The Saccharomyces cerevisiae Mob2p-Cbk1p kinase complex promotes polarized growth and acts with the mitotic exit network to facilitate daughter cell-specific localization of Ace2p transcription factor.
Weiss Eric L,Kurischko Cornelia,Zhang Chao,Shokat Kevan,Drubin David G,Luca Francis C
The Journal of cell biology
The Saccharomyces cerevisiae mitotic exit network (MEN) is a conserved signaling network that coordinates events associated with the M to G1 transition. We investigated the function 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 (Bidlingmaier, S., E.L. Weiss, C. Seidel, D.G. Drubin, and M. Snyder. 2001. Mol. Cell. Biol. 21:2449-2462). Cbk1p is also required for Ace2p-dependent transcription of genes involved in mother/daughter separation after cytokinesis. Here we show that 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. We found that 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 (a) maintain polarized cell growth, (b) prevent the nuclear export of Ace2p from the daughter cell nucleus after mitotic exit, and (c) 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.
High-resolution genetic mapping with ordered arrays of Saccharomyces cerevisiae deletion mutants.
Jorgensen Paul,Nelson Bryce,Robinson Mark D,Chen Yiqun,Andrews Brenda,Tyers Mike,Boone Charles
We present a method for high-resolution genetic mapping that takes advantage of the ordered set of viable gene deletion mutants, which form a set of colinear markers covering almost every centimorgan of the Saccharomyces cerevisiae genome, and of the synthetic genetic array (SGA) system, which automates the construction of double mutants formed by mating and meiotic recombination. The Cbk1 kinase signaling pathway, which consists minimally of CBK1, MOB2, KIC1, HYM1, and TAO3 (PAG1), controls polarized morphogenesis and activation of the Ace2 transcription factor. Deletion mutations in the Cbk1 pathway genes are tolerated differently by common laboratory strains of S. cerevisiae, being viable in the W303 background but dead in the S288C background. Genetic analysis indicated that the lethality of Cbk1 pathway deletions in the S288C background was suppressed by a single allele specific to the W303 background. SGA mapping (SGAM) was used to locate this W303-specific suppressor to the SSD1 locus, which contains a known polymorphism that appears to compromise SSD1 function. This procedure should map any mutation, dominant or recessive, whose phenotype is epistatic to wild type, that is, a phenotype that can be scored from a mixed population of cells obtained by germination of both mutant and wild-type spores. In principle, SGAM should be applicable to the analysis of multigenic traits. Large-scale construction of ordered mutations in other model organisms would broaden the application of this approach.
Mob2p interacts with the protein kinase Orb6p to promote coordination of cell polarity with cell cycle progression.
Hou Ming-Chin,Wiley David J,Verde Fulvia,McCollum Dannel
Journal of cell science
The molecular mechanisms that temporally and spatially coordinate cell morphogenesis with the cell cycle remain poorly understood. Here we describe the characterization of fission yeast Mob2p, a novel protein required for regulating cell polarity and cell cycle control. Deletion of mob2 is lethal and causes cells to become spherical, with depolarized actin and microtubule cytoskeletons. A decrease in Mob2p protein level results in a defect in the activation of bipolar growth. This phenotype is identical to that of mutants defective in the orb6 protein kinase gene, and we find that Mob2p physically interacts with Orb6p. In addition, overexpression of Mob2p, like that of Orb6p, results in a delay in the onset of mitosis. Mob2p localizes to the cell periphery and cytoplasm throughout the cell cycle and to the division site during late anaphase and telophase. Mob2p is unable to localize to the cell middle in mutants defective in actomyosin ring and septum formation. Our results suggest that Mob2p, along with Orb6p, is required for coordinating polarized cell growth during interphase with the onset of mitosis.
DMob4/Phocein regulates synapse formation, axonal transport, and microtubule organization.
