The DBF2 gene of the budding yeast Saccharomyces cerevisiae encodes a cell cycle-regulated protein kinase that plays an important role in the telophase/G1 transition. As a component of the multisubunit CCR4 transcriptional complex, DBF2 is also involved in the regulation of gene expression. MOB1, an essential protein required for a late mitotic event in the cell cycle, genetically and physically interacts with DBF2. DBF2 binds MOB1 in vivo and can bind it in vitro in the absence of other yeast proteins. Expression of MOB1 is also cell cycle regulated, its expression peaking slightly before that of DBF2 at the G2/M boundary. While overexpression of DBF2 suppressed phenotypes associated with mob1 temperature-sensitive alleles, it could not suppress a mob1 deletion. In contrast, overexpression of MOB1 suppresses phenotypes associated with a dbf2-deleted strain and suppresses the lethality associated with a dbf2 dbf20 double deletion. A mob1 temperature-sensitive allele with a dbf2 disruption was also found to be synthetically lethal. These results are consistent with DBF2 acting through MOB1 and aiding in its function. Moreover, the ability of temperature-sensitive mutated versions of the MOB1 protein to interact with DBF2 is severely reduced, confirming that binding of DBF2 to MOB1 is required for a late mitotic event. While MOB1 and DBF2 are capable of physically associating in a complex that does not include CCR4, MOB1 interacts with other components of the CCR4 transcriptional complex. Models concerning the role of DBF2 and MOB1 in controlling the telophase/G1 transition are discussed (Komarnitsky, 1998).
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 mob2Delta 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 (Luca, 1998).
In Saccharomyces cerevisiae, mothers and daughters have distinct fates. 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 (Colman-Lerner, 2001).
Exit from mitosis in budding yeast requires inactivation of cyclin-dependent kinases through mechanisms triggered by the protein phosphatase Cdc14. Cdc14 activity, in turn, is regulated by a group of proteins, the mitotic exit network (MEN), which includes Lte1, Tem1, Cdc5, Cdc15, Dbf2/Dbf20, and Mob1. The direct biochemical interactions between the components of the MEN remain largely unresolved. This study investigates the mechanisms that underlie activation of the protein kinase Dbf2. Dbf2 kinase activity depends on Tem1, Cdc15, and Mob1 in vivo. In vitro, recombinant protein kinase Cdc15 activated recombinant Dbf2, but only when Dbf2 was bound to Mob1. Conserved phosphorylation sites Ser-374 and Thr-544 (present in the human, Caenorhabditis elegans, and Drosophila melanogaster relatives of Dbf2) were required for DBF2 function in vivo, and activation of Dbf2-Mob1 by Cdc15 in vitro. Although Cdc15 phosphorylates Dbf2, Dbf2-Mob1, and Dbf2(S374A/T544A)-Mob1, the pattern of phosphate incorporation into Dbf2 Is substantially altered by either the S374A T544A mutations or omission of Mob1. Thus, Cdc15 promotes the exit from mitosis by directly switching on the kinase activity of Dbf2. It is proposed that Mob1 promotes this activation process by enabling Cdc15 to phosphorylate the critical Ser-374 and Thr-544 phosphoacceptor sites of Dbf2 (Mah, 2001).
The Saccharomyces cerevisiae mitotic exit network (MEN) is a conserved signaling network that coordinates events associated with the M to G1 transition. The function of two S. cerevisiae proteins related to the MEN proteins Mob1p and Dbf2p kinase has been investigated. 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, polarized morphogenesis is critical for bud site selection, bud development, and cell separation. The latter is mediated by Ace2p transcription factor, which controls the daughter cell-specific expression of cell separation genes. A set of proteins that include Cbk1p kinase, its binding partner Mob2p, Tao3p (Pag1p), and Hym1p regulate both Ace2p activity and cellular morphogenesis. These proteins seem to form a signaling network, which has been designated RAM for regulation of Ace2p activity and cellular morphogenesis. To find additional RAM components, genetic screens were conducted for bilateral mating and cell separation mutants and alleles of the PAK-related kinase Kic1p were identified in addition to Cbk1p, Mob2p, Tao3p, and Hym1p. Deletion of each RAM gene results in a loss of Ace2p function and causes cell polarity defects that are distinct from formin or polarisome mutants. Two-hybrid and coimmunoprecipitation experiments reveal a complex network of interactions among the RAM proteins, including Cbk1p-Cbk1p, Cbk1p-Kic1p, Kic1p-Tao3p, and Kic1p-Hym1p interactions, in addition to the previously documented Cbk1p-Mob2p and Cbk1p-Tao3p interactions. A novel leucine-rich repeat-containing protein Sog2p was also identified that interacts with Hym1p and Kic1p. Cells lacking Sog2p exhibit the characteristic cell separation and cell morphology defects associated with perturbation in RAM signaling. Each RAM protein localizes to cortical sites of growth during both budding and mating pheromone response. Hym1p is Kic1p- and Sog2p-dependent and Sog2p and Kic1p are interdependent for localization, indicating a close functional relationship between these proteins. Only Mob2p and Cbk1p are detectable in the daughter cell nucleus at the end of mitosis. The nuclear localization and kinase activity of the Mob2p-Cbk1p complex are dependent on all other RAM proteins, suggesting that Mob2p-Cbk1p functions late in the RAM network. These data suggest that the functional architecture of RAM signaling is similar to the S. cerevisiae mitotic exit network and Schizosaccharomyces pombe septation initiation network and is likely conserved among eukaryotes (Nelson, 2003).
The molecular mechanisms that temporally and spatially coordinate cell morphogenesis with the cell cycle remain poorly understood. Fission yeast Mob2p is 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 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. These results suggest that Mob2p, along with Orb6p, is required for coordinating polarized cell growth during interphase with the onset of mitosis (Hou, 2003).
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. Mob1p is shown to be required not only for the subcellular localization of Sid2p but also for its kinase activity. A region was identified 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. It is further shown 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. These 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, these 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 (Hou, 2004).
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. A human Mob1 family member, Mob1A, was cloned and its three-dimensional structure was determined 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 MOB1 in S. cerevisiae 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 (Stavridi, 2003).
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. This study reports 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. The 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 has been hypothesized to be the interaction surface according to the X-ray structure and has been identified as functionally important in yeast. The 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 (Ponchon, 2004).
Human NDR1 (nuclear Dbf2-related) is a widely expressed nuclear serine-threonine kinase that has been implicated in cell proliferation and/or tumor progression. The human NDR2 serine-threonine kinase, which shares approximately 87% sequence identity with NDR1, has been characterized. 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. NDR1 and NDR2 partially colocalize with human Mob2 in HeLa cells and the NDR-Mob interactions were confirmed 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 (Devore, 2004).
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, 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, hMOB1, the closest relative of yeast Mob1 and Mob2, is shown to stimulate 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 reduce 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: the data indicate that the binding of human MOB1 to the N-terminal domain of NDR induces the release of this autoinhibition (Bichsel, 2004).
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