Docking of PRAK/MK5 to the Atypical MAPKs ERK3 and ERK4 Defines a Novel MAPK Interaction Motif

ERK3 and ERK4 are atypical MAPKs in which the canonical TXY motif within the activation loop of the classical MAPKs is replaced by SEG. Both ERK3 and ERK4 bind, translocate, and activate the MAPK-activated protein kinase (MK) 5. The classical MAPKs ERK1/2 and p38 interact with downstream MKs (RSK1–3 and MK2–3, respectively) through conserved clusters of acidic amino acids, which constitute the common docking (CD) domain. In contrast to the classical MAPKs, the interaction between ERK3/4 and MK5 is strictly dependent on phosphorylation of the SEG motif of these kinases. Here we report that the conserved CD domain is dispensable for the interaction of ERK3 and ERK4 with MK5. Using peptide overlay assays, we have defined a novel MK5 interaction motif (FRIEDE) within both ERK4 and ERK3 that is essential for binding to the C-terminal region of MK5. This motif is located within the L16 extension lying C-terminal to the CD domain in ERK3 and ERK4 and a single isoleucine to lysine substitution in FRIEDE totally abrogates binding, activation, and translocation of MK5 by both ERK3 and ERK4. These findings are the first to demonstrate binding of a physiological substrate via this region of the L16 loop in a MAPK. Furthermore, the link between activation loop phosphorylation and accessibility of the FRIEDE interaction motif suggests a switch mechanism for these atypical MAPKs in which the phosphorylation status of the activation loop regulates the ability of both ERK3 and ERK4 to bind to a downstream effector.Mitogen-activated protein kinase (MAPK)² phosphorylation cascades play important roles in the regulation of diverse cellular functions such as cell proliferation, differentiation, migration, and apoptosis (1, 2). A characteristic and conserved feature of this family of signaling pathways is their organization into modules comprising a sequential three-tiered kinase cascade. This contains a MAPK kinase kinase, a MEK, and the MAPK itself. Four such MAPK signaling modules have been described in mammals: ERK1 and ERK2, the c-Jun N-terminal kinases 1–3, the p38 kinases (p38α/β/γ/δ), and ERK5 (3–7). The MAPK kinase kinases phosphorylate and activate the MEKs, which in turn activate the MAPKs by dual phosphorylation on both the threonine and the tyrosine residue of a highly conserved TXY motif in the kinase activation loop. MAPKs are Ser/Thr kinases, which phosphorylate a wide range of substrates with the minimal consensus sequence (S/T)P (2).ERK4 and its close relative ERK3 are regarded as atypical members of the MAPK family. In contrast to the classical MAPKs, ERK3 and ERK4 harbor an SEG motif in the activation loop and thus lack a second phosphoacceptor site. In addition, protein kinases all possess a conserved APE motif located just C-terminal to the phosphoacceptor sites within subdomain VIII, in which the conserved glutamate is important for maintaining the stability of the kinase domain. In both ERK3 and ERK4, this motif is substituted by SPR, and ERK3 and ERK4 are the only two protein kinases in the human genome with an arginine residue in this position (8). Although they display significant sequence homology (44% identity) with ERK1 and ERK2 within their kinase domains, both ERK3 and ERK4 have unique C-terminal extensions, which account for the large differences in size observed between ERK1/2 (∼360 amino acids) and ERK3/ERK4 (721/587 amino acids). Whereas classical MAPKs have been highly conserved throughout evolution, with examples found in both unicellular and multicellular organisms, ERK3 and ERK4 are only present in vertebrates. Finally, in contrast to many of the classical MAPKs, the regulation, substrate specificity, and physiological functions of ERK3 and ERK4 are poorly understood. Although ERK3 and ERK4 are very similar to each other, there are significant differences between them. For instance, whereas ERK4, like most classical MAPKs, is a stable protein, ERK3 is highly unstable and subject to rapid proteosomal degradation. Thus, ERK3 activity may be regulated at the level of cellular abundance, and taken together these features indicate that ERK3 and ERK4 may perform specialized functions and enjoy different