Schulte Joost,Sepp Katharine J,Jorquera Ramon A,Wu Chaohong,Song Yun,Hong Pengyu,Littleton J Troy
The Journal of neuroscience : the official journal of the Society for Neuroscience
The monopolar spindle-one-binder (Mob) family of kinase-interacting proteins regulate cell cycle and cell morphology, and their dysfunction has been linked to cancer. Models for Mob function are primarily based on studies of Mob1 and Mob2 family members in yeast. In contrast, the function of the highly conserved metazoan Phocein/Mob3 subfamily is unknown. We identified the Drosophila Phocein homolog (DMob4) as a regulator of neurite branching in a genome-wide RNA interference screen for neuronal morphology mutants. To further characterize DMob4, we generated null and hypomorphic alleles and performed in vivo cell biological and physiological analysis. We find that DMob4 plays a prominent role in neural function, regulating axonal transport, membrane excitability, and organization of microtubule networks. DMob4 mutant neuromuscular synapses also show a profound overgrowth of synaptic boutons, similar to known Drosophila endocytotic mutants. DMob4 and human Phocein are >80% identical, and the lethality of DMob4 mutants can be rescued by a human phocein transgene, indicating a conservation of function across evolution. These findings suggest a novel role for Phocein proteins in the regulation of axonal transport, neurite elongation, synapse formation, and microtubule organization.
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.
Human Mob proteins regulate the NDR1 and NDR2 serine-threonine kinases.
Devroe Eric,Erdjument-Bromage Hediye,Tempst Paul,Silver Pamela A
The Journal of biological chemistry
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.
Mechanism of activation of NDR (nuclear Dbf2-related) protein kinase by the hMOB1 protein.
Bichsel Samuel J,Tamaskovic Rastislav,Stegert Mario R,Hemmings Brian A
The Journal of biological chemistry
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.
Regulation of DNA damage responses and cell cycle progression by hMOB2.
Gomez Valenti,Gundogdu Ramazan,Gomez Marta,Hoa Lily,Panchal Neelam,O'Driscoll Mark,Hergovich Alexander
Mps one binder proteins (MOBs) are conserved regulators of essential signalling pathways. Biochemically, human MOB2 (hMOB2) can inhibit NDR kinases by competing with hMOB1 for binding to NDRs. However, biological roles of hMOB2 have remained enigmatic. Here, we describe novel functions of hMOB2 in the DNA damage response (DDR) and cell cycle regulation. hMOB2 promotes DDR signalling, cell survival and cell cycle arrest after exogenously induced DNA damage. Under normal growth conditions in the absence of exogenously induced DNA damage hMOB2 plays a role in preventing the accumulation of endogenous DNA damage and a subsequent p53/p21-dependent G1/S cell cycle arrest. Unexpectedly, these molecular and cellular phenotypes are not observed upon NDR manipulations, indicating that hMOB2 performs these functions independent of NDR signalling. Thus, to gain mechanistic insight, we screened for novel binding partners of hMOB2, revealing that hMOB2 interacts with RAD50, facilitating the recruitment of the MRE11-RAD50-NBS1 (MRN) DNA damage sensor complex and activated ATM to DNA damaged chromatin. Taken together, we conclude that hMOB2 supports the DDR and cell cycle progression.
Two NDR kinase-MOB complexes function as distinct modules during septum formation and tip extension in Neurospora crassa.
Maerz Sabine,Dettmann Anne,Ziv Carmit,Liu Yi,Valerius Oliver,Yarden Oded,Seiler Stephan
NDR kinases are important for growth and differentiation and require interaction with MOB proteins for activity and function. We characterized the NDR kinases and MOB activators in Neurospora crassa and identified two NDR kinases (COT1 and DBF2) and four MOB proteins (MOB1, MOB2A, MOB2B and MOB3/phocein) that form two functional NDR-MOB protein complexes. The MOB1-DBF2 complex is not only essential for septum formation in vegetative cells and during conidiation, but also functions during sexual fruiting body development and ascosporogenesis. The two MOB2-type proteins interact with both COT1 isoforms and control polar tip extension and branching by regulating COT1 activity. The conserved region directly preceding the kinase domain of COT1 is sufficient for the formation of COT1-MOB2 heterodimers, but also for kinase homodimerization. An additional N-terminal extension that is poorly conserved, but present in most fungal NDR kinases, is required for further stabilization of both types of interactions and for stimulating COT1 activity. COT1 lacking this region is degraded in a mob-2 background. We propose a specific role of MOB3/phocein during vegetative cell fusion, fruiting body development and ascosporogenesis that is unrelated to the three other MOB proteins and NDR kinase signalling.
The Hippo/MST Pathway Member SAV1 Plays a Suppressive Role in Development of the Prehierarchical Follicles in Hen Ovary.
