Constitutively active NDR1-PIF kinase functions independent of MST1 and hMOB1 signalling

The human MST1/hMOB1/NDR1 tumour suppressor cascade regulates important cellular processes, such as centrosome duplication. hMOB1/NDR1 complex formation appears to be essential for NDR1 activation by autophosphorylation on Ser281 and hydrophobic motif (HM) phosphorylation at Thr444 by MST1. To dissect these mechanistic relationships in MST1/hMOB1/NDR signalling, we designed NDR1 variants carrying modifications that mimic HM phosphorylation and/or abolish hMOB1/NDR1 interactions. Significantly, the analyses of these variants revealed that NDR1-PIF, an NDR1 variant containing the PRK2 hydrophobic motif, remains hyperactive independent of hMOB1/NDR1-PIF complex formation. In contrast, as reported for the T444A phospho-acceptor mutant, NDR1 versions carrying single phospho-mimicking mutations at the HM phosphorylation site, namely T444D or T444E, do not display increased kinase activities. Collectively, these observations suggest that in cells Thr444 phosphorylation by MST1 depends on the hMOB1/NDR1 association, while Ser281 autophosphorylation of NDR1 can occur independently. By testing centrosome-targeted NDR1 variants in NDR1- or MST1-depleted cells, we further observed that centrosome-enriched NDR1-PIF requires neither hMOB1 binding nor MST1 signalling to function in centrosome overduplication. Taken together, our biochemical and cell biological characterisation of NDR1 versions provides novel unexpected insights into the regulatory mechanisms of NDR1 and NDR1's role in centrosome duplication.     

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.     

Cell elongation and branching are regulated by differential phosphorylation states of the nuclear Dbf2-related kinase COT1 in Neurospora crassa

Dysfunction of the Neurospora crassa nuclear Dbf2-related kinase COT1 leads to cessation of tip extension and massive induction of new sites of growth. To determine the role phosphorylation plays in COT1 function, we mutated COT1 residues corresponding to positions of highly conserved nuclear Dbf2-related phosphorylation sites. Analyses of the point-mutation cot-1 strains (mimicking non- and constitutively phosphorylated states) indicate the involvement of COT1 phosphorylation in the regulation of hyphal elongation and branching as well as asexual development by altering cell wall integrity and actin organization. Phosphorylation of COT1's activation segment (at Ser417) is required for proper in vitro kinase activity, but has only a limited effect on hyphal growth. In marked contrast, even though phosphorylation of the C-terminal hydrophobic motif (at Thr589) is crucial for all COT1 functions in vivo , the lack of Thr589 phosphorylation did not significantly affect in vitro COT1 kinase activity. Nevertheless, its regulatory role has been made evident by the significant increase observed in COT1 kinase activity when this residue was substituted in a manner mimicking constitutive phosphorylation. We conclude that COT1 regulates elongation and branching in an independent manner, which is determined by its phosphorylation state.     

Phosphorylation and activation of nuclear Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKP-N/PPM1E) by Ca2+/calmodulin-dependent protein kinase I (CaMKI)

Nuclear Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKP-N/PPM1E) is an enzyme that dephosphorylates and downregulates multifunctional Ca(2+)/calmodulin-dependent protein kinases (CaMKs) as well as AMP-dependent protein kinase. In our previous study, we found that zebrafish CaMKP-N (zCaMKP-N) underwent proteolytic processing and translocated to cytosol in a proteasome inhibitor-sensitive manner. In the present study, we found that zCaMKP-N is regulated by phosphorylation at Ser-480. When zCaMKP-N was incubated with the activated CaMKI, time-dependent phosphorylation of the enzyme was observed. This phosphorylation was significantly reduced when Ser-480 was replaced by Ala, suggesting that CaMKI phosphorylates Ser-480 of zCaMKP-N. Phosphorylation-mimic mutants, S480D and S480E, showed higher phosphatase activities than those of wild type and S480A mutant in solution-based phosphatase assay using various substrates. Furthermore, autophosphorylation of CaMKII after ionomycin treatment was more severely attenuated in Neuro2a cells when CaMKII was cotransfected with the phosphorylation-mimic mutant of zCaMKP-N than with the wild-type or non-phosphorylatable zCaMKP-N. These results strongly suggest that phosphorylation of zCaMKP-N at Ser-480 by CaMKI activates CaMKP-N catalytic activity and thereby downregulates multifunctional CaMKs in the cytosol.     

Mechanism of Ca2+-mediated regulation of NDR protein kinase through autophosphorylation and phosphorylation by an upstream kinase

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.     

