The Structure of an NDR/LATS Kinase–Mob Complex Reveals a Novel Kinase–Coactivator System and Substrate Docking Mechanism

Eukaryotic cells commonly use protein kinases in signaling systems that relay information and control a wide range of processes. These enzymes have a fundamentally similar structure, but achieve functional diversity through variable regions that determine how the catalytic core is activated and recruited to phosphorylation targets. “Hippo” pathways are ancient protein kinase signaling systems that control cell proliferation and morphogenesis; the NDR/LATS family protein kinases, which associate with “Mob” coactivator proteins, are central but incompletely understood components of these pathways. Here we describe the crystal structure of budding yeast Cbk1–Mob2, to our knowledge the first of an NDR/LATS kinase–Mob complex. It shows a novel coactivator-organized activation region that may be unique to NDR/LATS kinases, in which a key regulatory motif apparently shifts from an inactive binding mode to an active one upon phosphorylation. We also provide a structural basis for a substrate docking mechanism previously unknown in AGC family kinases, and show that docking interaction provides robustness to Cbk1’s regulation of its two known in vivo substrates. Co-evolution of docking motifs and phosphorylation consensus sites strongly indicates that a protein is an in vivo regulatory target of this hippo pathway, and predicts a new group of high-confidence Cbk1 substrates that function at sites of cytokinesis and cell growth. Moreover, docking peptides arise in unstructured regions of proteins that are probably already kinase substrates, suggesting a broad sequential model for adaptive acquisition of kinase docking in rapidly evolving intrinsically disordered polypeptides.     

MOB control: reviewing a conserved family of kinase regulators

The family of Mps One binder (MOB) co-activator proteins is highly conserved from yeast to man. At least two different MOB proteins have been identified in every eukaryote analysed to date. Initially, yeast genetics revealed essential roles for Mob1p and Mob2p in the regulation of mitotic exit and cell morphogenesis. Studies in flies then showed that dMOB1/MATS is a core component of Hippo signalling. Loss of dMOB1 resulted in increased cell proliferation and decreased cell death, suggesting that MOB1 acts as tumour suppressor protein. Recent work focused primarily on mammalian cells has shown how hMOB1 can regulate NDR/LATS kinases, a function that can to be counteracted by hMOB2. Here we summarise and discuss our current knowledge of this emerging protein family, with emphasis on subcellular localisation, protein-protein interactions and biological functions in apoptosis, mitosis, morphogenesis, cell proliferation and centrosome duplication.     

NDR kinases regulate essential cell processes from yeast to humans

Members of the NDR (nuclear Dbf2-related) protein-kinase family are essential components of pathways that control important cellular processes, such as morphological changes, mitotic exit, cytokinesis, cell proliferation and apoptosis. Recent progress has shed light on the mechanisms that underlie the regulation and function of the NDR family members. Combined data from yeast, worms, flies, mice and human cells now highlight the conserved and important roles of the different NDR kinases in distinct cellular processes.     

Drosophila C-terminal Src kinase negatively regulates organ growth and cell proliferation through inhibition of the Src, Jun N-terminal kinase, and STAT pathways

Src family kinases regulate multiple cellular processes including proliferation and oncogenesis. C-terminal Src kinase (Csk) encodes a critical negative regulator of Src family kinases. We demonstrate that the Drosophila melanogaster Csk ortholog, dCsk, functions as a tumor suppressor: dCsk mutants display organ overgrowth and excess cellular proliferation. Genetic analysis indicates that the dCsk(-/-) overgrowth phenotype results from activation of Src, Jun kinase, and STAT signal transduction pathways. In particular, blockade of STAT function in dCsk mutants severely reduced Src-dependent overgrowth and activated apoptosis of mutant tissue. Our data provide in vivo evidence that Src activity requires JNK and STAT function.     

WWP1 E3 Ligase Targets LATS1 for Ubiquitin-Mediated Degradation in Breast Cancer Cells

