Evidence that C-terminal non-kinase domain of Pbs2p has a role in high osmolarity-induced nuclear localization of Hog1p

Mitogen-activated protein kinase (MAPK) cascade is a ubiquitous signaling module that transmits extracellular stimuli through the cytoplasm to the nucleus. In baker's yeast external high osmolarity activates high osmolarity glycerol (HOG) MAPK pathway which consists of two upstream branches (SHO1 and SLN1) and common downstream elements Pbs2p MAPKK and Hog1p MAPK. Activation of this pathway causes rapid nuclear accumulation of Hog1p, essentially leading to the expression of target genes. Previously we have isolated a PBS2 homologue (DPBS2) from osmo-tolerant and salt-tolerant yeast Debaryomyces hansenii that partially complemented pbs2 mutation in Saccharomyces cerevisiae. Here we show that by replacing C-terminal region of Dpbs2p with the homologous region of Pbs2p we could abrogate partial complementation exhibited by Dpbs2p and this was achieved due to increase in nuclear translocation of Hog1p. Thus, our result showed that in HOG pathway, MAPKK has important role in nuclear translocation of Hog1p.     

p38 MAPK inhibition is critically involved in VEGFR-2-mediated endothelial cell survival

Vascular endothelial growth factor (VEGF) promotes vasculogenesis, arteriogenesis, and angiogenesis by stimulating proliferation, migration, and cell survival of endothelial cells. VEGF mediates its actions through activation of two receptor tyrosine kinases, VEGFR-1 and VEGFR-2. Serum starvation led to apoptosis of human umbilical vein endothelial cells (HUVEC), which was accompanied by activation of p38 MAPK and caspase-3. Stimulation of both VEGF-receptors resulted in a considerable decrease of apoptosis, which was associated with the inhibition of p38 MAPK and caspase-3 activity. Selective stimulation of VEGFR-2 showed similar results, whereas the isolated activation of VEGFR-1 was without effect. Incubation of HUVEC with SB203580, a p38 MAPK inhibitor, resulted in similar effects as VEGF-stimulation: p38 MAPK and caspase-3 enzyme activity were reduced and apoptosis was prevented. These data indicate that activation of VEGFR-2 prevents endothelial cell apoptosis by inhibiting p38 MAPK phosphorylation and thus, reducing caspase-3 activity.     

NeuA O-acetylesterase activity is specific for CMP-activated O-acetyl sialic acid in Streptococcus suis serotype 2

The two ubiquitously expressed sphingosine kinases (SphK) 1 and 2 are key regulators of the sphingolipid signaling pathway. Despite the formation of an identical messenger, i.e. sphingosine 1-phosphate (S1P), they exert strikingly different functions. Particularly, SphK2 is necessary for the phosphorylation of the sphingosine analog fingolimod (FTY720), which is protective in rodent stroke models. Using gene deficient mice lacking either SphK1 or SphK2, we investigated the role of the two lipid kinases in experimental stroke.We performed 2 h transient middle cerebral artery occlusion (tMCAO) and analyzed lesion size and neurological function after 24 h. Treatment groups received 1 mg/kg FTY720. Neutrophil infiltration, microglia activation, mRNA and protein expression of SphK1, SphK2 and the S1P₁ receptor after tMCAO were studied.Genetic deletion of SphK2 but not SphK1 increased ischemic lesion size and worsened neurological function after tMCAO. The protective effect of FTY720 was conserved in SphK1^−/− mice but not in SphK2^−/− mice.This suggests that SphK2 activity is an important endogenous protective mechanism in cerebral ischemia and corroborates that the protective effect of FTY720 is mediated via phospho-FTY720.     

