Human nuclear Dbf2-related kinases (NDRs) are up-regulated in certain cancer types, yet their precise function(s) and regulatory mechanism(s) still remain to be defined. Here, we show that active (phosphorylated on Thr444) and inactive human NDRs are both mainly cytoplasmic. Moreover, NDR kinases colocalize at the plasma membrane with human MOBs (hMOBs), which are recently described coactivators of human NDR in vitro. Strikingly, membrane targeting of NDR results in a constitutively active kinase due to phosphorylation on Ser281 and Thr444 that is further activated upon coexpression of hMOBs. Membrane-targeted hMOBs also robustly promoted activation of NDR. We further demonstrate that the in vivo activation of human NDR by membrane-bound hMOBs is dependent on their interaction and occurs solely at the membrane. By using a chimeric molecule of hMOB, which allows inducible membrane translocation, we found that NDR phosphorylation and activation at the membrane occur a few minutes after association of hMOB with membranous structures. We provide insight into a potential in vivo mechanism of NDR activation through rapid recruitment to the plasma membrane mediated by hMOBs.The human genome encodes two highly related serine-threonine kinases, nuclear Dbf2-related kinase 1 (NDR1) and NDR2, which belong to a subfamily of kinases with roles in cell division and cell morphology (22). The two kinases represent a subclass of the AGC family of protein kinases and are partially related to protein kinase B (PKB) and protein kinase C (PKC). Human NDRs, as well as their yeast counterpart Dbf2, can be dramatically activated through inhibition of protein phosphatase 2A (PP2A) by okadaic acid (OA), demonstrating that NDR kinases require phosphorylation for activation (13,16). Indeed, NDR1 has been shown to become autophosphorylated on Ser281 in a Ca 2ϩ -dependent manner (23), while Thr444 is phosphorylated by a hydrophobic motif kinase (M. R. Stegert et al., submitted for publication). Phosphorylation on both residues is essential for full activation of NDR1 in vitro and in vivo (16,23). Recently, the phosphorylation of NDR2 on Ser282 and Thr442 was also shown to be required for full activation of enzymatic activity (20).The founding member of the Mps1 one binder (MOB) family is the yeast protein MOB1p (12), a molecule functioning as a coactivator of yeast Dbf2p and Dbf20p (10,11,13). As previously published, human MOB1A (hMOB1A) binds NDR1 and NDR2 (NDRs) at their N termini, leading to kinase activation in vitro (3). In addition, hMOB1B and hMOB2 also stimulated NDR activity in vitro (4). However, while the MOB regulation of the NDR kinases seems to be conserved from yeast to humans, the function(s) of mammalian NDRs is unknown. Although the basic mechanism of activation of human NDR protein kinase in vitro has been described in detail (3,16,20,23), the regulation of NDR in a cellular context still remains to be elucidated. Such information should bring insight into its cellular functions and, in particular, into its possible role ...

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.NDR 1 kinase belongs to a highly conserved family of kinases, a subclass of the AGC family of protein kinases (1, 2). The NDR family consists of the mammalian protein kinases NDR1 and NDR2, Drosophila melanogaster NDR, Caenorhabditis elegans SAX1, mammalian, D. melanogaster and C. elegans large tumor suppressor kinases, Neurospora crassa COT1, Ustilago maydis UKC1, Saccharomyces cerevisiae Cbk1, Dbf2, and Dbf20, Schizosaccharomyces pombe Orb6 and Sid2, and several plant kinases (1, 2). These kinases share a high sequence conservation, and some possess conserved functions, mainly involving regulation of cell morphology and the cell cycle (2-15).The kinase domain sequence of NDR is related to that of other members of the AGC group of kinases, e.g. protein kinases A, B, C, and G, PRK, p70 S6K , p90 RSK , and phosphoinositide-dependent kinase 1 (1). NDR contains all 12 subdomains of the kinase catalytic domain as described by Hanks and Hunter (16). However, the catalytic domains of all of the members of the NDR family are interrupted by an insert of 30 -60 amino acids between subdomains VII and VIII. This inserted sequence is not well conserved but is always rich in the basic amino acids, arginine and lysine. The catalytic domain insert has been shown to act as a non-consensus nuclear localization signal in the case of NDR1. NDR1 localizes predominantly to the nucleus in COS-1 cells, whereas mutant NDR1 with a deletion in the insert is localized to the cytosol (17). An additional special feature of the NDR family of kinases is a highly conserved N-terminal domain. In the case of NDR1, this domain consists of 81 amino acids and encompasses a region predicted to form an amphiphilic ␣-helix that binds to the EF-hand Ca 2ϩ -binding protein, S100B (18). Finally, the Cterminal extension of NDR kinase contains a broadly conserved hydrophobic motif ...