modes of regulation when compared with classical MAPKs (9–11).Despite the striking differences between ERK3 and ERK4 and the classical MAPKs, they do share one property with the ERK1/2, p38, and ERK5, namely the ability to interact with a group of downstream Ser/Thr protein kinases, termed MAPK-activated protein kinases (MAPKAPKs or MKs) (12, 13). In the case of ERK3 and ERK4, both proteins interact with, translocate, and activate the MK5 protein kinase. Several studies have drawn attention to the role of specific docking interactions that contribute to both substrate selectivity and regulation in MAPK pathways (14–17). These interactions involve docking domains, which specifically recognize small peptide docking motifs (D motifs) located on functional MAPK partner proteins including downstream substrates, scaffold proteins, as well as positive and negative regulators. The docking domains, although located within the kinase domains, are distinct from the active site. Similarly the D motifs, which these docking domains recognize, are also distinct from the phosphoacceptor sites within protein substrates (18). There are several classes of D motifs. The motifs found in MAPKAP kinases including MK5 have the consensus sequence LX_1–2(K/R)_2–5 where X is any amino acid (12). The corresponding docking domains within the MAPKs have also been characterized (16, 19, 20). The common docking (CD) domain is a cluster of negatively charged amino acids located in the L16 extension directly C-terminal to the kinase domain in the MAPK primary structure. A second domain termed ED (Glu-Asp) also contributes to binding specificity. This latter site is located near the CD domain in the MAPK tertiary structure. Whereas the CD domain is considered commonly important for all docking interactions, the ED site is thought to be important for the determination of specificity (16). Other residues and regions distinct from the ED and CD domains have also been shown to be important for docking.(21–25).This work has so far been largely confined to analysis of the classical MAPKs, and much less is known about the nature of substrate or regulatory docking interactions for the atypical MAPKs. We and others (9, 11, 26) have recently reported that the region encompassing residues 326–340 within both ERK3 and ERK4 is required for their ability to interact with and activate MK5. Furthermore, a truncated mutant of MK5, which lacks the 50 C-terminal residues (MK5 1–423), was unable to bind to ERK4 despite the fact that it retains its D domain. Finally, in contrast to conventional MAPKs, the interaction between ERK3 and ERK4 and MK5 requires activation loop phosphorylation of ERK3 and ERK4 (27, 28). Taken together these observations suggest that the mechanism by which the atypical MAPKs recognize and bind to at least one important class of effector kinases may be distinct to that found in the classical MAPKs such as ERK1/2 and p38.Here we demonstrate that two separate C-terminal regions, encompassing residues 383–393 and 460–465, respectively, are necessary for MK5 to interact with both ERK3 and ERK4. These regions are distinct from the D motif previously identified within MK5, suggesting that binding to ERK3 and ERK4 may be mediated by a different mechanism to that seen in the classical MAPKs. In support of this, the conserved CD domains within ERK3 and ERK4 are shown to be completely dispensable for MK5 interaction. Using peptide overlay assays, we have defined a minimal MK5 interaction motif FRIEDE in ERK4. Furthermore, we demonstrate that a single point mutation (ERK3 I334K or ERK4 I330K) within this FRIEDE motif is sufficient to disrupt the binding of both ERK3 and ERK4 to MK5 and consequently their ability to both translocate and activate MK5. The FRIEDE motif is located within the L16 extension C-terminal to the CD domain in both ERK3 and ERK4. Interestingly, molecular modeling of the corresponding region in ERK2 suggests that it undergoes a significant conformational change as a result of activation loop phosphorylation, making this part of the L16 extension more accessible (29). We propose that the FRIEDE motif represents a novel MAPK interaction motif, the function of which is linked to activation loop phosphorylation and MAPK activation.     

