Neurospora crassa is a filamentous fungus that grows on semisolid media by forming spreading colonies. Mutations at several loci prevent this spreading growth. cot-1 is a temperature sensitive mutant of N. crassa that exhibits restricted colonial growth. At temperatures above 32°C colonies are compact while at lower temperatures growth is indistinguishable from that of the wild type. Restricted colonial growth is due to a defect in hyphal tip elongation and a concomitant increase in hyphal branching. A genomic cosmid clone containing the wild type allele of cot-1 was isolated by complementation. Sequence analyses suggested that cot-1 encodes a member of the cAMP-dependent protein kinase family. Strains in which cot-1 was disrupted are viable but display restricted colonial growth. Duplication, by ectopic integration of a promoter-containing fragment which includes the first one-third (209 codons) of the structural gene, unexpectedly results in restricted colonial growth. These results suggest that an active COT1 kinase is required for one or more events essential for hyphal elongation (Yarden, 1992).
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).
The molecular mechanisms that coordinate cell morphogenesis with the cell cycle remain largely unknown. This process was investigated in fission yeast where changes in polarized cell growth are coupled with cell cycle progression. The orb6 gene is required during interphase to maintain cell polarity and encodes a serine/threonine protein kinase, belonging to the myotonic dystrophy kinase/cot1/warts family. A decrease in Orb6 protein levels leads to loss of polarized cell shape and to mitotic advance, whereas an increase in Orb6 levels maintains polarized growth and delays mitosis by affecting the p34(cdc2) mitotic kinase. Thus the Orb6 protein kinase coordinates maintenance of cell polarity during interphase with the onset of mitosis. orb6 interacts genetically with orb2, which encodes the Pak1/Shk1 protein kinase, a component of the Ras1 and Cdc42-dependent signaling pathway. These results suggest that Orb6 may act downstream of Pak1/Shk1, forming part of a pathway coordinating cell morphogenesis with progression through the cell cycle (Verde, 1998).
During the early stages of budding, cell wall remodeling and polarized secretion are concentrated at the bud tip (apical growth). The CBK1 gene, encoding a putative serine/threonine protein kinase, was identified in a screen designed to isolate mutations that affect apical growth. Analysis of cbk1Delta cells reveals that Cbk1p is required for efficient apical growth, proper mating projection morphology, bipolar bud site selection in diploid cells, and cell separation. Epitope-tagged Cbk1p localizes to both sides of the bud neck in late anaphase, just prior to cell separation. CBK1 and another gene, HYM1, have been identified in a screen for genes involved in transcriptional repression and proposed to function in the same pathway. Deletion of HYM1 causes phenotypes similar to those observed in cbk1Delta cells and disrupts the bud neck localization of Cbk1p. Whole-genome transcriptional analysis of cbk1Delta suggests that the kinase regulates the expression of a number of genes with cell wall-related functions, including two genes required for efficient cell separation: the chitinase-encoding gene CTS1 and the glucanase-encoding gene SCW11. The Ace2p transcription factor is required for expression of CTS1 and has been shown to physically interact with Cbk1p. Analysis of ace2Delta cells reveals that Ace2p is required for cell separation but not for polarized growth. These results suggest that Cbk1p and Hym1p function to regulate two distinct cell morphogenesis pathways: an ACE2-independent pathway that is required for efficient apical growth and mating projection formation and an ACE2-dependent pathway that is required for efficient cell separation following cytokinesis. Cbk1p is most closely related to the (1) Neurospora crassa Cot-1; (2) Schizosaccharomyces pombe Orb6; (3) Caenorhabditis elegans, Drosophila, and human Ndr, and (4) Drosophila and mammalian WARTS/LATS kinases. Many Cbk1-related kinases have been shown to regulate cellular morphology (Bidlingmaier, 2001).
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).
Protein kinases in the Cot-1/Orb6/Ndr/Warts family are important regulators of cell morphogenesis and proliferation. Cbk1p, a member of this family in Saccharomyces cerevisiae and a homolog of Fry, has been shown to be required for normal morphogenesis in vegetatively growing cells and in haploid cells responding to mating pheromone. A mutant of PAG1 (a homolog of fry), a novel gene in S. cerevisiae, displayed defects similar to those of cbk1 mutants. pag1 and cbk1 mutants share a common set of suppressors, including the disruption of SSD1, a gene encoding an RNA binding protein, and the overexpression of Sim1p, an extracellular protein. These genetic results suggest that PAG1 and CBK1 act in the same pathway. Furthermore, Pag1p and Cbk1p localize to the same polarized peripheral sites and they coimmunoprecipitate with each other. Pag1p is a conserved protein. The homologs of Pag1p in other organisms are likely to form complexes with the Cbk1p-related kinases and function with those kinases in the same biological processes (Du, 2002).