The Large Tumor Suppressor 1 (LATS1) is a serine/threonine kinase and tumor suppressor found down-regulated in various human cancers. LATS1 has recently been identified as a central player of the emerging Hippo signaling pathway, which plays important roles in organ size control, tumorigenesis, and stem cell differentiation and renewal, etc. Although mounting evidence supports a role of LATS1 in tumor suppression and tumorigenesis, how LATS1 is regulated at the molecular level is not fully understood. Recently several positive regulators of LATS1 (Mst1/2, MOB1, Kibra, etc) have been identified but how LATS1 is negatively regulated is still largely unknown. We have recently identified Itch, a member of the NEDD4-like family E3 ubiquitin ligases, as a novel negative regulator of LATS1. However, whether other ubiquitin ligases modulate LATS1 stability and function is unclear. By screening many E3 ligases of the NEDD4-like family using over-expression and short-interference RNA knockdown approaches, we have identified WWP1 E3 ligase as another novel negative regulator of LATS1. We have provided in vitro and in vivo evidence that WWP1 is essential for LATS1 stability and negatively regulate LATS1 by promoting LATS1 degradation through polyubiquitination and the 26S proteasome pathway. Importantly, we also showed that degradation of LATS1 is critical in mediating WWP1-induced increased cell proliferation in breast cancer cells. Since WWP1 is an oncogene and LATS1 is a tumor suppressor gene in breast cancer, our studies provide a promising therapeutic strategy in which developed drugs targeting WWP1 cause activation of LATS1 in suppressing breast cancer cell growth.     

Mammalian NDR protein kinases: from regulation to a role in centrosome duplication

The NDR (nuclear Dbf2-related) family of kinases is highly conserved from yeast to human, and has been classified as a subgroup of the AGC group of protein kinases based on the sequence of the catalytic domain. Like all other members of the AGC class of protein kinases, NDR kinases require the phosphorylation of conserved Ser/Thr residues for activation. Importantly, NDR family members have two unique stretches of primary sequence: an N-terminal regulatory (NTR) domain and an insert of several residues between subdomains VII and VIII of the kinase domain. The kinase domain insert functions as an auto-inhibitory sequence (AIS), while binding of the co-activator MOB (Mps-one binder) proteins to the NTR domain releases NDR kinases from inhibition of autophosphorylation. However, despite such advances in our understanding of the molecular activation mechanism(s) and physiological functions of NDR kinases in yeast and invertebrates, most biological NDR substrates still remain to be identified. Nevertheless, by showing that the centrosomal subpopulation of human NDR1/2 is required for proper centrosome duplication, the first biological role of human NDR1/2 kinases has been defined recently. How far NDR-driven centrosome overduplication could actually contribute to cellular transformation will also be discussed.     

Mechanism of activation of NDR (nuclear Dbf2-related) protein kinase by the hMOB1 protein

NDR (nuclear Dbf2-related) kinase belongs to a family of kinases that is highly conserved throughout the eukaryotic world. We showed previously that NDR is regulated by phosphorylation and by the Ca(2+)-binding protein, S100B. The budding yeast relatives of Homo sapiens NDR, Cbk1, and Dbf2, were 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, we show 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. We show that this sequence is autoinhibitory, and our data indicate that the binding of human MOB1 to the N-terminal domain of NDR induces the release of this autoinhibition.     

Human NDR kinases control G(1)/S cell cycle transition by directly regulating p21 stability

The G(1) phase of the cell cycle is an important integrator of internal and external cues, allowing a cell to decide whether to proliferate, differentiate, or die. Multiple protein kinases, among them the cyclin-dependent kinases (Cdks), control G(1)-phase progression and S-phase entry. With the regulation of apoptosis, centrosome duplication, and mitotic chromosome alignment downstream of the HIPPO pathway components MST1 and MST2, mammalian NDR kinases have been implicated to function in cell cycle-dependent processes. Although they are well characterized in terms of biochemical regulation and upstream signaling pathways, signaling mechanisms downstream of mammalian NDR kinases remain largely unknown. We identify here a role for human NDR in regulating the G(1)/S transition. In G(1) phase, NDR kinases are activated by a third MST kinase (MST3). Significantly, interfering with NDR and MST3 kinase expression results in G(1) arrest and subsequent proliferation defects. Furthermore, we describe the first downstream signaling mechanisms by which NDR kinases regulate cell cycle progression. Our findings suggest that NDR kinases control protein stability of the cyclin-Cdk inhibitor protein p21 by direct phosphorylation. These findings establish a novel MST3-NDR-p21 axis as an important regulator of G(1)/S progression of mammalian cells.     

