA, a less abundant, shorter, 3.4 kb primary transcript (Lats1S) also is expressed in the vertebrate retina. Compared to Lats1L, the sequence of Lats1S mRNA has a deletion of exons 6, 7, and 8 that corresponds to 792 bp of the open reading frame. Thus, 264 aa of the C-terminal region of the long transcript are missing in the Lats1S protein. The encoded truncated protein lacks four of eleven conserved kinase domains and the C-terminus. These results suggest that the 3.4 kb transcript is a splice variant of the 7.5 kb transcript. Direct evidence was found that both the retinal 7.5 and 3.4 kb mRNAs are translated into 170 kDa and 120 kDa proteins, respectively. The expression of both isoforms in vertebrate cells raises the possibility that these Lats1 proteins may act as negative key regulators of the cell cycle, each of them performing a unique role (Akhmedov, 2005).

TAZ promotes cell proliferation and epithelial-mesenchymal transition and is inhibited by the hippo pathway

TAZ is a WW domain containing a transcription coactivator that modulates mesenchymal differentiation and development of multiple organs. The TAZ transcription coactivator is closely related to YAP, which is the mammalian ortholog of the Drosophila Yki, a key component in the Hippo pathway. This study shows that TAZ is phosphorylated by the Lats tumor suppressor kinase, a key component of the Hippo pathway, whose alterations result in organ and tissue hypertrophy in Drosophila and contribute to tumorigenesis in humans. Lats phosphorylates TAZ on several serine residues in the conserved HXRXXS motif and creates 14-3-3 binding sites, leading to cytoplasmic retention and functional inactivation of TAZ. Ectopic expression of TAZ stimulates cell proliferation, reduces cell contact inhibition, and promotes epithelial-mesenchymal transition (EMT). Elimination of the Lats phosphorylation sites results in a constitutively active TAZ, enhancing the activity of TAZ in promoting cell proliferation and EMT. The results elucidate a molecular mechanism for TAZ regulation and indicate a potential function of TAZ as an important target of the Hippo pathway in regulating cell proliferation tumorigenesis (Lei, 2008).

Negative regulation of YAP by LATS1 underscores evolutionary conservation of the Drosophila Hippo pathway

The Hippo pathway defines a novel signaling cascade regulating cell proliferation and survival in Drosophila, which involves the negative regulation of the transcriptional coactivator Yorkie by the kinases Hippo and Warts. The human ortholog of Yorkie, YAP, maps to a minimal amplification locus in mouse and human cancers, and it mediates dramatic transforming activity in MCF10A primary mammary epithelial cells. This study shows that LATS proteins (mammalian orthologs of Warts) interact directly with YAP in mammalian cells and that ectopic expression of LATS1, but not LATS2, effectively suppresses the YAP phenotypes. Furthermore, shRNA-mediated knockdown of LATS1 phenocopies YAP overexpression. Because this effect can be suppressed by simultaneous YAP knockdown, it suggests that YAP is the primary target of LATS1 in mammalian cells. Expression profiling of genes induced by ectopic expression of YAP or by knockdown of LATS1 reveals a subset of potential Hippo pathway targets implicated in epithelial-to-mesenchymal transition, suggesting that this is a key feature of YAP signaling in mammalian cells (Zhang, 2009).

Lats kinase is involved in the intestinal apical membrane integrity in the nematode Caenorhabditis elegans

The roles of Lats kinases in the regulation of cell proliferation and apoptosis have been well established. This study reports new roles for Lats kinase in the integrity of the apical membrane structure. WTS-1, the C. elegans Lats homolog, localizes primarily to the subapical region in the intestine. A loss-of-function mutation in wts-1 results in an early larval arrest and defects in the structure of the intestinal lumen. An electron microscopy study of terminally arrested wts-1 mutant animals revealed numerous microvilli-containing lumen-like structures within the intestinal cells. The wts-1 phenotype was not caused by cell proliferation or apoptosis defects. Instead, the wts-1 mutant animals exhibited gradual mislocalization of apical actin and apical junction proteins, suggesting that wts-1 normally suppresses the formation of extra apical membrane structures. Heat-shock-driven pulse-chase expression experiments showed that WTS-1 regulates the localization of newly synthesized apical actins. RNAi of the exocyst complex genes suppressed the mislocalization phenotype of wts-1 mutation. Collectively, the data presented in this study suggest that Lats kinase plays important roles in the integrity of the apical membrane structure of intestinal cells (Kang, 2009).