Fission yeast cells identify growing regions at the opposite ends of the cell, producing the rod-like shape. The positioning of the growth zone(s) and the polarized growth require CLIP170-like protein Tip1 and the Ndr kinase Orb6, respectively. The mor2/cps12 mutation disrupts the localization of F-actin at the cell ends, producing spherical cells and concomitantly inducing a G(2) delay at 36°C. Mor2 is important for the localization of F-actin at the cell end(s) but not at the medial region, and is essential for the restriction of the growth zone(s) where Tip1 targets. Mor2 is homologous to the Drosophila Furry protein, which is required to maintain the integrity of cellular extensions, and is localized at both cell ends and the medial region of the cell in an actin-dependent fashion. Cellular localization of Mor2 and Orb6 was interdependent. The tyrosine kinase Wee1 is necessary for the G(2) delay and maintenance of viability of the mor2 mutant. These results indicate that Mor2 plays an essential role in cell morphogenesis in concert with Orb6, and the mutation activates the mechanism coordinating morphogenesis with cell cycle progression (Hirata, 2002).
The Saccharomyces cerevisiae mitotic exit network (MEN) is a conserved signaling network that coordinates events associated with the M to G1 transition. The function 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 C. elegans sax-1 gene regulates several aspects of neuronal cell shape. sax-1 mutants have expanded cell bodies and ectopic neurites in many classes of neurons, suggesting that SAX-1 functions to restrict cell and neurite growth. The ectopic neurites in sensory neurons of sax-1 mutants resemble the defects caused by decreased sensory activity. However, the activity-dependent pathway, mediated in part by the UNC-43 calcium/calmodulin-dependent kinase II, functions in parallel with SAX-1 to suppress neurite initiation. sax-1 encodes a serine/threonine kinase in the Ndr family that is related to the Orb6 (Schizosaccharomyces pombe), Warts/Lats (Drosophila), and COT-1 (Neurospora) kinases that function in cell shape regulation. These kinases have similarity to Rho kinases but lack consensus Rho-binding domains. Dominant negative mutations in the C. elegans RhoA GTPase cause neuronal cell shape defects similar to those of sax-1 mutants, and genetic interactions between rhoA and sax-1 suggest shared functions. These results suggest that SAX-1/Ndr kinases are endogenous inhibitors of neurite initiation and cell spreading (Zallen, 2000).
Mechanosensory neurons provide accurate information about stimulus location by restricting their sensory dendrites to nonoverlapping regions, a pattern called tiling. C. elegans sax-1 and sax-2 regulate mechanosensory tiling by controlling the termination point of sensory dendrites. During development, the posterior PLM mechanosensory dendrite overlaps transiently with the anterior ALM mechanosensory neuron. This overlap is eliminated during a discrete period of paused or slowed PLM process growth, between an early period of rapid outgrowth and a later period of maintenance growth. In sax-2 mutants, the PLM sensory dendrite fails to slow between the active growth and maintenance growth phases, leading to sustained overlap of anterior and posterior mechanosensory processes. sax-2 encodes a large conserved protein with HEAT/Armadillo repeats that functions with sax-1, an NDR cell morphology-regulating kinase. High-level expression of sax-2 leads to premature neurite termination, suggesting that SAX-2 can directly inhibit neurite growth (Gallegos, 2004).
A set of male-specific sensory neurons in Caenorhabditis elegans establish axonal connections during postembryonic development. In the adult male, 9 bilateral pairs of ray sensory neurons innervate an acellular fan that serves as a presumptive tactile and olfactory organ during copulation. Ray axon commissures were visualized with a ray neuron-specific reporter gene and both known and new mutations that affect the establishment of connections to the pre-anal ganglion were studied. The UNC-6/netrin-UNC-40/DCC pathway provides the primary dorsoventral guidance cue to ray axon growth cones. Some axon growth cones also respond to an anteroposterior cue, following a segmented pathway, and most or all also have a tendency to fasciculate. Two newly identified genes, rax-1 and rax-4, are highly specific to the ray neurons and appear to be required for ray axon growth cones to respond to the dorsoventral cue. Among other genes identified, rax-2 and rax-3 affect anteroposterior signaling or fate specification and rax-5 and rax-6 affect ray identities. A mutation was identfied in sax-2; the sax-2/Furry and sax-1/Tricornered pathway affects ectopic neurite outgrowth and establishment of normal axon synapses. Mutations in genes for muscle proteins that affect axon pathways by distorting the conformation of the body wall. Thus ray axon pathfinding relies on a variety of general and more ray neuron-specific genes and provides a potentially fruitful system for further studies of how migrating axon growth cones locate their targets. This system is applicable to the study of mechanisms underlying topographic mapping of sensory neurons into target circuitry where the next stage of information processing is carried out (Jia, 2006; full text of article).