The ability of Sgs1 to interact with DNA topoisomerase III is essential for damage-induced recombination

SGS1 encodes a protein having DNA helicase activity, and a mutant allele of SGS1 was identified as a suppressor of the slow growth phenotype of top3 mutants. In this study, we examined whether Sgs1 prevents formation of DNA double strand breaks (DSBs) or is involved in DSB repair following exposure to methyl methanesulfonate (MMS). An analysis by pulsed-field gel electrophoresis and epistasis analyses indicated that Sgs1 is required for DSB repair that involves Rad52. In addition, analyses on the relationship between Sgs1 and proteins involved in DSB repair suggested that Sgs1 and Mre11 function via independent pathways both of which require Rad52. In sgs1 mutants, interchromosomal heteroallelic recombination and sister chromatid recombination (SCR) were not induced upon exposure to MMS, though both were induced in wild type cells, indicating the involvement of Sgs1 in heteroallelic recombination and SCR. Surprisingly, the ability of Sgs1 to bind to DNA topoisomerase III (Top3) was absolutely required for the induction of heteroallelic recombination and SCR and suppression of MMS sensitivity but its helicase activity was not, suggesting that Top3 plays a more important role in both recombinations than the DNA helicase activity of Sgs1.     

The MK5/PRAK kinase and Myc form a negative feedback loop that is disrupted during colorectal tumorigenesis

Expression of the Myc oncoprotein is downregulated in response to stress signals to allow cells to cease proliferation and escape apoptosis, but the mechanisms involved in this process are poorly understood. Cell cycle arrest in response to DNA damage requires downregulation of Myc via a p53-independent signaling pathway. Here we have used siRNA?screening of the human kinome to identify MAPKAPK5 (MK5, PRAK) as a negative regulator of Myc expression. MK5 regulates translation of Myc, since it is required for expression of miR-34b and miR-34c that bind to the 3'UTR of MYC. MK5 activates miR-34b/c expression via phosphorylation of FoxO3a, thereby promoting nuclear localization of FoxO3a and enabling it to induce miR-34b/c expression and arrest proliferation. Expression of MK5 in turn is directly activated by Myc, forming a negative feedback loop. MK5 is downregulated in colon carcinomas, arguing that this feedback loop is disrupted during colorectal tumorigenesis.     

Galpha(12) and Galpha(13) interact with Ser/Thr protein phosphatase type 5 and stimulate its phosphatase activity

The Galpha subunits of the G(12) family of heterotrimeric G proteins, defined by Galpha(12) and Galpha(13), are involved in many signaling pathways and diverse cellular functions. In an attempt to elucidate downstream effectors of Galpha(12) for cellular functions, we have performed a yeast two-hybrid screening of a rat brain cDNA library and revealed that Ser/Thr protein phosphatase type 5 (PP5) is a novel effector of Galpha(12) and Galpha(13). PP5 is a newly identified phosphatase and consists of a C-terminal catalytic domain and an N-terminal regulatory tetratricopeptide repeat (TPR) domain [2]. Arachidonic acid was recently shown to activate PP5 phosphatase activity by binding to its TPR domain, however the precise regulatory mechanism of PP5 phosphatase activity is not fully determined. In this study, we show that active forms of Galpha(12) and Galpha(13) specifically interact with PP5 through its TPR domain and activate its phosphatase activity about 2.5-fold. Active forms of Galpha(12) and Galpha(13) also enhance the arachidonic acid-stimulated PP5 phosphatase activity about 2.5-fold. Moreover, we demonstrate that the active form of Galpha(12) translocates PP5 to the cell periphery and colocalizes with PP5. These results propose a new signaling pathway of G(12) family G proteins.     

Deletion within the amino-terminal region of Gs alpha impairs its ability to interact with beta gamma subunits and to activate adenylate cyclase.

Proteolytic experiments performed on transducin and Go alpha subunit strongly suggest that the amino-terminal residues of the alpha chain are involved in the interaction with beta gamma subunits. To test the possibility that the same region in Gs may fulfill a similar function, we introduced a deletion in the amino-terminal domain of Gs alpha. The properties of the wild type and the deleted alpha chains were characterized on in vitro translated proteins or after reconstitution of cyc- membranes by in vitro-translated alpha subunits. The mutant (delta 2-29) Gs alpha could still bind guanosine 5'-3-O-(thio)triphosphate, as revealed by its resistance to trypsin proteolysis and was still able to interact with the membrane. However, (delta 2-29) Gs alpha was not ADP-ribosylated by cholera toxin. In contrast to Gs alpha, addition of beta gamma subunits did not increase the rate of sedimentation of (delta 2-29) Gs alpha in sucrose gradients. Binding experiments on reconstituted membranes showed that the coupling to beta-adrenergic receptors was very low with (delta 2-29) Gs alpha. Finally, the mutant did not restore activation of adenylate cyclase of cyc- membranes. We propose that the primary functional defect is the loss of interaction with beta gamma subunits, which secondarily impairs beta gamma-dependent properties such as receptor coupling and cholera toxin-catalyzed ADP-ribosylation. However, it remains to be established that the lack of adenylate cyclase activation also results from this impaired interaction with beta gamma subunits.     

