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.     

Crystal structure of a human Mob1 protein: toward understanding Mob-regulated cell cycle pathways

The Mob protein family comprises a group of highly conserved eukaryotic proteins whose founding member functions in the mitotic exit network. At the molecular level, Mob proteins act as kinase-activating subunits. We cloned a human Mob1 family member, Mob1A, and determined its three-dimensional structure by X-ray crystallography. The core of Mob1A consists of a four-helix bundle that is stabilized by a bound zinc atom. The N-terminal helix of the bundle is solvent exposed and together with adjacent secondary structure elements forms an evolutionarily conserved surface with a strong negative electrostatic potential. Several conditional mutant alleles of S. cerevisiae MOB1 target this surface and decrease its net negative charge. Interestingly, the kinases with which yeast Mob proteins interact have two conserved basic regions within their N-terminal lobe. Thus, Mob proteins may regulate their target kinases through electrostatic interactions mediated by conserved charged surfaces.     

MOB1, an Essential Yeast Gene Required for Completion of Mitosis and Maintenance of Ploidy

Mob1p is an essential Saccharomyces cerevisiae protein, identified from a two-hybrid screen, that binds Mps1p, a protein kinase essential for spindle pole body duplication and mitotic checkpoint regulation. Mob1p contains no known structural motifs; however MOB1 is a member of a conserved gene family and shares sequence similarity with a nonessential yeast gene, MOB2. Mob1p is a phosphoprotein in vivo and a substrate for the Mps1p kinase in vitro. Conditional alleles of MOB1 cause a late nuclear division arrest at restrictive temperature. MOB1 exhibits genetic interaction with three other yeast genes required for the completion of mitosis, LTE1, CDC5, and CDC15 (the latter two encode essential protein kinases). Most haploid mutant mob1 strains also display a complete increase in ploidy at permissive temperature. The mechanism for the increase in ploidy may occur through MPS1 function. One mob1 strain, which maintains stable haploidy at both permissive and restrictive temperature, diploidizes at permissive temperature when combined with the mps1–1 mutation. Strains containing mob2Δ also display a complete increase in ploidy when combined with the mps1-1 mutation. Perhaps in addition to, or as part of, its essential function in late mitosis, MOB1 is required for a cell cycle reset function necessary for the initiation of the spindle pole body duplication.     

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.     

hMOB2 deficiency impairs homologous recombination-mediated DNA repair and sensitises cancer cells to PARP inhibitors

Monopolar spindle-one binder (MOBs) proteins are evolutionarily conserved and contribute to various cellular signalling pathways. Recently, we reported that hMOB2 functions in preventing the accumulation of endogenous DNA damage and a subsequent p53/p21-dependent G1/S cell cycle arrest in untransformed cells. However, the question of how hMOB2 protects cells from endogenous DNA damage accumulation remained enigmatic. Here, we uncover hMOB2 as a regulator of double-strand break (DSB) repair by homologous recombination (HR). hMOB2 supports the phosphorylation and accumulation of the RAD51 recombinase on resected single-strand DNA (ssDNA) overhangs. Physiologically, hMOB2 expression supports cancer cell survival in response to DSB-inducing anti-cancer compounds. Specifically, loss of hMOB2 renders ovarian and other cancer cells more vulnerable to FDA-approved PARP inhibitors. Reduced MOB2 expression correlates with increased overall survival in patients suffering from ovarian carcinoma. Taken together, our findings suggest that hMOB2 expression may serve as a candidate stratification biomarker of patients for HR-deficiency targeted cancer therapies, such as PARP inhibitor treatments.     

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.     

CDK-dependent phosphorylation of Mob2 is essential for hyphal development in Candida albicans

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.     