Lats kinases have been studied for their roles in mitosis and cytokinesis. Consistent with their known functions in mammals, it has been reported that LATS1 localizes to the mitotic apparatus, centrosome and midbody during cell division. LATS2 is also known to localize to the centrosome during the cell cycle. Yet other reports have suggested that Lats kinases are localized to subcellular compartments unrelated to mitosis or cytokinesis. For example, in flies it has been reported that Wts preferentially localizes near the membrane, where it may influence tissue polarity, growth and gene expression. Cbk1 is also known to localize to sites of polarized growth and regulate bud emergence, growth and maintenance of cell integrity in yeast cells. In addition, many components of the Lats pathway (Mst kinase, Mer, Ex, Mob1 and Yap) localize to the membrane. The current data clearly show that WTS-1 is localized to the subapical region in the intestine. The subapical membrane in epithelial cells is generally important for polarization because active exocytosis and membrane growth occur at subapical membranes. Consistent with the normal function of WTS-1 in inhibiting the mislocalization of intestinal apical actin, knockdown of the exocyst complex proteins, which are known to be important for protein targeting to the basolateral membrane, suppresses the ectopic localization of apical actin in the wts-1 mutant animals. These facts strongly suggest that wts-1 acts as a guardian to ensure that the apical actin and junctional proteins are properly transported and maintained near the plasma membrane to preserve normal lumen structures. Collectively, these data strongly suggest that an evolutionarily conserved function of Lats is to regulate apical protein localization to maintain cell integrity in epithelial cells. It would be of interest to examine whether the regulation of protein localization by wts-1 is conserved in other species, such as D. melanogaster and mammals. In addition, the phenotype associated with the wts-1 mutation in C. elegans is related, but not identical, to the phenotype seen with mutations in crumbs and stardust in D. melanogaster, both of which are crucial elements in the regulation of epithelial cell polarity. It would be of interest to examine the roles of homologs of these genes in the maintenance of cellular polarity in C. elegans (Kang, 2009).

How does WTS-1 regulate the integrity of the intestine? One possible mechanism is by regulating the localization of a subset of proteins through phosphorylation. It is known that PAR-1 phosphorylates BAZ, the PAR-3 homolog in Drosophila, causing Bazooka to bind 14-3-3 and thereby inhibiting the basolateral localization of Bazooka. aPKC also phosphorylates PAR-1 to regulate the localization of PAR-1. Likewise, wts-1 might phosphorylate target proteins that regulate cellular integrity. Interestingly, Cbk1 in budding yeasts binds and phosphorylates Sec2, a guanyl-nucleotide exchange factor (GEF), which is involved in polarized growth and secretion in yeast. There are many GEF homologs in C. elegans, and it would be of interest to examine whether any of these can be phosphorylated by WTS-1. Another possibility is that Lats kinase might be directly involved in the actin filament assembly. LATS1 in mammals is known to bind the actin-cytoskeleton-associated proteins zyxin and LIMK1. During mitosis and cytokinesis, LATS1 colocalizes with F-actin. During cytokinesis, LATS1 negatively regulates LIMK1, subsequently affecting cofilin and actin polymerization. Likewise, it is possible that Lats kinase modulates actin organization at the membrane by regulating the localization or the activity of actin-associated proteins, and mediates the organization of microvilli structure at the apical membrane. Further studies are needed to determine which proteins are primarily responsible for the wts-1 action in C. elegans (Kang, 2009).

