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.     

Hydrophobic motif phosphorylation coordinates activity and polar localization of the Neurospora crassa nuclear Dbf2-related kinase COT1

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.     

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.     

The Neurospora crassa colonial temperature-sensitive 3 (cot-3) gene encodes protein elongation factor 2

At elevated temperatures, the Neurospora crassa mutant colonial, temperature-sensitive 3 (cot-3) forms compact, highly branched colonies. Growth of the cot-3 strain under these conditions also results in the loss of the lower molecular weight (LMW) isoform of the Ser/Thr protein kinase encoded by the unlinked cot-1 gene, whose function is also involved in hyphal elongation. The unique cot-3 gene has been cloned by complementation and shown to encode translation elongation factor 2 (EF-2). As expected for a gene with a general role in protein synthesis, cot-3 mRNA is abundantly expressed throughout all asexual phases of the N. crassa life cycle. The molecular basis of the cot-3 mutation was determined to be an ATT to AAT transversion, which causes an Ile to Asn substitution at residue 278. Treatment with fusidic acid (a specific inhibitor of EF-2) inhibits hyphal elongation and induces hyperbranching in a manner which mimics the cot-3 phenotype, and also leads to a decrease in the abundance of the LMW isoform of COT1. This supports our conclusion that the mutation in cot-3 which results in abnormal hyphal elongation/branching impairs EF-2 function and confirms that the abundance of a LMW isoform of COT1 kinase is dependent on the function of this general translation factor.     

Cellular distribution of COT1 kinase in Neurospora crassa

The Neurospora crassa cot-1 gene encodes a Ser/Thr protein kinase, which is involved in hyphal elongation. Many vacuoles, abnormally shaped mitochondria, and nuclei, along with differences in the structure of the cell wall and hyphal septa, were observed in hyphae of the cot-1 mutant shortly after a shift to the restrictive temperature. Immunolocalization experiments indicated that COT1 was associated with the cytoplasmic membrane; COT1 was also detected in the cytoplasm. The membrane-associated COT1 was absent from the cot-1 mutant when shifted to the restrictive temperature, as was a lower molecular weight isoform of COT1. We propose that COT1 may be involved in several cellular processes, and the spatial and temporal regulation of COT1 activity involves trafficking of the kinase within the fungal cell and its possible interaction with additional proteins.     

Dbf4 is direct downstream target of ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related (ATR) protein to regulate intra-S-phase checkpoint

Dbf4/Cdc7 (Dbf4-dependent kinase (DDK)) is activated at the onset of S-phase, and its kinase activity is required for DNA replication initiation from each origin. We showed that DDK is an important target for the S-phase checkpoint in mammalian cells to suppress replication initiation and to protect replication forks. We demonstrated that ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related (ATR) proteins directly phosphorylate Dbf4 in response to ionizing radiation and replication stress. We identified novel ATM/ATR phosphorylation sites on Dbf4 and showed that ATM/ATR-mediated phosphorylation of Dbf4 is critical for the intra-S-phase checkpoint to inhibit DNA replication. The kinase activity of DDK, which is not suppressed upon DNA damage, is required for fork protection under replication stress. We further demonstrated that ATM/ATR-mediated phosphorylation of Dbf4 is important for preventing DNA rereplication upon loss of replication licensing through the activation of the S-phase checkpoint. These studies indicate that DDK is a direct substrate of ATM and ATR to mediate the intra-S-phase checkpoint in mammalian cells.     

A mutation within the catalytic domain of COT1 kinase confers changes in the presence of two COT1 isoforms and in Ser/Thr protein kinase and phosphatase activities in Neurospora crassa.

Neurospora crassa grows by forming spreading colonies. cot-1 belongs to a class of N. crassa colonial temperature-sensitive (cot) mutants and encodes a Ser/Thr protein kinase. We have mapped the cot-1 mutation to a single base change resulting in a His to Arg substitution at amino acid 351, which resides within the catalytic domain. Antibodies raised against COT1 detected and immunoprecipitated a predominant 73-kDa polypeptide in N. crassa extracts, whose abundance was constant under all growth conditions tested. An additional, lower MW COT1 isoform (67-kDa) present in the wild-type was not detected in cot-1 grown at the restrictive temperature. Similarly, this isoform was not detected in cot-3 or cot-5 strains, when grown at restrictive temperatures. Reduced levels of Ser/Thr kinase activity and an increase in type 1 and type 2B phosphatase (calcineurin) activities were measured in a cot-1 background. Apparent changes in the phosphorylation state of the p150(Glued) subunit of the dynactin cytoskeletal motor component (encoded by ro-3, a suppressor of cot-1) and evidence of in vitro physical interactions between COT1 and calcineurin indicate a functional linkage among COT1 kinase, type 2B phosphatase, and dynactin.     