Drosophila Mob family proteins interact with the related tricornered (Trc) and warts (Wts) kinases

The function of Tricornered (Trc), the Drosophila Ndr (Nuclear Dbf2-related) serine/threonine protein kinase, is required for the normal morphogenesis of a variety of polarized outgrowths including epidermal hairs, bristles, arista laterals, and dendrites. In yeast the Trc homolog Cbk1 needs to bind Mob2 to activate the RAM pathway. In this report, we provide genetic and biochemical data that Drosophila Trc also interacts with and is activated by Drosophila Dmob proteins. In addition, Drosophila Mob proteins appear to interact with the related Warts/Lats kinase, which functions as a tumor suppressor in flies and mammals. Interestingly, the overgrowth tumor phenotype that results from mutations in Dmob1 (mats) was only seen in genetic mosaics and not when the entire animal was mutant. We conclude that unlike in yeast, in Drosophila individual Mob proteins interact with multiple kinases and that individual NDR family kinases interact with multiple Mob proteins. We further provide evidence that Mo25, the Drosophila homolog of the RAM pathway hym1 gene does not function along with Trc.     

Human Mob proteins regulate the NDR1 and NDR2 serine-threonine kinases

Human NDR1 (nuclear Dbf2-related) is a widely expressed nuclear serine-threonine kinase that has been implicated in cell proliferation and/or tumor progression. Here we present molecular characterization of the human NDR2 serine-threonine kinase, which shares approximately 87% sequence identity with NDR1. NDR2 is expressed in most human tissues with the highest expression in the thymus. In contrast to NDR1, NDR2 is excluded from the nucleus and exhibits a punctate cytoplasmic distribution. The differential localization of NDR1 and NDR2 suggests that each kinase may serve distinct functions. Thus, to identify proteins that interact with NDR1 or NDR2, epitope-tagged kinases were immunoprecipitated from Jurkat T-cells. Two uncharacterized proteins that are homologous to the Saccharomyces cerevisiae kinase regulators Mob1 and Mob2 were identified. We demonstrate that NDR1 and NDR2 partially colocalize with human Mob2 in HeLa cells and confirm the NDR-Mob interactions in cell extracts. Interestingly, NDR1 and NDR2 form stable complexes with Mob2, and this association dramatically stimulates NDR1 and NDR2 catalytic activity. In summary, this work identifies a unique class of human kinase-activating subunits that may be functionally analagous to cyclins.     

LATS1 and LATS2 Phosphorylate CDC26 to Modulate Assembly of the Tetratricopeptide Repeat Subcomplex of APC/C

In budding yeast, the Mitotic Exit Network (MEN) regulates anaphase promoting complex/cyclosome (APC/C) via the Dbf2-Cdc14 signaling cascade. Dbf2 kinase phosphorylates and activates Cdc14 phosphatase, which removes the inhibitory phosphorylation of the APC/C cofactor Cdh1. Although each component of the MEN was highly conserved during evolution, there is presently no evidence supporting direct phosphorylation of CDC14 by large tumor suppressor kinase 1 (LATS1), the human counterpart of Dbf2; hence, it is unclear how LATS1 regulates APC/C. Here, we demonstrate that LATS1 phosphorylates the Thr7 (T7) residue of the APC/C component CDC26 directly. Nocodazole-induced phosphorylation of T7 was reduced by knockdown of LATS1 and LATS2 in HeLa cells, indicating that both of these kinases contribute to the phosphorylation of CDC26 in vivo. The T7 residue of CDC26 is critical for its interaction with APC6, a tetratricopeptide repeat-containing subunit of APC/C, and mutation of this residue to Asp (T7D) reduced the interaction of CDC26 with APC6. Replacement of endogenous CDC26 in HeLa cells with exogenous phosphor-mimic T7D-mutated CDC26 increased the elution size of APC/C subunits in a gel filtration assay, implying a change in the APC/C assembly upon phosphorylation of CDC26. Furthermore, T7D-mutated CDC26 promoted the ubiquitination of polo-like kinase 1, a well-known substrate of APC/C. Overall, these results suggest that LATS1/2 are novel kinases involved in APC/C phosphorylation and indicate a direct regulatory link between LATS1/2 and APC/C.     