Lyu Zhichao,Qin Ning,Tyasi Thobela Louis,Zhu Hongyan,Liu Dehui,Yuan Shuguo,Xu Rifu
The Hippo/MST signaling pathway is a critical player in controlling cell proliferation, self-renewal, differentiation, and apoptosis of most tissues and organs in diverse species. Previous studies have shown that Salvador homolog 1 (SAV1), a scaffolding protein which functions in the signaling system is expressed in mammalian ovaries and play a vital role in governing the follicle development. But the exact biological effects of chicken SAV1 in prehierarchical follicle development remain poorly understood. In the present study, we demonstrated that the SAV1 protein is predominantly expressed in the oocytes and undifferentiated granulosa cells in the various sized prehierarchical follicles of hen ovary, and the endogenous expression level of SAV1 mRNA appears down-regulated from the primordial follicles to the largest preovulatory follicles (F2-F1) by immunohistochemistry and real-time RT-PCR, respectively. Moreover, we found the intracellular SAV1 physically interacts with each of the pathway members, including STK4/MST1, STK3/MST2, LATS1 and MOB2 using western blotting. And SAV1 significantly promotes the phosphorylation of LATS1 induced by the kinase of STK4 or STK3 in vitro. Furthermore, SAV1 knockdown by small interfering RNA (siRNA) significantly increased proliferation of granulosa cells from the prehierarchical follicles (6-8 mm in diameter) by BrdU-incorporation assay, in which the expression levels of GDF9, StAR and FSHR mRNA was notably enhanced. Meanwhile, these findings were consolidated by the data of SAV1 overexpression. Taken together, the present results revealed that SAV1 can inhibit proliferation of the granulosa cells whereby the expression levels of GDF9, StAR and FSHR mRNA were negatively regulated. Accordingly, SAV1, as a member of the hippo/MST signaling pathway plays a suppressive role in ovarian follicle development by promoting phosphorylation and activity of the downstream LATS1, may consequently lead to prevention of the follicle selection during ovary development.
Strong parent-of-origin effects in the association of KCNQ1 variants with type 2 diabetes in American Indians.
Hanson Robert L,Guo Tingwei,Muller Yunhua L,Fleming Jamie,Knowler William C,Kobes Sayuko,Bogardus Clifton,Baier Leslie J
Parent-of-origin effects were observed in an Icelandic population for several genetic variants associated with type 2 diabetes, including those in KLF14 (rs4731702), MOB2 (rs2334499), and KCNQ1 (rs2237892, rs231362). We analyzed parent-of-origin effects for these variants, along with two others in KCNQ1 identified in previous genome-wide association studies (rs2237895, rs2299620), in 7,351 Pima Indians from 4,549 nuclear families; 34% of participants had diabetes. In a subset of 287 normoglycemic individuals, acute insulin secretion was measured by an intravenous glucose tolerance test. Statistically significant (P < 0.05) parent-of-origin effects were seen for association with type 2 diabetes for all variants. The strongest effect was seen at rs2299620 in KCNQ1; the C allele was associated with increased diabetes when maternally derived (odds ratio [OR], 1.92; P = 4.1 × 10(-12)), but not when paternally derived (OR, 0.93; P = 0.47; P = 9.9 × 10(-6) for difference in maternal and paternal effects). A maternally derived C allele also was associated with a 28% decrease in insulin secretion (P = 0.002). This study confirms parent-of-origin effects in the association with type 2 diabetes for variants in KLF14, MOB2, and KCNQ1. In Pima Indians, the effect of maternally derived KCNQ1 variants appears to be mediated through decreased insulin secretion and is particularly strong, accounting for 4% of the variance in liability to diabetes.
The N-terminal region of the Neurospora NDR kinase COT1 regulates morphology via its interactions with MOB2A/B.