Inhibition of Cdc7/Dbf4 kinase activity affects specific phosphorylation sites on MCM2 in cancer cells

The Cdc7/Dbf4 kinase is required for initiation of DNA replication and also plays a role in checkpoint function in response to replication stress. Exactly how Cdc7/Dbf4 mediates those activities remains to be elucidated. Cdc7/Dbf4 physically interacts with and phosphorylates the minichromosome maintenance complex (MCM), such as MCM2, MCM4 and MCM6. Cdc7/Dbf4 activity is required for association of Cdc45 followed by recruitment of DNA polymerase on the chromatin. Using high resolution mass spectrometry, we identified six phosphorylation sites on MCM2, two of them have not been described before. We provide evidence that Cdc7/Dbf4 mediates phosphorylation on serine 108 and serine 40 on human MCM2 in vitro and in vivo in cancer cells in the absence of DNA damage. Antibodies specific to pS108 or pS40 confirmed the sites and established useful read-outs for inhibition of Cdc7/Dbf4. This report demonstrates the utility of an in vitro to in vivo workflow utilizing immunoprecipitation and mass spectrometry to map phosphorylation sites on endogenous kinase substrates. The approach can be readily generalized to identify target modulation read-outs for other potential kinase cancer targets.     

Interleukin-1 stimulated activation of the COT catalytic subunit through the phosphorylation of Thr290 and Ser62

The protein kinase COT/Tpl2 is activated by interleukin-1 (IL-1), TNFalpha and lipopolysaccharide, and its activation by these agonists involves the IkappaB kinase beta (IKKbeta) catalysed phosphorylation of the p105 regulatory subunit. Here, we show that COT activation also requires catalytic subunit phosphorylation, since IL-1beta induced a 5-10-fold activation of a COT mutant unable to bind p105. Activation was paralleled by the phosphorylation of Thr290 and Ser62 and unaffected by the IKKbeta inhibitor PS1145 at concentrations which prevented the degradation of IkappaBalpha. Mutagenesis experiments indicated that COT activation is initiated by Thr290 phosphorylation catalysed by an IL-1-stimulated protein kinase distinct from IKKbeta, while Ser62 phosphorylation is an autophosphorylation event required for maximal activation.     

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.     

p40MO15 associates with a p36 subunit and requires both nuclear translocation and Thr176 phosphorylation to generate cdk-activating kinase activity in Xenopus oocytes.

p40MO15, a cdc2-related protein, is the catalytic subunit of the kinase (CAK, cdk-activating kinase) responsible for Thr161/Thr160 phosphorylation and activation of cdk1/cdk2. We have found that strong overexpression of p40MO15 only moderately increases CAK activity in Xenopus oocytes, indicating that a regulatory CAK subunit (possibly a cyclin-like protein) limits the ability to generate CAK activity in p40MO15 overexpressing oocytes. This 36 kDa subunit was microsequenced after extensive purification of CAK activity. Production of Xenopus CAK activity was strongly reduced in enucleated oocytes overexpressing p40MO15 and p40MO15 shown to contain a nuclear localization signal required for nuclear translocation and generation of CAK activity. p40MO15 was found to be phosphorylated on Ser170 and Thr176 by proteolytic degradation, radiosequencing of tryptic peptides and mutagenesis. Thr176 phosphorylation is required and Ser170 phosphorylation is dispensable for p40MO15 to generate CAK activity upon association with the 36 kDa regulatory subunit. Finally, Thr176 and Ser170 phosphorylations are not intramolecular autophosphorylation reactions. Taken together, the above results identify protein-protein interactions, nuclear translocation and phosphorylation (by an unidentified kinase) as features of p40MO15 that are required for the generation of active CAK.     

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.     