NDR protein kinases are involved in the regulation of cell cycle progression and morphology. NDR1/NDR2 protein kinase is activated by phosphorylation on the activation loop phosphorylation site Ser281/Ser282 and the hydrophobic motif phosphorylation site Thr444/Thr442. Autophosphorylation of NDR is responsible for phosphorylation on Ser281/Ser282, whereas Thr444/Thr442 is targeted by an upstream kinase. Here we show that MST3, a mammalian Ste20-like protein kinase, is able to phosphorylate NDR protein kinase at Thr444/ Thr442. In vitro, MST3 selectively phosphorylated Thr442 of NDR2, resulting in a 10-fold stimulation of NDR activity. MOB1A (Mps one binder 1A) protein further increased the activity, leading to a fully active kinase. In vivo, Thr442 phosphorylation after okadaic acid stimulation was potently inhibited by MST3KR, a kinasedead mutant of MST3. Knockdown of MST3 using short hairpin constructs abolished Thr442 hydrophobic motif phosphorylation of NDR in HEK293F cells. We conclude that activation of NDR is a multistep process involving phosphorylation of the hydrophobic motif site Thr444/2 by MST3, autophosphorylation of Ser281/2, and binding of MOB1A.The NDR and LATS family of serine/threonine protein kinases participates in the regulation of cell cycle progression and cell morphology (14,43,53). These kinases share a conserved N-terminal regulatory domain that interacts with MOB (3, 6, 18, 33, 50) and S100B (31) proteins. The conserved catalytic (kinase) domain has an insertion of 30 amino acids between subdomains VII and VIII containing the activation segment phosphorylation site Ser281/Ser282 and, in the case of mammalian NDR1 and NDR2, an autoinhibitory sequence (3). The C-terminal regulatory domain encompasses the regulatory hydrophobic motif phosphorylation site Thr444 (32). Recent data show that NDR1 and NDR2 protein kinase activities are stimulated by Ca 2ϩ /S100B-and MOB1-binding-induced autophosphorylation on S281/S282 in vitro and in vivo (3, 31). Phosphorylation of the hydrophobic motif phosphorylation site T444/T442 is required for maximal activation and involves an upstream kinase (42,44).Genetic evidence suggests that Ste20 (Sterile)-like protein kinases function as upstream kinases of the NDR family. For example, one of the Saccharomyces cerevisiae Ste20-like kinases, Cdc15p, phosphorylates Dbf2p (28), and the Schizosaccharomyces pombe Ste20-like kinase Pak1p/Shk1p genetically interacts with Orb6p (49). Furthermore, the Ste20-like kinase Kic1p functionally interacts with Cbk1p, the closest relative of NDR from Saccharomyces cerevisiae (33), and the Drosophila melanogaster Ste20-like kinase HIPPO phosphorylates WARTS/LATS kinase (16,20,34,45,51). The closest mammalian homologues of Kic1p and Hippo are the mammalian Ste20-like protein kinases MST1/KRS1, MST2/KRS2, MST3, MST4/MASK, and SOK/YSK (29), which are involved in the regulation of cell morphology, proliferation, and apoptosis (7,9,22,25,38). This group of kinases contains an N-terminal kinase domain as well as an autoinhibitory C-...