Mechanism of activation of protein kinase D2(PKD2) by the CCK(B)/gastrin receptor

Recently, we cloned a novel serine/threonine kinase termed protein kinase D2 (PKD2). PKD2 can be activated by phorbol esters both in vivo and in vitro but also by gastrin via the cholecystokinin/CCK(B) receptor in human gastric cancer cells stably transfected with the CCK(B)/gastrin receptor (AGS-B cells). Here we identify the mechanisms of gastrin-induced PKD2 activation in AGS-B cells. PKD2 phosphorylation in response to gastrin was rapid, reaching a maximum after 10 min of incubation. Our data demonstrate that gastrin-stimulated PKD2 activation involves a heterotrimeric G alpha(q) protein as well as the activation of phospholipase C. Furthermore, we show that PKD2 can be activated by classical and novel members of the protein kinase C (PKC) family such as PKC alpha, PKC epsilon, and PKC eta. These PKCs are activated by gastrin in AGS-B cells. Thus, PKD2 is likely to be a novel downstream target of specific PKCs upon the stimulation of AGS-B cells with gastrin. Our data suggest a two-step mechanism of activation of PKD2 via endogenously produced diacylglycerol and the activation of PKCs.     

Differential activation of the rice sucrose nonfermenting1-related protein kinase2 family by hyperosmotic stress and abscisic acid

To date, a large number of sequences of protein kinases that belong to the sucrose nonfermenting1-related protein kinase2 (SnRK2) family are found in databases. However, only limited numbers of the family members have been characterized and implicated in abscisic acid (ABA) and hyperosmotic stress signaling. We identified 10 SnRK2 protein kinases encoded by the rice (Oryza sativa) genome. Each of the 10 members was expressed in cultured cell protoplasts, and its regulation was analyzed. Here, we demonstrate that all family members are activated by hyperosmotic stress and that three of them are also activated by ABA. Surprisingly, there were no members that were activated only by ABA. The activation was found to be regulated via phosphorylation. In addition to the functional distinction with respect to ABA regulation, dependence of activation on the hyperosmotic strength was different among the members. We show that the relatively diverged C-terminal domain is mainly responsible for this functional distinction, although the kinase domain also contributes to these differences. The results indicated that the SnRK2 protein kinase family has evolved specifically for hyperosmotic stress signaling and that individual members have acquired distinct regulatory properties, including ABA responsiveness by modifying the C-terminal domain.     

The nuclear Dbf2-related kinase COT1 and the mitogen-activated protein kinases MAK1 and MAK2 genetically interact to regulate filamentous growth, hyphal fusion and sexual development in Neurospora crassa

Ndr kinases, such as Neurospora crassa COT1, are important for cell differentiation and polar morphogenesis, yet their input signals as well as their integration into a cellular signaling context are still elusive. Here, we identify the cot-1 suppressor gul-4 as mak-2 and show that mutants of the gul-4/mak-2 mitogen-activated protein (MAP) kinase pathway suppress cot-1 phenotypes along with a concomitant reduction in protein kinase A (PKA) activity. Furthermore, mak-2 pathway defects are partially overcome in a cot-1 background and are associated with increased MAK1 MAPK signaling. A comparative characterization of N. crassa MAPKs revealed that they act as three distinct modules during vegetative growth and asexual development. In addition, common functions of MAK1 and MAK2 signaling during maintenance of cell-wall integrity distinguished the two ERK-type pathways from the p38-type OS2 osmosensing pathway. In contrast to separate functions during vegetative growth, the concerted activity of the three MAPK pathways is essential for cell fusion and for the subsequent formation of multicellular structures that are required for sexual development. Taken together, our data indicate a functional link between COT1 and MAPK signaling in regulating filamentous growth, hyphal fusion, and sexual development.     

Mechanism of mitogen-activated protein kinase phosphatase-3 activation by ERK2

The mitogen-activated protein kinase phosphatase 3 (MKP3)-catalyzed hydrolysis of aryl phosphates in the absence and presence of extracellular signal-regulated kinase 2 (ERK2) was investigated in order to provide insights into the molecular basis of the ERK2-induced MKP3 activation. In the absence of ERK2, the MKP3-catalyzed hydrolysis of simple aryl phosphates does not display any dependence on pH, viscosity, and the nature of the leaving group. Increased catalytic activity and enhanced affinity for oxyanions are observed for MKP3 in the presence of ERK2. In addition, normal bell-shaped pH dependence on the reaction catalyzed by MKP3 is restored in the presence of ERK2. Collectively, these results suggest that the rate-limiting step in the absence of ERK2 for the MKP3 reaction corresponds to a substrate-induced conformational change in MKP3 involving active site rearrangement and general acid loop closure. The binding of ERK2 to the N-terminal domain of MKP3 facilitates the repositioning of active site residues and speeds up the loop closure in MKP3 such that chemistry becomes rate-limiting in the presence of ERK2. Remarkably, it is found that the extent of ERK2-induced MKP3 activation is substrate dependent, with smaller activation observed for bulkier substrates. Unlike simple aryl phosphates, the MKP3-catalyzed hydrolysis of bulky polycyclic substrates exhibits bell-shaped pH rate profiles in the absence of ERK2. Furthermore, it is found that glycerol can also activate the MKP3-catalyzed reaction, increase the affinity of MKP3 for oxyanion, and restore the bell-shaped pH rate profile for the MKP3-catalyzed reaction. Thus, the rate of repositioning of catalytic groups and the reorienting of the electrostatic environment in the MKP3 active site can be enhanced not only by ERK2 but also by high affinity substrates or by glycerol.     