Direct phosphorylation and activation of a Nim1-related kinase Gin4 by Elm1 in budding yeast

In budding yeast, Gin4, a Nim1-related kinase, plays an important role in proper organization of the septin ring at the mother-bud neck, a filamentous structure that is critical for diverse cellular processes including mitotic entry and cytokinesis. How Gin4 kinase activity is regulated is not known. Here we showed that a neck-associated Ser/Thr kinase Elm1, which is important for septin assembly, is critical for proper modification of Gin4 and its physiological substrate Shs1. In vitro studies with purified recombinant proteins demonstrated that Elm1 directly phosphorylates and activates Gin4, which in turn phosphorylates Shs1. Consistent with these observations, acute inhibition of Elm1 activity abolished mitotic Gin4 phosphorylation and Gin4-dependent Shs1 modification in vivo. In addition, a gin4 mutant lacking the Elm1-dependent phosphorylation sites exhibited an impaired localization to the bud-neck and, as a result, induced a significant growth defect with an elongated bud morphology. Thus, Elm1 regulates the septin assembly-dependent cellular events by directly phosphorylating and activating the Gin4-dependent pathway(s).     

Human NDR kinases are rapidly activated by MOB proteins through recruitment to the plasma membrane and phosphorylation

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.     

Dbf2 is essential for cytokinesis and correct mitotic spindle formation in Candida albicans

We have characterized the DBF2 gene, encoding a protein kinase of the NDR family in Candida albicans, and demonstrate that this gene is essential for cell viability. Conditional mutants were constructed by using the MET3 promoter to analyse the phenotype of cells lacking this kinase. The absence of Dbf2 resulted in cells arrested as large-budded pairs that failed to contract the actomyosin ring, a function similar to that described for its Saccharomyces cerevisiae orthologue. In addition to its role in cytokinesis, Dbf2 regulates mitotic spindle organization and nuclear segregation as Dbf2-depleted cells have abnormal microtubules and severe defects in nuclear migration to the daughter cell, which results in a cell cycle block during mitosis. Taken together, these results imply that Dbf2 performs several functions during exit from mitosis and cytokinesis. Consistent with a role in spindle organization, the protein localizes to the mitotic spindle during anaphase, and it interacts physically with tubulin, as indicated by immunoprecipitation experiments. Finally, DBF2 depletion also resulted in impaired true hyphal growth.     

Mapping of MST1 kinase sites of phosphorylation. Activation and autophosphorylation

MST1 is a member of the Sterile-20 family of cytoskeletal, stress, and apoptotic kinases. MST1 is activated by phosphorylation at previously unidentified sites. This study examines the role of phosphorylation at several sites and effects on kinase activation. We define Thr(183) in subdomain VIII as a primary site of phosphoactivation. Thr(187) is also critical for kinase activity. Phosphorylation of MST1 in subdomain VIII was catalyzed by active MST1 via intermolecular autophosphorylation, enhanced by homodimerization. Active MST1 (wild-type or T183E), but not inactive Thr(183)/Thr(187) mutants, was also highly autophosphorylated at the newly identified Thr(177) and Thr(387) residues. Cells expressing active MST1 were mostly detached, whereas with inactive MST1, adhesion was normal. Active MKK4, JNK, caspase-3, and caspase-9 were detected in the detached cells. These cells also contained all autophosphorylated and essentially all caspase-cleaved MST1. Similar phenotypes were elicited by a caspase-insensitive D326N mutant, suggesting that kinase activity, but not cleavage of MST1, is required. Interestingly, an S327E mutant mimicking Ser(327) autophosphorylation was also caspase-insensitive, but only when expressed in caspase-3-deficient cells. Together, these data suggest a model whereby MST1 activation is induced by existing, active MST kinase, which phosphorylates Thr(183) and possibly Thr(187). Dimerization promotes greater phosphorylation. This leads to induction of the JNK signaling pathway, caspase activation, and apoptosis. Further activation of MST1 by caspase cleavage is best promoted by caspase-3, although this appears to be unnecessary for signaling and morphological responses.     