Saccharomyces cerevisiae Mob1p is required for cytokinesis and mitotic exit

The Saccharomyces cerevisiae mitotic exit network (MEN) is a conserved set of genes that mediate the transition from mitosis to G(1) by regulating mitotic cyclin degradation and the inactivation of cyclin-dependent kinase (CDK). Here, we demonstrate that, in addition to mitotic exit, S. cerevisiae MEN gene MOB1 is required for cytokinesis and cell separation. The cytokinesis defect was evident in mob1 mutants under conditions in which there was no mitotic-exit defect. Observation of live cells showed that yeast myosin II, Myo1p, was present in the contractile ring at the bud neck but that the ring failed to contract and disassemble. The cytokinesis defect persisted for several mitotic cycles, resulting in chains of cells with correctly segregated nuclei but with uncontracted actomyosin rings. The cytokinesis proteins Cdc3p (a septin), actin, and Iqg1p/ Cyk1p (an IQGAP-like protein) appeared to correctly localize in mob1 mutants, suggesting that MOB1 functions subsequent to actomyosin ring assembly. We also examined the subcellular distribution of Mob1p during the cell cycle and found that Mob1p first localized to the spindle pole bodies during mid-anaphase and then localized to a ring at the bud neck just before and during cytokinesis. Localization of Mob1p to the bud neck required CDC3, MEN genes CDC5, CDC14, CDC15, and DBF2, and spindle pole body gene NUD1 but was independent of MYO1. The localization of Mob1p to both spindle poles was abolished in cdc15 and nud1 mutants and was perturbed in cdc5 and cdc14 mutants. These results suggest that the MEN functions during the mitosis-to-G(1) transition to control cyclin-CDK inactivation and cytokinesis.     

The fission yeast MO25 protein functions in polar growth and cell separation

Proteins of the MO25 family are widely conserved but their function has not been characterized in detail. Human MO25 is a cofactor of LKB1, a conserved protein kinase with roles in cell polarity in nematodes, flies and mammalian cells. Furthermore, the budding yeast MO25 homologue, Hym1, is important for cell separation and morphogenesis. We have characterized Pmo25p, the MO25 homologue in the fission yeast Schizosaccharomyces pombe. Pmo25p is an essential protein required for polar growth; in its absence the actin cytoskeleton becomes depolarized and cells adopt a round morphology. In addition, pmo25 mutants are defective in cell separation. Both functions of Pmo25p appear to be mediated by the Orb6p–Mob2p kinase complex. Pmo25p shows no distinct localization during interphase, but it is recruited to one of the two spindle pole bodies during anaphase and to the division site during cytokinesis. The septation initiation network (SIN) regulates the localization of Pmo25p, suggesting that it regulates Pmo25p function during cell division.     

Initiation of cytokinesis is controlled through multiple modes of regulation of the Sid2p-Mob1p kinase complex

The Sid2p-Mob1p kinase complex is an important component of the septation initiation network (SIN) in the fission yeast Schizosaccharomyces pombe. However, regulation of this complex is still elusive. Here we show that Mob1p is required not only for the subcellular localization of Sid2p but also for its kinase activity. We identified a region at the amino terminus of Sid2p that is required for Mob1p binding and spindle pole body (SPB) localization. Deletion of this region abolishes Mob1p binding and diminishes SPB localization, whereas this region alone is sufficient to associate with Mob1p and SPBs. We further show that a similar region of the N terminus of the Sid2p-related protein kinase Orb6p binds to the Mob1p-related protein Mob2p, suggesting that this may be a conserved mode of interaction for this family of kinases. Phosphorylation of Ser402 and especially Thr578 is important for Sid2p function. Sid2p with a mutation of Thr578 to Ala (T578A) can no longer rescue sid2-250 mutant cells, and this results in reduction of Mob1p binding. Sid2p mutants mimicking phosphorylation at this site (T578D and T578E) can rescue sid2-250 cells, enhance Sid2p kinase activity, and partially rescue growth defects of upstream sin mutants. Interestingly, Sid2p, but not Mob1p, is self-associated. Our experiments suggest that self-associated Sid2p is inactive. This self-association is mediated by a region that overlaps with Mob1p and SPB binding sites. Overexpression of Mob1p is able to disrupt the self-association of Sid2p. Taken together, our results suggest that Sid2p kinase may utilize multiple modes of regulation including self-association, Mob1p binding, and phosphorylation to achieve its full activity at an appropriate time and place in the cell.     