The multiple lumen-like-structure phenotype of the wts-1 mutant animals is similar to the phenotype observed in human patients with microvillus inclusion disease (MVID). In addition, the fact that these patients die before 20 months of age is analogous to the L1 to L2 larval arrest phenotype of wts-1. Interestingly, defects in protein trafficking are thought to be the major pathogenic factor underlying MVID. For example, abnormal apical membrane protein trafficking or misregulated endocytosis can lead to MVID. Further studies are needed to determine whether the malfunction of Lats kinases and the genes that are involved in protein targeting/sorting are involved in MVID pathogenesis (Kang, 2009).

Hippo pathway regulation by cell morphology and stress fibers

The Hippo signaling pathway plays an important role in regulation of cell proliferation. Cell density regulates the Hippo pathway in cultured cells; however, the mechanism by which cells detect density remains unclear. This study demonstrates that changes in cell morphology are a key factor. Morphological manipulation of single cells without cell-cell contact resulted in flat spread or round compact cells with nuclear or cytoplasmic Yap, respectively. Stress fibers increased in response to expanded cell areas, and F-actin regulated Yap downstream of cell morphology. Cell morphology- and F-actin-regulated phosphorylation of Yap, and the effects of F-actin were suppressed by modulation of Lats (Warts in Drosophila). These results suggest that cell morphology is an important factor in the regulation of the Hippo pathway, which is mediated by stress fibers consisting of F-actin acting upstream of, or on Lats, and that cells can detect density through their resulting morphology. This cell morphology (stress-fiber)-mediated mechanism probably cooperates with a cell-cell contact (adhesion)-mediated mechanism involving the Hippo pathway to achieve density-dependent control of cell proliferation (Wada, 2011).

A kinase shRNA screen links LATS2 and the pRB tumor suppressor

pRB-mediated inhibition of cell proliferation is a complex process that depends on the action of many proteins. However, little is known about the specific pathways that cooperate with the Retinoblastoma protein (pRB) and the variables that influence pRB's ability to arrest tumor cells. Here two short hairpin RNA (shRNA) screens are described that identify kinases that are important for pRB to suppress cell proliferation and pRB-mediated induction of senescence markers. The results reveal an unexpected effect of LATS2, a component of the Hippo pathway, on pRB-induced phenotypes. Partial knockdown of LATS2 strongly suppresses some pRB-induced senescence markers. Further analysis shows that LATS2 cooperates with pRB to promote the silencing of E2F target genes, and that reduced levels of LATS2 lead to defects in the assembly of DREAM (DP, RB [retinoblastoma], E2F, and MuvB) repressor complexes at E2F-regulated promoters. Kinase assays show that LATS2 can phosphorylate DYRK1A, and that it enhances the ability of DYRK1A to phosphorylate the DREAM subunit LIN52. Intriguingly, the LATS2 locus is physically linked with RB1 on 13q, and this region frequently displays loss of heterozygosity in human cancers. Thee results reveal a functional connection between the pRB and Hippo tumor suppressor pathways, and suggest that low levels of LATS2 may undermine the ability of pRB to induce a permanent cell cycle arrest in tumor cells (Tschöp, 2011).

Lats2 Modulates Adipocyte Proliferation and Differentiation via Hippo Signaling

First identified in Drosophila and highly conserved in mammals, the Hippo pathway controls organ size. Lats2 is one of the core kinases of the Hippo pathway and plays major roles in cell proliferation by interacting with the downstream transcriptional cofactors YAP and TAZ. Although the function of the Hippo pathway and Lats2 is relatively well understood in several tissues and organs, less is known about the function of Lats2 and Hippo signaling in adipose development. This study shows that Lats2 is an important modulator of adipocyte proliferation and differentiation via Hippo signaling. Upon activation, Lats2 phosphorylates YAP and TAZ, leading to their retention in the cytoplasm, preventing them from activating the transcription factor TEAD in the nucleus. Because TAZ remains in the cytoplasm, PPARgamma regains its transcriptional activity. Furthermore, cytoplasmic TAZ acts as an inhibitor of Wnt signaling by suppressing DVL2, thereby preventing beta-catenin from entering the nucleus to stimulate TCF/LEF transcriptional activity. The above effects contribute to the phenotype of repressed proliferation and accelerated differentiation in adipocytes. Thus, Lats2 regulates the balance between proliferation and differentiation during adipose development. Interestingly, this study provides evidence that Lats2 not only negatively modulates cell proliferation but also positively regulates cell differentiation (An, 2013).