A novel non-canonical mechanism of regulation of MST3 (mammalian Sterile20-related kinase 3)

The canonical pathway of regulation of the GCK (germinal centre kinase) III subgroup member, MST3 (mammalian Sterile20-related kinase 3), involves a caspase-mediated cleavage between N-terminal catalytic and C-terminal regulatory domains with possible concurrent autophosphorylation of the activation loop MST3(Thr(178)), induction of serine/threonine protein kinase activity and nuclear localization. We identified an alternative 'non-canonical' pathway of MST3 activation (regulated primarily through dephosphorylation) which may also be applicable to other GCKIII (and GCKVI) subgroup members. In the basal state, inactive MST3 co-immunoprecipitated with the Golgi protein GOLGA2/gm130 (golgin A2/Golgi matrix protein 130). Activation of MST3 by calyculin A (a protein serine/threonine phosphatase 1/2A inhibitor) stimulated (auto)phosphorylation of MST3(Thr(178)) in the catalytic domain with essentially simultaneous cis-autophosphorylation of MST3(Thr(328)) in the regulatory domain, an event also requiring the MST3(341-376) sequence which acts as a putative docking domain. MST3(Thr(178)) phosphorylation increased MST3 kinase activity, but this activity was independent of MST3(Thr(328)) phosphorylation. Interestingly, MST3(Thr(328)) lies immediately C-terminal to a STRAD (Sterile20-related adaptor) pseudokinase-like site identified recently as being involved in binding of GCKIII/GCKVI members to MO25 scaffolding proteins. MST3(Thr(178)/Thr(328)) phosphorylation was concurrent with dissociation of MST3 from GOLGA2/gm130 and association of MST3 with MO25, and MST3(Thr(328)) phosphorylation was necessary for formation of the activated MST3-MO25 holocomplex.     

Thr2446 is a novel mammalian target of rapamycin (mTOR) phosphorylation site regulated by nutrient status

The mammalian target of rapamycin (mTOR) is a key regulator of protein translation. Signaling via mTOR is increased by growth factors but decreased during nutrient deprivation. Previous studies have identified Ser2448 as a nutrient-regulated phosphorylation site located in the mTOR catalytic domain, insulin stimulates Ser2448 phosphorylation via protein kinase B (PKB), while Ser2448 phosphorylation is attenuated with amino acid starvation. Here we have identified Thr2446 as a novel nutrient-regulated phosphorylation site on mTOR. Thr2446 becomes phosphorylated when CHO-IR cells are nutrient-deprived, but phosphorylation is reduced by insulin stimulation. Nutrient deprivation activates AMP-activated protein kinase (AMPK). To test whether this could be involved in regulating phoshorylation of mTOR, we treated cultured murine myotubes with 5'-aminoimidazole-4-carboxamide ribonucleoside (AICAR) or dinitrophenol (DNP). Both treatments activated AMPK and also caused a concomitant increase in phosphorylation of Thr2446 and a parallel decrease in insulin's ability to phosphorylate p70 S6 kinase. In vitro kinase assays using peptides based on the sequence in amino acids 2440-2551 of mTOR found that PKB and AMPK are capable of phosphorylating sites in this region. However, phosphorylation by PKB is restricted when Thr2446 is mutated to an acidic residue mimicking phosphorylation. Conversely, AMP-kinase-induced phosphorylation is reduced when Ser2448 is phosphorylated. These data suggest differential phosphorylation Thr2446 and Ser2448 could act as a switch mechanism to integrate signals from nutrient status and growth factors to control the regulation of protein translation.     

LATS1/WARTS phosphorylates MYPT1 to counteract PLK1 and regulate mammalian mitotic progression

In the mitotic exit network of budding yeast, Dbf2 kinase phosphorylates and regulates Cdc14 phosphatase. In contrast, no phosphatase substrates of LATS1/WARTS kinase, the mammalian equivalent of Dbf2, has been reported. To address this discrepancy, we performed phosphoproteomic screening using LATS1 kinase. Screening identified MYPT1 (myosin phosphatase-targeting subunit 1) as a new substrate for LATS1. LATS1 directly and preferentially phosphorylated serine 445 (S445) of MYPT1. An MYPT1 mutant (S445A) failed to dephosphorylate Thr 210 of PLK1 (pololike kinase 1), thereby activating PLK1. This suggests that LATS1 promotes MYPT1 to antagonize PLK1 activity. Consistent with this, LATS1-depleted HeLa cells or fibroblasts from LATS1 knockout mice showed increased PLK1 activity. We also found deoxyribonucleic acid (DNA) damage-induced LATS1 activation caused PLK1 suppression via the phosphorylation of MYPT1 S445. Furthermore, LATS1 knockdown cells showed reduced G2 checkpoint arrest after DNA damage. These results indicate that LATS1 phosphorylates a phosphatase as does the yeast Dbf2 and demonstrate a novel role of LATS1 in controlling PLK1 at the G2 DNA damage checkpoint.