Ziv Carmit,Feldman Daria,Aharoni-Kats Liran,Chen She,Liu Yi,Yarden Oded
Nuclear Dbf2p-related (NDR) protein kinases are important for cell differentiation and polar morphogenesis in various organisms, yet some of their functions are still elusive. Dysfunction of the Neurospora crassa NDR kinase COT1 leads to cessation of tip extension and hyperbranching. NDR kinases require the physical interaction between the kinase's N-terminal region (NTR) and the MPS1-binding (MOB) proteins for their activity and functions. To study the interactions between COT1 and MOB2 proteins, we mutated several conserved residues and a novel phosphorylation site within the COT1 NTR. The phenotypes of these mutants suggest that the NTR is required for COT1 functions in regulating hyphal elongation and branching, asexual conidiation and germination. Interestingly, while both MOB2A and MOB2B promote proper hyphal growth, they have distinct COT1-dependent roles in regulation of macroconidiation. Immunoprecipitation experiments indicate physical association of COT1 with both MOB2A and MOB2B, simultaneously. Furthermore, the binding of the two MOB2 proteins to COT1 is mediated by different residues at the COT1 NTR, suggesting a hetero-trimer is formed. Thus, although MOB2A/B may have some overlapping functions in regulating hyphal tip extension, their function is not redundant and they are both required for proper fungal development.
MST2- and Furry-mediated activation of NDR1 kinase is critical for precise alignment of mitotic chromosomes.
Chiba Shuhei,Ikeda Masanori,Katsunuma Kokichi,Ohashi Kazumasa,Mizuno Kensaku
Current biology : CB
The precise alignment of chromosomes on the metaphase plate prior to the onset of anaphase is essential for ensuring equal segregation of sister chromatids into two daughter cells, and defects in this process potentially cause chromosome instability and tumor progression [1-3]. NDR1 is an evolutionarily conserved serine/threonine kinase whose activity is regulated by MST kinases, Furry (Fry), and MOB . Although the NDR1 signaling pathway is implicated in cell division and morphogenesis in yeast and invertebrates [4-16], the mechanisms of NDR1 activation and the functional significance of the NDR1 pathway in mammalian cells are largely unknown. Here, we show that NDR1 is required for accurate chromosome alignment at metaphase in HeLa cells; depletion of NDR1, Fry, or MST2 caused mitotic chromosome misalignment. Chromosome misalignment in MST2-depleted cells was corrected by expression of active NDR1. The kinase activity of NDR1 increased in early mitotic phase and was dependent on Fry and MST2. We also provide evidence that Fry binds to microtubules, localizes on the spindle, acts as a scaffold that binds to both NDR1 and MOB2, and synergistically activates NDR1 with MOB2. Our findings suggest that MST2-, Fry-, and MOB2-mediated activation of NDR1 is crucial for the fidelity of mitotic chromosome alignment in mammalian cells.
ChMob2 binds to ChCbk1 and promotes virulence and conidiation of the fungal pathogen Colletotrichum higginsianum.
Schmidpeter Johannes,Dahl Marlis,Hofmann Jörg,Koch Christian
BACKGROUND:Mob family proteins are conserved between animals, plants and fungi and are essential for the activation of NDR kinases that control crucial cellular processes like cytokinesis, proliferation and morphology. RESULTS:We identified a hypomorphic allele of ChMOB2 in a random insertional mutant (vir-88) of the hemibiotrophic ascomycete fungus Colletotrichum higginsianum. The mutant is impaired in conidiation, host penetration and virulence on Arabidopsis thaliana. ChMob2 binds to and co-localizes with the NDR/LATS kinase homolog ChCbk1. Mutants in the two potential ChCbk1 downstream targets ChSSD1 and ChACE2 show defects in pathogenicity. The genome of C. higginsianum encodes two more Mob proteins. While we could not detect any effect on pathogenicity in ΔChmob3 mutants, ChMob1 is involved in conidiation, septae formation and virulence. CONCLUSION:This study shows that ChMob2 binds to the conserved NDR/LATS Kinase ChCbk1 and plays an important role in pathogenicity of Colletotrichum higginsianum on Arabidopsis thaliana.
The Structure of an NDR/LATS Kinase-Mob Complex Reveals a Novel Kinase-Coactivator System and Substrate Docking Mechanism.
Gógl Gergő,Schneider Kyle D,Yeh Brian J,Alam Nashida,Nguyen Ba Alex N,Moses Alan M,Hetényi Csaba,Reményi Attila,Weiss Eric L
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.
ICEKp2: description of an integrative and conjugative element in Klebsiella pneumoniae, co-occurring and interacting with ICEKp1.