Oxidative stress employs phosphatidyl inositol 3-kinase and ERK signalling pathways to activate cAMP phosphodiesterase-4D3 (PDE4D3) through multi-site phosphorylation at Ser239 and Ser579

RAW macrophages, which express the PDE4D3 and PDE4D5 cAMP phosphodiesterase isoforms, exhibited increased PDE4 activity when challenged with H2O2 in a fashion that was negated by treatment with the cell permeant antioxidant, N-acetyl cysteine and by diphenyleneiodonium chloride, an inhibitor of NADPH oxidase. In Cos1 cells transfected to express PDE4D3, challenge with H2O2 caused a rapid increase in both the activity and phosphorylation of PDE4D3. Lysates from H2O2-treated COS cells caused the phosphorylation of purified, recombinant PDE4D3 at two sites. One was the established ERK phosphorylation site at Ser579, located at the extreme C-terminus of the catalytic unit, and the other was a novel site at Ser239, located at the extreme N-terminus of the catalytic unit. Double Ser239Ala:Ser579Ala mutation of PDE4D3 prevented its H2O2-dependent phosphorylation both in vitro and in intact COS cells. Phosphorylation of PDE4D3 at Ser579 was ablated by treating COS cells with the MEK inhibitor, PD98059, which also negated activation. The activity of the Ser239Ala:Ser579Ala double mutant, and the Ser579Ala single PDE4D3 mutant was unaffected by H2O2 challenge of COS cells, whilst the Ser239Ala mutant was inhibited. Wortmannin inhibited the H2O2-dependent phosphorylation of PDE4D3 in COS cells by around 50%, whilst it fully ablated phosphorylation at Ser239 as well as ablating activation of PDE4D3. Neither immunodepletion of p70S6 kinase nor siRNA-mediated knockdown of mTor inhibited the H2O2-dependent phosphorylation of PDE4D3 at Ser239. Activation of PDE4D3 by challenge with H2O2 was not additive with activation through protein kinase A (PKA)-mediated phosphorylation of PDE4D3. Challenge with H2O2 did not alter PKA-mediated phosphorylation of PDE4D3 at Ser54. H2O2 dependent phosphorylation of PDE4D3, at Ser239 and Ser579, did not alter the sensitivity of PDE4D3 to inhibition by the selective PDE4 inhibitor, rolipram. An unknown protein kinase acting downstream of phosphatidyl inositol 3-kinase phosphorylates PDE4D3 at Ser239. This switches the effect of phosphorylation by ERK at Ser579 from inhibition to activation. We propose that phosphorylation at Ser239 attenuates interaction between either UCR2 or the UCR1/UCR2 module and the PDE4 catalytic unit so as to re-programme the functional outcome effect of phosphorylation by ERK. We identify a novel process through which reactive oxygen species activate long PDE4 isoforms so as to reduce cAMP levels and thereby promote inflammatory responses.     

Activation loop phosphorylation of the atypical MAP kinases ERK3 and ERK4 is required for binding, activation and cytoplasmic relocalization of MK5

Mitogen-activated protein (MAP) kinases are typical examples of protein kinases whose enzymatic activity is mainly controlled by activation loop phosphorylation. The classical MAP kinases ERK1/ERK2, JNK, p38 and ERK5 all contain the conserved Thr-Xxx-Tyr motif in their activation loop that is dually phosphorylated by members of the MAP kinase kinases family. Much less is known about the regulation of the atypical MAP kinases ERK3 and ERK4. These kinases display structural features that distinguish them from other MAP kinases, notably the presence of a single phospho-acceptor site (Ser-Glu-Gly) in the activation loop. Here, we show that ERK3 and ERK4 are phosphorylated in their activation loop in vivo. This phosphorylation is exerted, at least in part, in trans by an upstream cellular kinase. Contrary to classical MAP kinases, activation loop phosphorylation of ERK3 and ERK4 is detected in resting cells and is not further stimulated by strong mitogenic or stress stimuli. However, phosphorylation can be modulated indirectly by interaction with the substrate MAP kinase-activated protein kinase 5 (MK5). Importantly, we found that activation loop phosphorylation of ERK3 and ERK4 stimulates their intrinsic catalytic activity and is required for the formation of stable active complexes with MK5 and, consequently, for efficient cytoplasmic redistribution of ERK3/ERK4-MK5 complexes. Our results demonstrate the importance of activation loop phosphorylation in the regulation of ERK3/ERK4 function and highlight differences in the regulation of atypical MAP kinases as compared to classical family members.     