ERK1/2 and p38-MAPK signalling pathways, through MSK1, are involved in NF-kappaB transactivation during oxidative stress in skeletal myoblasts

Skeletal muscle is highly adapted to respond to oxidative imbalances, since it is continuously subjected to an increased production of reactive oxygen species (ROS) during exercise. Oxidative stress, however, has been associated with skeletal muscle atrophy and damage in many diseases. In this study, we examined whether MAPK and NF-κB pathways participate in the response of skeletal myoblasts to oxidative stress, and whether there is a cross talk between these pathways. H₂O₂ induced a strong activation of ERKs, JNKs and p38-MAPK in a time- and dose-dependent profile. ERK and JNK activation by H₂O₂, but not that of p38-MAPK, was mediated by Src kinase and, at least in part, by EGFR. H₂O₂ also stimulated a mild translocation of NF-κB to the nucleus, as well as a moderate phosphorylation of its endogenous cytoplasmic inhibitor IκB (at Ser32/36), without any significant decrease in IκB total levels. Moreover, oxidative stress induced a strong phosphorylation of NF-κB p65 subunit at Ser536 and Ser276. Inhibition of MAPK pathways by selective inhibitors did not appear to affect H₂O₂-induced nuclear translocation of NF-κB or the phosphorylation of IκB. In contrast, phosphorylation of p65 at Ser276 was found to be mediated by MSK1, a substrate of both ERKs and p38-MAPK. In conclusion, it seems that, during oxidative stress, NF-κB translocation to the nucleus is most likely not related with the MAPK activation, while p65 phosphorylations are in part mediated by MAPKs pathways, probably modifying signal specificity.     

Phosphatidylcholine transfer activity of yeast Sec14p is not essential for its function in vivo

Yeast phosphatidylinositol (PI)/phosphatidylcholine (PC) transfer protein, Sec14p, is essential for protein transport from the Golgi apparatus and for the cell viability. It is instrumental in maintaining the lipid composition of the Golgi membranes to be compatible with vesicle biogenesis and the secretory process by coordination of PC and PI metabolism. To address the question to which extent PC transfer ability of Sec14p is required for its essential in vivo function we generated a Sec14p mutant unable to transfer PC between membranes in the in vitro assay. Yeast cells with this modified Sec14p(D115G) as a sole Sec14p were viable with improved secretory activity compared to sec14 deficient strain. Thus, in vitro PC transfer ability of Sec14p is not required for its essential function(s) in living cells, however, yeast cells having PC transfer deficient Sec14p(D115G) as a sole Sec14p display regulatory abnormalities, including increased phospholipase D mediated PC turnover.     

Evidence that the actin site is impaired by Ca2+-activated degradation of the heavy chain of dystrophic myosin

At pathophysiological concentrations of Ca²⁺, the heavy chain of dystrophic myosin was degraded by an endogenous protease. This was not the case for normal myosin. However, normal myosin was a substrate of Ca²⁺-activated neutral protease (CAF) from platelets. This indicated that the endogenous protease in preps of dystrophic myosin was CAF. The pathophysiological effect of heavy chain degradation was restricted to the actin site. Under Vmax conditions hydrolytic activities remained within the normal range, whereas the Kapp of actin for myosin increased 3-fold following extensive heavy chain degradation of dystrophic myosin. Removal of those heavy chain fragments which were soluble at low inoic strength restored Kapp to normal levels.     

A phosphorylation site in the Toll-like receptor 5 TIR domain is required for inflammatory signalling in response to flagellin

Flagellin, the major structural subunit of bacterial flagella, potently induces inflammatory responses in mammalian cells by activating Toll-like receptor (TLR) 5. Like other TLRs, TLR5 recruits signalling molecules to its intracellular TIR domain, leading to inflammatory responses. Phosphatidylinositol 3-kinase (PI3K) has been reported to play a role in early TLR signalling. We identified a putative binding site for PI3K at tyrosine 798 in the TLR5 TIR domain, at a site analogous to the PI3K recruitment domain in the interleukin-1 receptor. Mutation of this residue did not affect homodimerization, but prevented inflammatory responses to flagellin. While we did not detect direct interaction of PI3K with TLR5, we demonstrated by mass spectrometry that Y798 is phosphorylated in flagellin-treated HEK 293T cells. Together, these results suggest that phosphorylation of Y798 in TLR5 is required for signalling, but not for TLR5 dimerization.     