Farzand Robeena,Rajakumar Kumar,Zamudio Roxana,Oggioni Marco R,Barer Michael R,O'Hare Helen M
Klebsiella pneumoniae is a human pathogen, prominent in antimicrobial-resistant and nosocomial infection. The integrative and conjugative element ICEKp1 is present in a third of clinical isolates and more prevalent in invasive disease; it provides genetic diversity and enables the spread of virulence-associated genes. We report a second integrative conjugative element that can co-occur with ICEKp1 in K. pneumoniae. This element, ICEKp2, is similar to the Pseudomonas aeruginosa pathogenicity island PAPI. We identified ICEKp2 in K. pneumoniae sequence types ST11, ST258 and ST512, which are associated with carbapenem-resistant outbreaks in China and the US, including isolates with and without ICEKp1. ICEKp2 was competent for excision, but self-mobilisation to recipient Escherichia coli was not detected. In an isolate with both elements, ICEKp2 positively influenced the efficiency of plasmid mobilisation driven by ICEKp1. We propose a putative mechanism, in which a Mob2 ATPase of ICEKp2 may contribute to the ICEKp1 conjugation machinery. Supporting this mechanism, mob2, but not a variant with mutations in the ATPase motif, restored transfer efficiency to an ICEKp2 knockout. This is the first demonstration of the interaction between integrative and conjugative genetic elements in a single Gram-negative bacterium with implications for understanding evolution by horizontal gene transfer.
Hydrophobic motif phosphorylation coordinates activity and polar localization of the Neurospora crassa nuclear Dbf2-related kinase COT1.
Maerz Sabine,Dettmann Anne,Seiler Stephan
Molecular and cellular biology
Nuclear Dbf2p-related (NDR) kinases and associated proteins are recognized as a conserved network that regulates eukaryotic cell polarity. NDR kinases require association with MOB adaptor proteins and phosphorylation of two conserved residues in the activation segment and hydrophobic motif for activity and function. We demonstrate that the Neurospora crassa NDR kinase COT1 forms inactive dimers via a conserved N-terminal extension, which is also required for the interaction of the kinase with MOB2 to generate heterocomplexes with basal activity. Basal kinase activity also requires autophosphorylation of the COT1-MOB2 complex in the activation segment, while hydrophobic motif phosphorylation of COT1 by the germinal center kinase POD6 fully activates COT1 through induction of a conformational change. Hydrophobic motif phosphorylation is also required for plasma membrane association of the COT1-MOB2 complex. MOB2 further restricts the membrane-associated kinase complex to the hyphal apex to promote polar cell growth. These data support an integrated mechanism of NDR kinase regulation in vivo, in which kinase activation and cellular localization of COT1 are coordinated by dual phosphorylation and interaction with MOB2.
Monopolar spindle-one-binder protein 2 regulates the activity of large tumor suppressor/yes-associated protein to inhibit the motility of SMMC-7721 hepatocellular carcinoma cells.
Zhang Weicheng,Shen Jingyuan,Gu Fengming,Zhang Ying,Wu Wenjuan,Weng Jiachun,Liao Yuexia,Deng Zijing,Yuan Qing,Zheng Lu,Zhang Yu,Shen Weigan
Accumulating evidence implicates monopolar spindle-one-binder protein (MOB)2 as an inhibitor of nuclear-Dbf2-related kinase (NDR) by competing with MOB1 for interaction with NDR1/2. NDR/large tumor suppressor (LATS) kinases may function similarly to yes-associated protein (YAP) kinases and be considered as members of the Hippo core cassette. MOB2 appears to serve roles in cell survival, cell cycle progression, responses to DNA damage and cell motility. However, the underlying mechanisms involved remain unclarified. In the present study, it was demonstrated that the knockout of MOB2 by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 promoted migration and invasion, induced phosphorylation of NDR1/2 and decreased phosphorylation of YAP in SMMC-7721 cells when compared with the blank vector-transduced cells. By contrast, the overexpression of MOB2 resulted in the opposite results. Mechanistically, MOB2 regulated the alternative interaction of MOB1 with NDR1/2 and LATS1, which resulted in increased phosphorylation of LATS1 and MOB1 and thereby led to the inactivation of YAP and consequently inhibition of cell motility. The results of the present study provide evidence of MOB2 serving a positive role in LATS/YAP activation by activating the Hippo signaling pathway.