Identification of conserved domains in Salmonella muenchen flagellin that are essential for its ability to activate TLR5 and to induce an inflammatory response in vitro

The bacterial surface protein flagellin is widely distributed and well conserved among distant bacterial species. We and other investigators have reported recently that purified flagellin from Salmonella dublin or recombinant flagellin of Salmonella muenchen origin binds to the eukaryotic toll receptor TLR5 and activates the nuclear translocation of NF-kappaB and mitogen-activated protein kinase, resulting in the release of a host of pro-inflammatory mediators in vitro and in vivo. The amino acid sequence alignment of flagellins from various Gram-negative bacteria shows that the C and N termini are well conserved. It is possible that sequences within the N and C termini or both may regulate the pro-inflammatory activity of flagellin. Here we set out to map more precisely the regions in both termini that are required for TLR5 activation and pro-inflammatory signaling. Systematic deletion of amino acids from either terminus progressively reduced eukaryotic pro-inflammatory activation. However, deletion of amino acids 95-108 (motif N) in the N terminus and 441-449 (motif C) in the C terminus abolished pro-inflammatory activity completely. Site-directed mutagenesis analysis provided further evidence for the importance of motifs N and C. We also present evidence for the functional role of motifs N and C with the TLR5 receptor using a reporter assay system. Taken together, our results demonstrate that the pro-inflammatory activity of flagellin results from the interaction of motif N with the TLR5 receptor on the cell surface.     

Toll-like receptor 4 (TLR4) is essential for Hsp70-like protein 1 (HSP70L1) to activate dendritic cells and induce Th1 response

Toll-like receptors (TLRs) play important roles in initiation of innate and adaptive immune responses. Emerging evidence suggests that TLR agonists can serve as potential adjuvant for vaccination. Heat shock proteins (HSPs), functionally serving as TLR4 agonists, have been proposed to act as Th1 adjuvant. We have identified a novel Hsp70 family member, termed Hsp70-like protein 1 (Hsp70L1), shown that Hsp70L1 is a potent T helper cell (Th1) polarizing adjuvant that contributes to antitumor immune responses. However, the underlying mechanism for how Hsp70L1 exerts its Th1 adjuvant activity remains to be elucidated. In this study, we found that Hsp70L1 binds directly to TLR4 on the surface of DCs, activates MAPK and NF-κB pathways, up-regulates I-a(b), CD40, CD80, and CD86 expression and promotes production of TNF-α, IL-1β, and IL-12p70. Hsp70L1 failed to induce such phenotypic maturation and cytokine production in TLR4-deficient DCs, indicating a role for TLR4 in mediating Hsp70L1-induced DC activation. Furthermore, more efficient induction of carcinoembryonic antigen (CEA)-specific Th1 immune response was observed in mice immunized by wild-type DCs pulsed with Hsp70L1-CEA(576-669) fusion protein as compared with TLR4-deficient DCs pulsed with same fusion protein. In addition, TLR4 antagonist impaired induction of CEA-specific human Th1 immune response in a co-culture system of peripheral blood lymphocytes (PBLs) from HLA-A2.1(+) healthy donors and autologous DCs pulsed with Hsp70L1-CEA(576-669) in vitro. Taken together, these results demonstrate that TLR4 is a key receptor mediating the interaction of Hsp70L1 with DCs and subsequently enhancing the induction of Th1 immune response by Hsp70L1/antigen fusion protein.     

MO25alpha/beta interact with STRADalpha/beta enhancing their ability to bind, activate and localize LKB1 in the cytoplasm

Mutations in the LKB1 protein kinase result in the inherited Peutz Jeghers cancer syndrome. LKB1 has been implicated in regulating cell proliferation and polarity although little is known about how this enzyme is regulated. We recently showed that LKB1 is activated through its interaction with STRADalpha, a catalytically deficient pseudokinase. Here we show that endogenous LKB1-STRADalpha complex is associated with a protein of unknown function, termed MO25alpha, through the interaction of MO25alpha with the last three residues of STRADalpha. MO25alpha and STRADalpha anchor LKB1 in the cytoplasm, excluding it from the nucleus. Moreover, MO25alpha enhances the formation of the LKB1-STRADalpha complex in vivo, stimulating the catalytic activity of LKB1 approximately 10-fold. We demonstrate that the related STRADbeta and MO25beta isoforms are also able to stabilize LKB1 in an active complex and that it is possible to isolate complexes of LKB1 bound to STRAD and MO25 isoforms, in which the subunits are present in equimolar amounts. Our results indicate that MO25 may function as a scaffolding component of the LKB1-STRAD complex and plays a crucial role in regulating LKB1 activity and cellular localization.     