In sax-2(bx130), ray neurons send out axons that grow normally and form commissures to the pre-anal ganglion (PAG). In addition, they grow ectopic neurites that extend for varying distances. These aberrant outgrowths continue to form during the adult stage, becoming continuously more abundant as the animals age. Ectopic extensions behave like axons in growing anteriorly, but they fail to fasciculate or turn toward the ventral side. Additional neurons were examined and it was found that the four male-specific CEM neurons of the head, visualized with pkd-2::GFP, also have several abnormal posterior processes. The mutation bx130 mapped to and failed to complement the previously described gene sax-2. bx130 is rescued by the predicted gene F21H11.2. F21H11.2 encodes a large conserved protein with HEAT/Armadillo repeats (Jia, 2006).
sax-2 functions with sax-1, an NDR ser/thr kinase, in the maintenance of amphid neuronal morphology and mechanosensory neurite termination and tiling. Their Drosophila homologs, trc and fry, regulate the dendritic branching and tiling of Drosophila sensory neurons. sax-1(ky211) causes ectopic processes of ray axons similar to those in sax-2 mutants, suggesting that sax-1 and sax-2 function together to regulate ray axon morphology as they do for other sensory neurons. Interestingly, the unassigned mutation bx141 also causes the production of ectopic processes at late stage, albeit weakly. The similarity of sax-2(bx130) and bx141 mutant phenotypes suggests that bx141 either is a weak allele of sax-1 or sax-2 or may define an additional gene in this pathway (Jia, 2006).
Previous studies found little or no apparent disruption of neuron function in sax-2 and sax-1 mutants, consistent with a role in a maintenance pathway with little consequence for neuron activity. However, sax-2(bx130) males are defective in mating, suggesting ray neuron function is compromised. Therefore the density of presynaptic vesicles of ray neurons in the PAG was examined by scoring the expression of a PKD-2 promoter-driven SNB-1::GFP fusion protein (bxEx94). SNB-1 encodes C. elegans synaptobrevin, a synaptic vesicle protein expressed at synaptic sites. The presence of the sax-2(bx130) mutation dramatically reduces the density of GFP puncta in the PAG in the adult, suggesting a reduced density of synaptic vesicles. Thus sax-2 gene function may be necessary for normal synaptogenesis of the ray neurons (Jia, 2006).
Interestingly, a significant percentage of sax-2 animals also exhibit abnormal gonad morphology in both males and hermaphrodites. This phenotype was present for both alleles. In affected animals, the normally two-armed hermaphrodite gonad has either only the anterior arm or only the posterior arm. In the male, the testis fails to extend and forms a large bulb in the middle of the body. These observations suggest that sax-2 may also play a role in gonad morphogenesis (Jia, 2006).
Human, Drosophila, and C. elegans cDNA clones encoding homologues of Ndr protein kinase have been isolated and sequenced. The human and Drosophila cDNAs predict polypeptides of 54 kDa and 52 kDa, respectively, which share approximately 80% amino acid similarity. Northern analysis of human tissues has revealed a ubiquitously expressed 3.9-kb transcript. Recombinant GST-Ndr undergoes intramolecular autophosphorylation on serine and threonine residues in vitro but fails to transphosphorylate several standard protein kinase substrates. Transfection of the human cDNA into COS-1 cells results in the appearance of an intense nuclear staining in cells analyzed by indirect immunofluorescence; deletion mutagenesis identified a short basic peptide, KRKAETWKRNRR, responsible for the nuclear accumulation of Ndr. Thus, Ndr is a conserved and widely expressed nuclear protein kinase. The closest known relative of this previously uncharacterized kinase is Dbf2, a budding yeast protein kinase required for the completion of nuclear division (Millward, 1995).