NDR1 (nuclear Dbf2-related) is a serine/threonine protein kinase belonging to subfamily of kinases implicated in the regulation of cell division and morphology. Previously, we demonstrated that the activity of NDR1 is controlled by phosphorylation of two regulatory residues, Ser-281 and Thr-444. Moreover, we found that NDR1 becomes activated through a direct interaction with EF-hand Ca 2؉ -binding proteins of the S100 family. In this work, we characterize this regulatory mechanism in detail. We found that NDR1 autophosphorylates in vitro predominantly on Ser-281 and to a lesser extent on Thr-74 and Thr-444. All of these residues proved to be crucial also for NDR1 activity in vivo; however, in contrast to Ser-281 and Thr-444, Thr-74 seems to be involved only in binding to S100B rather than directly regulating NDR1 activity per se. When we added Ca 2؉ /S100B, we observed an increased autophosphorylation on Ser-281 and Thr-444, resulting in stimulation of NDR1 activity in vitro. Using phosphospecific antibodies, we found that Ser-281 also becomes autophosphorylated in vivo, whereas Thr-444 is targeted predominantly by an as yet unidentified upstream kinase. Significantly, the Ca 2؉ -chelating agent BAPTA-AM suppressed the activity and phosphorylation of NDR1 on both Ser-281 and Thr-444, and specifically, these effects were reversed when we added the sarcoplasmic-endoplasmic reticulum Ca 2؉ ATPase pump inhibitor thapsigargin. NDR1 (nuclear Dbf2-related) is a conserved and widely expressed nuclear serine/threonine kinase that belongs to a recently identified subfamily of kinases that play a crucial role in cell division and cell morphogenesis. We originally cloned this kinase from a human fetal brain cDNA library using Caenorhabditis elegans expressed sequence tag clone cm11b8 (1). Later, it was mapped to chromosome 6p21 next to the major histocompatibility complex class I gene cluster (2). Recently, we isolated a second NDR isoform, termed NDR2, which is localized on chromosome 12p11 next to the K-ras gene.1 The two isoforms display an identity of more than 87% at the protein and 77% at the DNA level. In addition to mammalian NDR1 and NDR2, the NDR family of protein kinases comprises orthologous proteins Tricornered (trc gene) from Drosophila melanogaster; Sax-1 from C. elegans; and several closely related kinases such as Warts/Lats kinases from mammals, D. melanogaster, and C. elegans; Cbk1, Dbf2, and Dbf20 from Saccharomyces cerevisiae; Orb6 from Schizosaccharomyces pombe; Ukc1 from Ustilago maydis; and Cot-1 from Neurospora crassa (for review, see Ref.3). These kinases share 40 -60% amino acid identity within their catalytic domains as well as conserved regions in their regulatory domains. The structural similarity within this family suggests that these kinases might perform related functions even in evolutionary distant organisms.In fact, recent reports confirmed the involvement of this group of kinases in various aspects of regulation of cell division and cell morphogenesis. For instance, mutations in trc resul...

The nuclear Dbf2-related (NDR) family of protein serine/threonine kinases comprises mammalian NDR and large tumor suppressor (LATS) kinases, their orthologs from Drosophila melanogaster and Caenorhabditis elegans, and a number of related kinases from yeast and plants. The members of this family were independently implicated in various aspects of the control of cell division and morphogenesis. They are crucial regulators of the actin and tubulin cytoskeletal organization during polarized growth and cytokinesis in yeast. Furthermore, they are key players in control of proliferation and morphology of many cell types in D. melanogaster and C. elegans. In mammalians, the LATS kinase is a tumor suppressor, negatively regulating the cyclin-dependent kinase CDK1, cell proliferation rate, and modulating cell survival.