A single site (Ser16) phosphorylation in phospholamban is sufficient in mediating its maximal cardiac responses to beta -agonists

Phospholamban (PLB) can be phosphorylated at Ser(16) by cyclic AMP-dependent protein kinase and at Thr(17) by Ca(2+)-calmodulin-dependent protein kinase during beta-agonist stimulation. A previous study indicated that mutation of S16A in PLB resulted in lack of Thr(17) phosphorylation and attenuation of the beta-agonist stimulatory effects in perfused mouse hearts. To further delineate the functional interplay between dual-site PLB phosphorylation, we generated transgenic mice expressing the T17A mutant PLB in the cardiac compartment of the null background. Lines expressing similar levels of T17A mutant, S16A mutant, or wild-type PLB in the null background were characterized in parallel. Cardiac myocyte basal mechanics and Ca(2+) kinetics were similar among the three groups. Isoproterenol stimulation was associated with phosphorylation of both Ser(16) and Thr(17) in wild-type PLB and Ser(16) phosphorylation in T17A mutant PLB, whereas there was no detectable phosphorylation of S16A mutant PLB. Phosphorylation of Ser(16) alone in T17A mutant PLB resulted in responses of the mechanical and Ca(2+) kinetic parameters to isoproterenol similar to those in wild-type myocytes, which exhibited dual-site PLB phosphorylation. However, those parameters were significantly attenuated in the S16A mutant myocytes. Thus, Ser(16) in PLB can be phosphorylated independently of Thr(17) in vivo, and phosphorylation of Ser(16) is sufficient for mediating the maximal cardiac responses to beta-adrenergic stimulation.     

ERK1 and ERK2 activation by chemotactic factors in human eosinophils is interleukin 5-dependent and contributes to leukotriene C(4) biosynthesis

Eosinophils, the major immune effector cells contributing to allergic inflammation and asthma, are profoundly affected by interleukin (IL) 5 with respect to their differentiation, viability, recruitment, and cytotoxic effector functions. IL-5 enhances eosinophil responsiveness to a variety of chemotactic factors via a process called priming, although the molecular mechanism is unknown. In this study, we report that, following IL-5 priming of eosinophils, chemotactic agents including fMet-Leu-Phe, IL-8, and RANTES, promote vigorous transient activation of ERK1 and ERK2. In contrast, these chemotactic factors stimulate weak or indiscernible ERK activation in unprimed eosinophils. Furthermore, this intracellular marker of priming is selective for IL-5-related cytokines, in that it is observed following exposure to IL-5 and granulocyte macrophage-colony stimulating factor but not to interferon-gamma, stem cell factor, tumor necrosis factor alpha, or IL-4. Interestingly, priming of chemoattractant-induced ERK activation is accompanied by an increase in association of tyrosine-phosphorylated proteins with the adapter protein Grb2. The biological relevance of ERK activation to IL-5 priming is supported by the observation that inhibition of ERK activity by treatment with the MEK inhibitors PD98059 or U0126 inhibited the release of leukotriene C(4) stimulated by fMet-Leu-Phe in IL-5-primed eosinophils. These data provide evidence for a previously undescribed fundamental mechanism by which stimulation of IL-5 family receptors induces a rapid phenotypic alteration in the signal transduction pathways of chemotactic receptors, enabling their activation of the ERK1 and ERK2 pathway and contributing to the capacity of these cells to synthesize LTC(4).     