Mps one binder 2 gene upregulation in the stellation of astrocytes induced by cAMP-dependent pathway.
Fang Kuan-Min,Liu Yi-Ying,Lin Cheng-Han,Fan Seng-Sheen,Tsai Chung-Hua,Tzeng Shun-Fen
Journal of cellular biochemistry
Astrocytes, the major glial population in the central nervous system (CNS), play an important role in neuronal homeostasis, neurogenesis, and synaptogenesis. The cells have a stellate shape with elaborated processes in the developing CNS. Cultured astrocytes become stellate when the cells undergo differentiation in response to stimuli. Nevertheless, the molecular mechanism for astrocytic stellation is poorly understood. Here, we showed that the addition of serum induced a flat polygonal shape in cultured astrocytes with a reduced level of Mps one binder 2 (Mob2) that is involved in neurite growth by forming stable complex with a nuclear Ser/Thr kinase Dbf2-related protein kinase 1 (NDR1). Furthermore, exposure to a membrane permeable cAMP analogue, dbcAMP, not only induced astrocytic stellation, but also caused an increase in Mob2 expression. Similarly, the upregulation of Mob2 mRNA expression was induced by exposure of astrocytes to pituitary adenylyl cyclase-activating polypeptide (PACAP). Pretreatment with a cAMP/protein kinase A (PKA) inhibitor, KT-5720, significantly blocked the effect of dbcAMP and PACAP on induced upregulation of Mob2 mRNA expression in astrocytes. In addition, the process withdrawal of dbcAMP-treated astrocytes was caused by the inhibition of Mob2 expression using lentivirus-mediated Mob2 shRNA delivery system. Based on our findings, we suggest that Mob2 is involved in PKA signaling-mediated astrocytic stellation.
Yeast Cbk1 and Mob2 activate daughter-specific genetic programs to induce asymmetric cell fates.
Colman-Lerner A,Chin T E,Brent R
In Saccharomyces cerevisiae, mothers and daughters have distinct fates. We show that Cbk1 kinase and its interacting protein Mob2 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.
Lre1 directly inhibits the NDR/Lats kinase Cbk1 at the cell division site in a phosphorylation-dependent manner.
Mancini Lombardi Ilde,Palani Saravanan,Meitinger Franz,Darieva Zoulfia,Hofmann Astrid,Sharrocks Andrew D,Pereira Gislene
Current biology : CB
BACKGROUND:The nuclear Dbf2 related (NDR) family of protein kinases play important roles in cell-cycle regulation, apoptosis, cell morphogenesis, and development in a variety of organisms. In budding yeast, the NDR kinase complex composed of Cbk1 and its regulatory subunit, Mob2, have an established role in the control of cell separation/abscission that follows cytokinesis. Whereas the activators of Cbk1-Mob2 have been more extensively described, the mechanisms that restrict or inhibit Cbk1-Mob2 catalytic activity remain largely unknown. RESULTS:We identified the protein Lre1 as a direct inhibitor of Cbk1-Mob2 catalytic activity. We show that Lre1 accumulates at the cell division site in late anaphase and associates with both Mob2 and Cbk1 in vivo and in vitro. Biochemical and functional analysis established that the ability of Lre1 to associate with Cbk1-Mob2 was reduced by mitotic Cdk1 activity and promoted by Cdc14 phosphatase at the end of mitosis. The inhibition of Cbk1-Mob2 by Lre1 was critical to promote the survival of cells lacking the actomyosin driven pathway of cytokinesis. CONCLUSIONS:We established Lre1 as a direct inhibitor of the NDR kinase Cbk1-Mob2, which is regulated in a cell-cycle-dependent manner. We propose that similar inhibitory proteins may also provide fine tuning for the activity of NDR kinases in other organisms.
Function of Drosophila mob2 in photoreceptor morphogenesis.