The NDR kinase–MOB complex FgCot1-Mob2 regulates polarity and lipid metabolism in Fusarium graminearum

Members of the NDR (nuclear Dbf2-related) protein-kinase family are essential for cell differentiation and polarized morphogenesis. However, their functions in plant pathogenic fungi are not well understood. Here, we characterized the NDR kinase FgCot1 and its activator FgMob2 in Fusarium graminearum, a major pathogen causing Fusarium head blight (FHB) in wheat. FgCot1 and FgMob2 formed a NDR kinase–MOB protein complex. Localization assays using FgCot1-GFP or FgMob2-RFP constructs showed diverse subcellular localizations, including cytoplasm, septum, nucleus and hyphal tip. ΔFgcot1 and ΔFgmob2 exhibited serious defects in hyphal growth, polarity, fungal development and cell wall integrity as well as reduced virulence in planta. In contrast, lipid droplet accumulation was significantly increased in these two mutants. Phosphorylation of FgCot1 at two highly conserved residues (S462 and T630) as well as five new sites synergistically contributed its role in various cellular processes. In addition, non-synonymous mutations in two MAPK (mitogen-activated protein kinase) proteins, FgSte11 and FgGpmk1, partially rescued the growth defect of ΔFgmob2, indicating a functional link between the FgCot1–Mob2 complex and the FgGpmk1 signalling pathway in regulating filamentous fungal growth. These results indicated that the FgCot1–Mob2 complex is critical for polarity, fungal development, cell wall organization, lipid metabolism and virulence in F. graminearum.     

NMR solution structure of Mob1, a mitotic exit network protein and its interaction with an NDR kinase peptide

Proteins of the Mob1/phocein family are found in all eukaryotic cells. In yeast, they are activating subunits of Dbf2-related protein kinases involved in cell cycle control. Despite the wide occurrence of these proteins, their biological functions remain poorly understood. Here we report the solution structure of the Mob1 protein from Xenopus laevis solved by heteronuclear multidimensional NMR. The structure reveals a fold constituted by a central left-handed four-helix bundle, one connecting helix, two flanking helices and a long flexible loop. The clustering of two Cys and two His residues, and zinc measurement by atomic absorption spectroscopy support the existence of a zinc ion binding site. Our NMR structure is in good agreement with the recently described X-ray structure of human Mob1-A. Chemical shift perturbations observed upon addition of a peptide encompassing the basic region of the N-terminal regulatory domain of NDR kinase were used to identify and map a specific interaction between Mob1 and this kinase. The chemical shift changes indicate that the main interaction occurs on the acidic and conserved surface of Mob1. This surface was previously hypothesized to be the interaction surface according to the X-ray structure and was identified as functionally important in yeast. Our data suggest that the NDR kinase is a functional Dbf2 homologue in animal cells and contributes to the understanding of the molecular function of Mob1 proteins.     

The N‐terminal region of the Neurospora NDR kinase COT1 regulates morphology via its interactions with MOB2A/B

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.     

Characterization of the roles of Blt1p in fission yeast cytokinesis

Spatial and temporal regulation of cytokinesis is essential for cell division, yet the mechanisms that control the formation and constriction of the contractile ring are incompletely understood. In the fission yeast Schizosaccharomyces pombe proteins that contribute to the cytokinetic contractile ring accumulate during interphase in nodes—precursor structures around the equatorial cortex. During mitosis, additional proteins join these nodes, which condense to form the contractile ring. The cytokinesis protein Blt1p is unique in being present continuously in nodes from early interphase through to the contractile ring until cell separation. Blt1p was shown to stabilize interphase nodes, but its functions later in mitosis were unclear. We use analytical ultracentrifugation to show that purified Blt1p is a tetramer. We find that Blt1p interacts physically with Sid2p and Mob1p, a protein kinase complex of the septation initiation network, and confirm known interactions with F-BAR protein Cdc15p. Contractile rings assemble normally in blt1∆ cells, but the initiation of ring constriction and completion of cell division are delayed. We find three defects that likely contribute to this delay. Without Blt1p, contractile rings recruited and retained less Sid2p/Mob1p and Clp1p phosphatase, and β-glucan synthase Bgs1p accumulated slowly at the cleavage site.