Inhibition of lipid phosphate phosphatase activity by VPC32183 suppresses the ability of diacylglycerol pyrophosphate to activate ERK(1/2) MAP kinases

The lipidic metabolite, diacylglycerol pyrophosphate (DGPP), in its dioctanoyl form (DGPP 8:0), has been described as an antagonist for mammalian lysophosphatidic acid (LPA) receptors LPA1 and LPA3. In this study we show that DGPP 8:0 does not antagonize LPA dependent activation of ERK(1/2) MAP kinases but strongly stimulated them in various mammalian cell lines. LPA and DGPP 8:0 stimulation of ERK(1/2) occurred through different pathways. The DGPP 8:0 effect appeared to be dependent on PKC, Raf and MEK but was insensitive to pertussis toxin and did not involve G protein activation. Finally we showed that DGPP 8:0 effect on ERK(1/2) was dependent on its dephosphorylation by a phosphatase activity sharing lipid phosphate phosphatase properties. The inhibition of this phosphatase activity by VPC32183, a previously characterized LPA receptor antagonist, blocked the DGPP 8:0 effect on ERK(1/2) activation. Moreover, down-regulation of lipid phosphate phosphatase 1 (LPP1) expression by RNA interference technique also reduced DGPP 8:0-induced ERK(1/2) activation. Consistently, over expression of LPP1 in HEK293 cells increases DGPP 8:0 hydrolysis and this increased activity was inhibited by VPC32183. In conclusion, DGPP 8:0 does not exert its effect by acting on a G protein coupled receptor, but through its dephosphorylation by LPP1, generating dioctanoyl phosphatidic acid which in turn activates PKC. These results suggest that LPP1 could have a positive regulatory function on cellular signaling processes such as ERK(1/2) activation.     

Phosphorylation dynamics of eukaryotic initiation factor 4E binding protein 1 (4E-BP1) is discordant with its potential to interact with eukaryotic initiation factor 4E (eIF4E)

Mammalian target of rapamycin (mTOR) controls cellular growth and proliferation by virtue of its ability to regulate protein translation. Eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1) — a key mTOR substrate, binds and sequesters eIF4E to impede translation initiation that is supposedly overcome upon 4E-BP1 phosphorylation by mTOR. Ambiguity surrounding the precise identity of mTOR regulated sites in 4E-BP1 and their invariable resistance to mTOR inactivation raises concerns about phospho-regulated model proposed for 4E:4E-BP1 interaction. Our attempt to mimic dephosphorylation associated with rapamycin response by introducing phospho deficient mutants for sites implicated in regulating 4E:4E-BP1 interaction individually or globally highlighted no obvious difference in the quantum of their association with CAP bound 4E when compared with their phosphomimicked counterparts or the wild type 4E-BP1. TOS or RAIP motif deletion variants compromised for raptor binding and resultant phosphodeficiency did little to influence their association with CAP bound 4E. Interestingly ectopic expression of ribosomal protein S6 kinase 1 (S6K1) that restored 4E-BP1 sensitivity to rapamycin/Torin reflected by instant loss of 4E-BP1 phosphorylation, failed to bring about any obvious change in 4E:4E-BP1 stoichiometry. Our data clearly demonstrate a potential disconnect between rapamycin response of 4E-BP1 and its association with CAP bound 4E.     

Functional specificity of the Xenopus T-domain protein Brachyury is conferred by its ability to interact with Smad1

Members of the T-box gene family play important and diverse roles in development and disease. Here, we study the functional specificities of the Xenopus T-domain proteins Xbra and VegT, which differ in their abilities to induce gene expression in prospective ectodermal tissue. In particular, VegT induces strong expression of goosecoid whereas Xbra cannot. Our results indicate that Xbra is unable to induce goosecoid because it directly activates expression of Xom, a repressor of goosecoid that acts downstream of BMP signaling. We show that the inability of Xbra to induce goosecoid is imposed by an N-terminal domain that interacts with the C-terminal MH2 domain of Smad1, a component of the BMP signal transduction pathway. Interference with this interaction causes ectopic activation of goosecoid and anteriorization of the embryo. These findings suggest a mechanism by which individual T-domain proteins may interact with different partners to elicit a specific response.     