Liu Ling-Yu,Lin Cheng-Han,Fan Seng-Sheen
Cell and tissue research
The Drosophila photoreceptor is a highly polarized cell; a mature photoreceptor cell in Drosophila contains a photosensitive structure (the rhabdomere) and a supporting membrane (stalk) at its apical membrane. In a screen to isolate genes involved in determining stalk and rhabdomere formation, this study has identified the Drosophila mob2 (Dmob2) gene. Dmob2 belongs to a Mob1/phocein domain protein family whose functions are involved in polarized cell growth and asymmetric cell fate determination in yeast. To study the role of Dmob2 in photoreceptor development, we have raised an antibody against the Dmob2 protein. An immunocytochemical study has shown that Dmob2 is mainly localized in the apical membrane of photoreceptor cells during early development. As development proceeds, Dmob2 is gradually confined to the rhabdomere base of the photoreceptor cells. RNA interference (RNAi) for knockdown Dmob2 expression during eye development impairs rhabdomere formation. Our study further shows that the subcellular localization of phosphorylated Moesin and Crumbs in the developing photoreceptor cell is disrupted in Dmob2 RNAi flies. This work thus reports a novel function of Dmob2 in photoreceptor cell development.
CDK-dependent phosphorylation of Mob2 is essential for hyphal development in Candida albicans.
Gutiérrez-Escribano Pilar,González-Novo Alberto,Suárez M Belén,Li Chang-Run,Wang Yue,de Aldana Carlos R Vázquez,Correa-Bordes Jaime
Molecular biology of the cell
Nuclear Dbf2-related (NDR) protein kinases are essential components of regulatory pathways involved in cell morphogenesis, cell cycle control, and viability in eukaryotic cells. For their activity and function, these kinases require interaction with Mob proteins. However, little is known about how the Mob proteins are regulated. In Candida albicans, the cyclin-dependent kinase (CDK) Cdc28 and the NDR kinase Cbk1 are required for hyphal growth. Here we demonstrate that Mob2, the Cbk1 activator, undergoes a Cdc28-dependent differential phosphorylation on hyphal induction. Mutations in the four CDK consensus sites in Mob2 to Ala significantly impaired hyphal development. The mutant cells produced short hyphae with enlarged tips that displayed an illicit activation of cell separation. We also show that Cdc28 phosphorylation of Mob2 is essential for the maintenance of polarisome components at hyphal tips but not at bud tips during yeast growth. Thus we have found a novel signaling pathway by which Cdc28 controls Cbk1 through the regulatory phosphorylation of Mob2, which is crucial for normal hyphal development.
Identification of Mob2, a novel regulator of larval neuromuscular junction morphology, in natural populations of Drosophila melanogaster.
Campbell Megan,Ganetzky Barry
Although evolutionary changes must take place in neural connectivity and synaptic architecture as nervous systems become more complex, we lack understanding of the general principles and specific mechanisms by which these changes occur. Previously, we found that morphology of the larval neuromuscular junction (NMJ) varies extensively among different species of Drosophila but is relatively conserved within a species. To identify specific genes as candidates that might underlie phenotypic differences in NMJ morphology among Drosophila species, we performed a genetic analysis on one of two phenotypic variants we found among 20 natural isolates of Drosophila melanogaster. We discovered genetic polymorphisms for both positive and negative regulators of NMJ growth segregating within the variant line. Focusing on one subline, that displayed NMJ overgrowth, we mapped the phenotype to Mob2 [Monopolar spindle (Mps) one binding protein 2)], a gene encoding a Nuclear Dbf2 (Dumbbell formation 2)-Related (NDR) kinase activator. We confirmed this identification by transformation rescue experiments and showed that presynaptic expression of Mob2 is necessary and sufficient to regulate NMJ growth. Mob2 interacts in a dominant, dose-dependent manner with tricornered but not with warts, to cause NMJ overgrowth, suggesting that Mob2 specifically functions in combination with the former NDR kinase to regulate NMJ development. These results demonstrate the feasibility and utility of identifying genetic variants affecting NMJ morphology in natural populations of Drosophila. These variants can lead to discovery of new genes and molecular mechanisms that regulate NMJ development while also providing new information that can advance our understanding of mechanisms that underlie nervous system evolution.
The promotion of neurite formation in Neuro2A cells by mouse Mob2 protein.
Lin Cheng-Han,Hsieh Mingli,Fan Seng-Sheen
The molecular mechanism of neuritogenesis has been extensively studied but remains unclear. In this study, we identified Mob2 protein which plays a significant role in promoting neurite formation in Neuro2A (N2A) cells. Our results showed that Mob2 was expressed in developing N2A cells. To study whether Mob2 was involved in neurite formation, we downregulated Mob2 expression using RNA interference and found that neurite formation decreased in low serum induced N2A cells. In addition, we found that overexpression of Mob2 promoted neurite formation in N2A cells. Furthermore, downregulation of Mob2 expression altered the rearrangement of the actin cytoskeleton and decreased the expression of phosphorylated Moesin. Together, these results provide information on the role of Mob2 in mediating neurite formation.