Ndr is a nuclear serine/threonine protein kinase that belongs to a subfamily of kinases implicated in the regulation of cell division and cell morphology. This subfamily includes the kinases LATS, Orb6, Cot-1, and Dbf2. Ndr is potently activated when intact cells are treated with okadaic acid, suggesting that Ndr is normally held in a state of low activity by protein phosphatase 2A. The regulatory phosphorylation sites of Ndr protein kinase were mapped; active Ndr is phosphorylated on Ser-281 and Thr-444. Mutation of either site to alanine strongly reduces both basal and okadaic acid-stimulated Ndr activity, while combined mutation abolishes Ndr activity completely. Importantly, each of these sites (and also the surrounding sequences) are conserved in the kinase relatives of Ndr, suggesting a general mechanism of activation for kinases of this subfamily. Ser-281 and Thr-444 are also similar to the regulatory phosphorylation sites in several targets of the phosphoinositide-dependent protein kinase PDK1. However, PDK1 does not appear to function as an upstream kinase for Ndr. Thus, Ndr and its close relatives may operate in a novel signaling pathway downstream of an as-yet-unidentified kinase with specificity similar to, but distinct from, PDK1 (Millward, 1999).
NDR, a nuclear serine/threonine kinase, belongs to the subfamily of Dbf2 kinases that is critical to the morphology and proliferation of cells. The activity of NDR kinase is modulated in a Ca(2+)/S100B-dependent manner by phosphorylation of Ser281 in the catalytic domain and Thr444 in the C-terminal regulatory domain. S100B, a member of the S100 subfamily of EF-hand proteins, binds to a basic/hydrophobic sequence at the junction of the N-terminal regulatory and catalytic domains [NDR(62-87)]. Unlike calmodulin-dependent kinases, regulation of NDR by S100B is not associated with direct autoinhibition of the active site, but rather involves a conformational change in the catalytic domain triggered by Ca(2+)/S100B binding to the junction region. To gain further insight into the mechanism of activation of the kinase, studies have been carried out on Ca(2+)/S100B in complex with the intact N-terminal regulatory domain, NDR(1-87). Multidimensional heteronuclear NMR analysis shows that the binding mode and stoichiometry of a peptide fragment of NDR [NDR(62-87)] is the same as for the intact N-terminal regulatory domain. The solution structure of Ca(2+)/S100B and NDR(62-87) has been determined. One target molecule is found to associate with each subunit of the S100B dimer. The peptide adopts three turns of helix in the bound state, and the complex is stabilized by both hydrophobic and electrostatic interactions. These structural studies, in combination with available biochemical data, have been used to develop a model for calcium-induced activation of NDR kinase by S100B (Bhattacharya, 2003).
Nuclear Dbf2-related (NDR) protein kinases are a family of AGC group kinases that are involved in the regulation of cell division and cell morphology. The human and mouse NDR2, a second mammalian isoform of NDR protein kinase, has been cloned and characterized. NDR1 and NDR2 share 86% amino acid identity and are highly conserved between human and mouse. However, they differ in expression pattern; mouse Ndr1 is expressed mainly in spleen, lung and thymus, whereas mouse Ndr2 shows highest expression in the gastrointestinal tract. NDR2 is potently activated in cells following treatment with the protein phosphatase 2A inhibitor okadaic acid, which also results in phosphorylation on the activation segment residue Ser-282 and the hydrophobic motif residue Thr-442. Ser-282 becomes autophosphorylated in vivo, whereas Thr-442 is targeted by an upstream kinase. This phosphorylation can be mimicked by replacing the hydrophobic motif of NDR2 with a PRK2-derived sequence, resulting in a constitutively active kinase. Similar to NDR1, the autophosphorylation of NDR2 protein kinase is stimulated in vitro by S100B, an EF-hand Ca(2+)-binding protein of the S100 family, suggesting that the two isoforms are regulated by the same mechanisms. Further, a predominant cytoplasmic localization of ectopically expressed NDR2 is demonstrated (Stegert, 2004).
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).
A novel member of the Ndr subfamily of serine/threonine protein kinases, Ndr2, has been identified as a gene product that is induced in the mouse amygdala during fear memory consolidation, and a possible function of this kinase in neural differentiation was examined. Expression of Ndr2 mRNA was detected in various cortical and subcortical brain regions, as well as non-neuronal tissues. Its expression in the amygdala increases 6 h after Pavlovian fear conditioning training and returns to control levels within 24 h. To study intracellular localization and functions of Ndr2, EGFP::Ndr2 fusion proteins were expressed in rat pheochromocytoma (PC12) cells and acutely isolated cortical neurons, thereby revealing an association of Ndr2 with the actin cytoskeleton in somata, neurites and filopodia, in spines and at sites of cell contact. Co-precipitation and pull-down experiments support this finding. Further evidence for an involvement of Ndr2 in actin-mediated cellular functions comes from the observation of decreased cell spreading and changes in neurite outgrowth that were associated with protein serine phosphorylation in transfected PC12 cells. Together, these data suggest that Ndr2 is an interesting candidate gene for the regulation of structural processes in differentiating and mature neuronal cells (Stork, 2004).
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