Mcm10 interacts with Rad4/Cut5(TopBP1) and its association with origins of DNA replication is dependent on Rad4/Cut5(TopBP1)

Initiation of DNA replication in eukaryotes is a highly conserved and ordered process involving the co-ordinated, stepwise association of distinct proteins at multiple origins of replication throughout the genome. Here, taking Schizosaccharomyces pombe as a model, the role of Rad4(TopBP1) in the assembly of the replication complex has been examined. Quantitative chromatin immunoprecipitation experiments confirm that Rad4(TopBP1) associates with origins of DNA replication and, in addition, demonstrate that the protein is not present within the active replisome. A direct interaction between Rad4(TopBP1) and Mcm10 is shown and this is reflected in the Rad4(TopBP1)-dependent origin association of Mcm10. Rad4(TopBP1) is also shown to interact with Sld2 and Sld3 and to be required for the stable origin association of these two proteins. Rad4(TopBP1) chromatin association at stalled replication forks was found to be dependent upon the checkpoint protein Rad9, which was not required for Rad4(TopBP1) origin association. Comparison of the levels of chromatin association at origins of replication and stalled replication forks and the differential requirement for Rad9 suggest functional differences for Rad4(TopBP1) at these distinct sites.     

Mad1’s ability to interact with Mad2 is essential to regulate and monitor meiotic synapsis in C. elegans

Meiotic homolog synapsis is essential to ensure accurate segregation of chromosomes during meiosis. In C. elegans, proper regulation of synapsis and a checkpoint that monitors synapsis relies on the spindle checkpoint components, Mad1 and Mad2, and Pairing Centers (PCs), cis-acting loci that interact with the nuclear envelope to mobilize chromosomes within the nucleus. Here, we test what specific functions of Mad1 and Mad2 are required to regulate and monitor synapsis. We find that a mutation that prevents Mad1’s localization to the nuclear periphery abolishes the synapsis checkpoint but has no effect on Mad2’s localization to the nuclear periphery or synapsis. By contrast, a mutation that prevents Mad1’s interaction with Mad2 abolishes the synapsis checkpoint, delays synapsis and fails to localize Mad2 to the nuclear periphery. These data indicate that Mad1’s primary role in regulating synapsis is through control of Mad2 and that Mad2 can bind other factors at the nuclear periphery. We also tested whether Mad2’s ability to adopt a specific conformation associated with its activity during spindle checkpoint function is required for its role in meiosis. A mutation that prevents Mad2 from adopting its active conformer fails to localize to the nuclear periphery, abolishes the synapsis checkpoint and exhibits substantial defects in meiotic synapsis. Thus, Mad2, and its regulation by Mad1, is an important regulator of meiotic synapsis in C. elegans.     

Bovine herpesvirus 5 (BHV-5) Us9 is essential for BHV-5 neuropathogenesis

Bovine herpesvirus 5 (BHV-5) is a neurovirulent alphaherpesvirus that causes fatal encephalitis in calves. In a rabbit model, the virus invades the central nervous system (CNS) anterogradely from the olfactory mucosa following intranasal infection. In addition to glycoproteins E and I (gE and gI, respectively), Us9 and its homologue in alphaherpesviruses are necessary for the viral anterograde spread from the presynaptic to postsynaptic neurons. The BHV-5 Us9 gene sequence was determined, and the predicted amino acid sequence of BHV-5 Us9 was compared with the corresponding Us9 sequences of BHV-1.1. Alignment results showed that they share 77% identity and 83% similarity. BHV-5 Us9 peptide-specific antibody recognized a doublet of 17- and 19-kDa protein bands in BHV-5-infected cell lysates and in purified virions. To determine the role of the BHV-5 Us9 gene in BHV-5 neuropathogenesis, a BHV-5 Us9 deletion recombinant was generated and its neurovirulence and neuroinvasive properties were compared with those of a Us9 rescue mutant of BHV-5 in a rabbit model. Following intranasal infection, the Us9 rescue mutant of BHV-5 displayed a wild-type level of neurovirulence and neural spread in the olfactory pathway, but the Us9 deletion mutant of BHV-5 was virtually avirulent and failed to invade the CNS. In the olfactory mucosa containing the olfactory receptor neurons, the Us9 deletion mutant virus replicated with an efficiency similar to that of the Us9 rescue mutant of BHV-5. However, the Us9 deletion mutant virus was not transported to the bulb. Confocal microscopy of the olfactory epithelium detected similar amounts of virus-specific antigens in the cell bodies of olfactory receptor neuron for both the viruses, but only the Us9 rescue mutant viral proteins were detected in the processes of the olfactory receptor neurons. When injected directly into the bulb, both viruses were equally neurovirulent, and they were transported retrogradely to areas connected to the bulb. Taken together, these results indicate that Us9 is essential for the anterograde spread of the virus from the olfactory mucosa to the bulb.