Pak1 protein kinase regulates activation and nuclear localization of Snf1-Gal83 protein kinase

Three kinases, Pak1, Tos3, and Elm1, activate Snf1 protein kinase in Saccharomyces cerevisiae. This cascade is conserved in mammals, where LKB1 activates AMP-activated protein kinase. We address the specificity of the activating kinases for the three forms of Snf1 protein kinase containing the beta-subunit isoforms Gal83, Sip1, and Sip2. Pak1 is the most important kinase for activating Snf1-Gal83 in response to glucose limitation, but Elm1 also has a significant role; moreover, both Pak1 and Elm1 affect Snf1-Sip2. These findings exclude the possibility of a one-to-one correspondence between the activating kinases and the Snf1 complexes. We further identify a second, unexpected role for Pak1 in regulating Snf1-Gal83: the catalytic activity of Pak1 is required for the nuclear enrichment of Snf1-Gal83 in response to carbon stress. The nuclear enrichment of Snf1 fused to green fluorescent protein (GFP) depends on both Gal83 and Pak1 and is abolished by a mutation of the activation loop threonine; in contrast, the nuclear enrichment of Gal83-GFP occurs in a snf1Delta mutant and depends on Pak1 only when Snf1 is present. Snf1-Gal83 is the only form of the kinase that localizes to the nucleus. These findings, that Pak1 both activates Snf1-Gal83 and controls its nuclear localization, implicate Pak1 in regulating nuclear Snf1 protein kinase activity.     

Regulation of NDR2 protein kinase by multi-site phosphorylation and the S100B calcium-binding protein

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. We describe the cloning and characterization of the human and mouse NDR2, a second mammalian isoform of NDR protein kinase. 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. We show that 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 was 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 we show a predominant cytoplasmic localization of ectopically expressed NDR2.     

Mechanism of protein kinase B activation by cyclic AMP-dependent protein kinase.

Activation of protein kinase B (PKB) by growth factors and hormones has been demonstrated to proceed via phosphatidylinositol 3-kinase (PI3-kinase). In this report, we show that PKB can also be activated by PKA (cyclic AMP [cAMP]-dependent protein kinase) through a PI3-kinase-independent pathway. Although this activation required phosphorylation of PKB, PKB is not likely to be a physiological substrate of PKA since a mutation in the sole PKA consensus phosphorylation site of PKB did not abolish PKA-induced activation of PKB. In addition, mechanistically, this activation was different from that of growth factors since it did not require phosphorylation of the S473 residue, which is essential for full PKB activation induced by insulin. These data were supported by the fact that mutation of residue S473 of PKB to alanine did not prevent it from being activated by forskolin. Moreover, phosphopeptide maps of overexpressed PKB from COS cells showed differences between insulin- and forskolin-stimulated cells that pointed to distinct activation mechanisms of PKB depending on whether insulin or cAMP was used. We looked at events downstream of PKB and found that PKA activation of PKB led to the phosphorylation and inhibition of glycogen synthase kinase-3 (GSK-3) activity, a known in vivo substrate of PKB. Overexpression of a dominant negative PKB led to the loss of inhibition of GSK-3 in both insulin- and forskolin-treated cells, demonstrating that PKB was responsible for this inhibition in both cases. Finally, we show by confocal microscopy that forskolin, similar to insulin, was able to induce translocation of PKB to the plasma membrane. This process was inhibited by high concentrations of wortmannin (300 nM), suggesting that forskolin-induced PKB movement may require phospholipids, which are probably not generated by class I or class III PI3-kinase. However, high concentrations of wortmannin did not abolish PKB activation, which demonstrates that translocation per se is not important for PKA-induced PKB activation.     