Spatial Organization of Hippo Signaling at the Plasma Membrane Mediated by the Tumor Suppressor Merlin/NF2

Although Merlin/NF2 was discovered two decades ago as a tumor suppressor underlying Neurofibromatosis type II, its precise molecular mechanism remains poorly understood. Recent studies in Drosophila revealed a potential link between Merlin and the Hippo pathway by placing Merlin genetically upstream of the kinase Hpo/Mst. This study shows that in both Drosophila and mammals, Merlin promotes downstream Hippo signaling without activating the intrinsic kinase activity of Hpo/Mst. Instead, Merlin directly binds and recruits the effector kinase Wts/Lats to the plasma membrane. Membrane recruitment, in turn, promotes Wts phosphorylation by the Hpo-Sav kinase complex. This study further shows that disruption of the actin cytoskeleton promotes Merlin-Wts interactions, which implicates Merlin in actin-mediated regulation of Hippo signaling. These findings elucidate an important molecular function of Merlin and highlight the plasma membrane as a critical subcellular compartment for Hippo signal transduction (Yin, 2013).

LATS2 suppresses oncogenic Wnt signaling by disrupting beta-catenin/BCL9 interaction

Abnormal activation of Wnt/β-catenin-mediated transcription is associated with a variety of human cancers. This study reports that LATS2 inhibits oncogenic Wnt/β-catenin-mediated transcription by disrupting the β-catenin/BCL9 interaction. LATS2 directly interacts with β-catenin and is present on Wnt target gene promoters. Mechanistically, LATS2 inhibits the interaction between BCL9 and β-catenin and subsequent recruitment of BCL9, independent of LATS2 kinase activity. LATS2 is downregulated and inversely correlated with the levels of Wnt target genes in human colorectal cancers. Moreover, nocodazole, an antimicrotubule drug, potently induces LATS2 to suppress tumor growth in vivo by targeting β-catenin/BCL9. These results suggest that LATS2 is not only a key tumor suppressor in human cancer but may also be an important target for anticancer therapy (Li, 2013).

SHANK2 is a frequently amplified oncogene with evolutionarily conserved roles in regulating Hippo signaling

Dysfunction of the Hippo pathway enables cells to evade contact inhibition and provides advantages for cancerous overgrowth. However, for a significant portion of human cancer, how Hippo signaling is perturbed remains unknown. To answer this question, a genome-wide screening was performed for genes that affect the Hippo pathway in Drosophila and cross-referenced the hit genes with human cancer genome. In this screen, Prosap was identified as a novel regulator of the Hippo pathway that potently affects tissue growth. Interestingly, a mammalian homolog of Prosap, SHANK2, is the most frequently amplified gene on 11q13, a major tumor amplicon in human cancer. Gene amplification profile in this 11q13 amplicon clearly indicates selective pressure for SHANK2 amplification. More importantly, across the human cancer genome, SHANK2 is the most frequently amplified gene that is not located within the Myc amplicon. Further studies in multiple human cell lines confirmed that SHANK2 overexpression causes deregulation of Hippo signaling through competitive binding for a LATS1 (see Drosophila Lats) activator, and as a potential oncogene, SHANK2 promotes cellular transformation and tumor formation in vivo. In cancer cell lines with deregulated Hippo pathway, depletion of SHANK2 restores Hippo signaling and ceases cellular proliferation. Taken together, these results suggest that SHANK2 is an evolutionarily conserved Hippo pathway regulator, commonly amplified in human cancer and potently promotes cancer. This study illustrated oncogenic function of SHANK2, one of the most frequently amplified gene in human cancer. Furthermore, given that in normal adult tissues, SHANK2's expression is largely restricted to the nervous system, SHANK2 may represent an interesting target for anticancer therapy (Xu, 2020).

date revised: 5 December 2023

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