The Possible Crosstalk of MOB2 With NDR1/2 Kinases in Cell Cycle and DNA Damage Signaling.
Gundogdu Ramazan,Hergovich Alexander
Journal of cell signaling
This article is the authors' opinion of the roles of the signal transducer Mps one binder 2 (MOB2) in the control of cell cycle progression and the DNA Damage Response (DDR). We recently found that endogenous MOB2 is required to prevent the accumulation of endogenous DNA damage in order to prevent the undesired, and possibly detrimental, activation of cell cycle checkpoints. In this regard, it is noteworthy that MOB2 has been linked biochemically to the regulation of the NDR1/2 (aka STK38/STK38L) protein kinases, which themselves have functions at different steps of the cell cycle. Therefore, we are speculating in this article about the possible connections of MOB2 with NDR1/2 kinases in cell cycle and DDR Signaling.
Growth-inhibitory effects of MOB2 on human hepatic carcinoma cell line SMMC-7721.
Leng Jian-Jun,Tan Hua-Min,Chen Ke,Shen Wei-Gan,Tan Jing-Wang
World journal of gastroenterology
AIM:To investigate the growth-inhibiting and apoptosis-inducing effects of the gene MOB2 on human hepatic carcinoma cell line SMMC-7721. METHODS:The full-length cDNA of the MOB2 gene was amplified from human umbilical vein endothelial cells. The correct full-length MOB2 cDNA was subcloned into the eukaryotic expression vector pEGFP-C1. After lipofection of the MOB2 gene into cancer cells, the levels of MOB2 protein in the cancer cells were detected by immunoblotting. To transfect the recombined plasmid vector pEGFP-CI-MOB2 into SMMC-7721 cells, the cells were cultured in Dulbecco's Modified Eagle's Medium with 10% fetal calf serum and glutamine, and then mixed with liposomes, Lipofectamine 2000 and the plasmid vector pEGFP-CI-MOB2. RESULTS:We observed the growth and proliferation of SMMC-7721 cells containing pEGFP-CI-MOB2 and analyzed their apoptosis and growth cycle phases by flow cytometry. We successfully transfected the recombined plasmid vector pEGFP-CI-MOB2 into SMMC-7721 cells and screened for a single clone cell containing MOB2. After transfection, MOB2 enhanced growth suppression, induced apoptosis, increased the ratio of G0/G1, significantly inhibited the advance of cell cycle phase, and arrested cells in G0/G1 phase. CONCLUSION:MOB2 overexpression induces apoptosis and inhibits the growth of human hepatic cancer cells, which may be useful in gene therapy for hepatic carcinoma.
Mob2 Insufficiency Disrupts Neuronal Migration in the Developing Cortex.
O'Neill Adam C,Kyrousi Christina,Einsiedler Melanie,Burtscher Ingo,Drukker Micha,Markie David M,Kirk Edwin P,Götz Magdalena,Robertson Stephen P,Cappello Silvia
Frontiers in cellular neuroscience
Disorders of neuronal mispositioning during brain development are phenotypically heterogeneous and their genetic causes remain largely unknown. Here, we report biallelic variants in a Hippo signaling factor--in a patient with one such disorder, periventricular nodular heterotopia (PH). Genetic and cellular analysis of both variants confirmed them to be loss-of-function with enhanced sensitivity to transcript degradation via nonsense mediated decay (NMD) or increased protein turnover via the proteasome. Knockdown of within the developing mouse cortex demonstrated its role in neuronal positioning. Cilia positioning and number within migrating neurons was also impaired with comparable defects detected following a reduction in levels of an upstream modulator of Mob2 function, Dchs1, a previously identified locus associated with PH. Moreover, reduced Mob2 expression increased phosphorylation of Filamin A, an actin cross-linking protein frequently mutated in cases of this disorder. These results reveal a key role for Mob2 in correct neuronal positioning within the developing cortex and outline a new candidate locus for PH development.