CRISPR/Cas9介导的LATS1/2敲除抑制卵巢癌细胞ES-2增殖

Hippo信号通路在哺乳动物肝脏发育、动态平衡、再生和疾病中发挥非常重要的作用。大肿瘤抑制基因1/2（large tumor suppressor 1/2, LATS1/2）激酶是Hippo信号通路的关键激酶,可以磷酸化YES相关蛋白（yes-associated protein,YAP）,从而调节YAP的核质定位和降解。本文采用CRISPR/Cas9方法构建慢病毒介导的Last1/2基因敲除的载体,通过包装、感染和嘌呤霉素筛选,获得LATS1/2部分敲除的人卵巢癌ES-2和H08910细胞,免疫印迹方法检测LATS1/2表达明显减少。细胞增殖实验检测LATS1/2缺失明显抑制ES-2和HO8910细胞增殖。软琼脂克隆形成实验表明,LATS1/2缺失抑制卵巢癌ES-2细胞的克隆形成能力。细胞划痕和Transwell实验证明,LATS1/2缺失明显抑制卵巢癌ES-2细胞迁移。流式细胞检测发现,LATS1/2敲除促进卵巢癌ES-2细胞凋亡并影响细胞周期。裸鼠成瘤实验表明,LATS1/2缺失明显抑制体内肿瘤组织增殖。分子机制研究表明, LATS1/2敲除促进卵巢癌ES-2细胞中胶原I型α1（collagen type I α1,ColIα1）基因表达量增加,在卵巢癌ES-2细胞中同时敲除LATS1/2和COL1A1,可以促进细胞克隆形成。综上结果,人卵巢癌ES-2细胞中LATS1/2缺失能促进COL1A1表达增加, 从而抑制细胞增殖、转移和克隆形成,并影响细胞周期和促进细胞凋亡。     

LATS2-Ajuba complex regulates gamma-tubulin recruitment to centrosomes and spindle organization during mitosis

LATS2 is a human homolog of Drosophila tumor suppressor lats/warts, and encodes a mitotic kinase whose physiological roles remain to be elucidated. We performed yeast two-hybrid screening and identified a LIM protein Ajuba, as a binding partner of LATS2. LATS2 was localized to the centrosomes throughout the cell cycle and was associated with Ajuba during mitosis, contributing to latter's mitotic phosphorylation. Depletion of LATS2 or Ajuba impaired centrosomal accumulation of gamma-tubulin and spindle formation at the onset of mitosis, suggesting that the LATS2-Ajuba complex regulates organization of the spindle apparatus through recruitment of gamma-tubulin to the centrosome.     

Cell Cycle Regulated Interaction of a Yeast Hippo Kinase and Its Activator MO25/Hym1

Hippo pathways are ancient signaling systems that contribute to cell growth and proliferation in a wide diversity of eukaryotes, and have emerged as a conserved regulator of organ size control in metazoans. In budding yeast, a Hippo signaling pathway called the Regulation of Ace2 and Morphogenesis (RAM) network promotes polarized cell growth and the final event in the separation of mother and daughter cells. A crucial regulatory input for RAM network control of cell separation is phosphorylation of a conserved hydrophobic motif (HM) site on the NDR/LATS family kinase Cbk1. Here we provide the first direct evidence that the Hippo-like kinase Kic1 in fact phosphorylates the HM site of Cbk1, and show that Kic1 is allosterically activated by Hym1, a highly conserved protein related to mammalian MO25. Using the structure of mammalian MO25 in complex with the Kic1-related pseudokinase STRAD, we identified conserved residues on Kic1 that are required for interaction with Hym1. We find that Kic1 and Hym1 protein levels remain constant throughout the cell cycle but the proteins’ association is regulated, with maximal interaction coinciding with peak Cbk1 HM site phosphorylation. We show that this association is necessary but not sufficient for this phosphorylation, suggesting another level of regulation is required to promote the complex to act upon its substrates. This work presents a previously undiscovered cell cycle regulated interaction between a Hippo kinase and a broadly conserved allosteric activator. Because of the conserved nature of this pathway in higher eukaryotes, this work may also provide insight into the modularity of Hippo signaling pathways.     

The eIF2alpha kinases PERK and PKR activate glycogen synthase kinase 3 to promote the proteasomal degradation of p53

Phosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha) is mediated by a family of kinases that respond to various forms of environmental stress. The eIF2alpha kinases are critical for mRNA translation, cell proliferation, and apoptosis. Activation of the tumor suppressor p53 results in cell cycle arrest and apoptosis in response to various types of stress. We previously showed that, unlike the majority of stress responses that stabilize and activate p53, induction of endoplasmic reticulum stress leads to p53 degradation through an Mdm2-dependent mechanism. Here, we demonstrate that the endoplasmic reticulum-resident eIF2alpha kinase PERK mediates the proteasomal degradation of p53 independently of translational control. This role is not specific for PERK, because the eIF2alpha kinase PKR also promotes p53 degradation in response to double-stranded RNA. We further establish that the eIF2alpha kinases induce glycogen synthase kinase 3 to promote the nuclear export and proteasomal degradation of p53. Our findings reveal a novel cross-talk between the eIF2alpha kinases and p53 with implications in cell proliferation and tumorigenesis.