Activation of a nuclear Cdc2-related kinase within a mitogen-activated protein kinase-like TDY motif by autophosphorylation and cyclin-dependent protein kinase-activating kinase

Male germ cell-associated kinase (MAK) and intestinal cell kinase (ICK) are nuclear Cdc2-related kinases with nearly identical N-terminal catalytic domains and more divergent C-terminal noncatalytic domains. The catalytic domain is also related to mitogen-activated protein kinases (MAPKs) and contains a corresponding TDY motif. Nuclear localization of ICK requires subdomain XI and interactions of the conserved Arg-272, but not kinase activity or, surprisingly, any of the noncatalytic domain. Further, nuclear localization of ICK is required for its activation. ICK is activated by dual phosphorylation of the TDY motif. Phosphorylation of Tyr-159 in the TDY motif requires ICK autokinase activity but confers only basal kinase activity. Full activation requires additional phosphorylation of Thr-157 in the TDY motif. Coexpression of ICK with constitutively active MEK1 or MEK5 fails to increase ICK phosphorylation or activity, suggesting that MEKs are not involved. ICK and MAK are related to Ime2p in budding yeast, and cyclin-dependent protein kinase-activating kinase Cak1p has been placed genetically upstream of Ime2p. Recombinant Cak1p phosphorylates Thr-157 in the TDY motif of recombinant ICK and activates its activity in vitro. Coexpression of ICK with wild-type CAK1 but not kinase-inactive CAK1 in cells also increases ICK phosphorylation and activity. Our studies establish ICK as the prototype for a new group of MAPK-like kinases requiring dual phosphorylation at TDY motifs.     

Structure of mitogen-activated protein kinase-activated protein (MAPKAP) kinase 2 suggests a bifunctional switch that couples kinase activation with nuclear export

MAPK-activated protein kinase 2 (MAPKAPK2), one of several kinases directly phosphorylated and activated by p38 MAPK, plays a central role in the inflammatory response. The activated MAPKAPK2 phosphorylates its nuclear targets CREB/ATF1, serum response factor, and E2A protein E47 and its cytoplasmic targets HSP25/27, LSP-1, 5-lipoxygenase, glycogen synthase, and tyrosine hydroxylase. The crystal structure of unphosphorylated MAPKAPK2, determined at 2.8 A resolution, includes the kinase domain and the C-terminal regulatory domain. Although the protein is inactive, the kinase domain adopts an active conformation with aspartate 366 mimicking the missing phosphorylated threonine 222 in the activation loop. The C-terminal regulatory domain forms a helix-turn-helix plus a long strand. Phosphorylation of threonine 334, which is located between the kinase domain and the C-terminal regulatory domain, may serve as a switch for MAPKAPK2 nuclear import and export. Phosphorylated MAPKAPK2 masks the nuclear localization signal at its C terminus by binding to p38. It unmasks the nuclear export signal, which is part of the second C-terminal helix packed along the surface of kinase domain C-lobe, and thereby carries p38 to the cytoplasm.     

Phosphoinositide-dependent protein kinase-1 (PDK1)-independent activation of the protein kinase C substrate, protein kinase D

Phosphoinoisitide dependent kinase l (PDK1) is proposed to phosphorylate a key threonine residue within the catalytic domain of the protein kinase C (PKC) superfamily that controls the stability and catalytic competence of these kinases. Hence, in PDK1-null embryonic stem cells intracellular levels of PKCalpha, PKCbeta1, PKCgamma, and PKCepsilon are strikingly reduced. Although PDK1-null cells have reduced endogenous PKC levels they are not completely devoid of PKCs and the integrity of downstream PKC effector pathways in the absence of PDK1 has not been determined. In the present report, the PDK1 requirement for controlling the phosphorylation and activity of a well characterised substrate for PKCs, the serine kinase protein kinase D, has been examined. The data show that in embryonic stem cells and thymocytes loss of PDK1 does not prevent PKC-mediated phosphorylation and activation of protein kinase D. These results reveal that loss of PDK1 does not functionally inactivate all PKC-mediated signal transduction.     

Mechanism of activation of protein kinase B by insulin and IGF-1.

Insulin activated endogenous protein kinase B alpha (also known as RAC/Akt kinase) activity 12-fold in L6 myotubes, while after transfection into 293 cells PKBalpha was activated 20- and 50-fold in response to insulin and IGF-1 respectively. In both cells, the activation of PKBalpha was accompanied by its phosphorylation at Thr308 and Ser473 and, like activation, phosphorylation of both of these residues was prevented by the phosphatidylinositol 3-kinase inhibitor wortmannin. Thr308 and/or Ser473 were mutated to Ala or Asp and activities of mutant PKBalpha molecules were analysed after transfection into 293 cells. The activity of wild-type and mutant PKBalpha was also measured in vitro after stoichiometric phosphorylation of Ser473 by MAPKAP kinase-2. These experiments demonstrated that activation of PKBalpha by insulin or insulin-like growth factor-1 (IGF-1) results from phosphorylation of both Thr308 and Ser473, that phosphorylation of both residues is critical to generate a high level of PKBalpha activity and that the phosphorylation of Thr308 in vivo is not dependent on phosphorylation of Ser473 or vice versa. We propose a model whereby PKBalpha becomes phosphorylated and activated in insulin/IGF-1-stimulated cells by an upstream kinase(s).