Yeast adaptor protein, Nbp2p, is conserved regulator of fungal Ptc1p phosphatases and is involved in multiple signaling pathways

Nbp2p is an Src homology 3 (SH3) domain-containing yeast protein that is involved in a variety of cellular processes. This small adaptor protein binds to a number of different proteins through its SH3 domain, and a region N-terminal to the SH3 domain binds to the protein phosphatase, Ptc1p. Despite its involvement in a large number of physical and genetic interactions, the only well characterized function of Nbp2p is to recruit Ptc1p to the high osmolarity glycerol pathway, which results in down-regulation of this pathway. In this study, we have discovered that Nbp2p orthologues exist in all Ascomycete and Basidiomycete fungal genomes and that all possess an SH3 domain and a conserved novel Ptc1p binding motif. The ubiquitous occurrence of these two features, which we have shown are both critical for Nbp2p function in Saccharomyces cerevisiae, implies that a conserved role of Nbp2p in all of these fungal species is the targeting of Ptc1p to proteins recognized by the SH3 domain. We also show that in a manner analogous to its role in the high osmolarity glycerol pathway, Nbp2p functions in the down-regulation of the cell wall integrity pathway through SH3 domain-mediated interaction with Bck1p, a component kinase of this pathway. Based on functional studies on the Schizosaccharomyces pombe and Neurospora crassa Nbp2p orthologues and the high conservation of the Nbp2p binding site in Bck1p orthologues, this function of Nbp2p appears to be conserved across Ascomycetes. Our results also clearly imply a function for the Nbp2p-Ptc1p complex other cellular processes.     

Pbcrk-1, the Plasmodium berghei orthologue of P. falciparum cdc-2 related kinase-1 (Pfcrk-1), is essential for completion of the intraerythrocytic asexual cycle

The molecular mechanisms underlying gametocytogenesis in malaria parasites are not understood. Plasmodium falciparum cdc2-related kinase 1 (pfcrk-1), a gene that is expressed predominantly in gametocytes, bears homology to the PITSLRE subfamily of cyclin-dependent kinases and has been hypothesized to function as a negative regulator of the cell cycle. We attempted to knock-out pbcrk-1, the P. berghei orthologue of pfcrk-1, but were unable to recover P. berghei parasites with a disrupted pbcrk-1 locus. In contrast, an integration event at this locus that did not result in a loss-of-function of the pbcrk-1 gene was readily observed. This strongly suggests that a functional pbcrk-1 gene product is essential to intraerythrocytic asexual multiplication.     

The Kex2p proregion is essential for the biosynthesis of an active enzyme and requires a C-terminal basic residue for its function

The Saccharomyces cerevisiae prohormone-processing enzyme Kex2p is biosynthesized as an inactive precursor extended by its N-terminal proregion. Here we show that deletion of the proregion renders Kex2p inactive both in vivo and in vitro. Absence of the proregion impaired glycosylation and stability and resulted in the retention of the enzyme in the endoplasmic reticulum. These phenotypes were partially complemented by expression of the proregion in trans. Trans complementation was specific to Kex2p proregion because expression of any of the seven mammalian prohormone convertase propeptides had no effect. These data are consistent with a model whereby Kex2p proregion functions as an intramolecular chaperone and indicate that covalent linkage to the protein is not an absolute requirement for proregion function. Furthermore, extensive mutagenesis revealed that, in addition to their function as proteolytic recognition sites, C-terminal basic residues play an active role in proregion-dependent Kex2p activation.     

Phosphatidyl ethanolamine is essential for targeting the arginine transporter Can1p to the plasma membrane of yeast

In continuation of our previous study, we show that phosphatidyl ethanolamine (PE) depletion affects, in addition to amino acid transporters, activities of at least two other proton motive force (pmf)-driven transporters (Ura4p and Mal6p). For Can1p, we demonstrate that the lack of PE results in a failure of the permease targeting to plasma membrane. Despite the pleiotropic effect of PE depletion, a specific role of PE in secretion of a defined group of permeases can be distinguished. Pmf-driven transporters are more sensitive to the lack of PE than other plasma membrane proteins.     

Evidence that the novobiocin-sensitive ATP-binding site of the heat shock protein 90 (hsp90) is necessary for its autophosphorylation

The 90kDa heat shock protein (Hsp90) is one of the most abundant protein and essential for all eukaryotic cells. Many proteins require the interaction with Hsp90 for proper function. Upon heat stress the expression level of Hsp90 is even enhanced. It is assumed, that under these conditions Hsp90 is required to protect other proteins from aggregation. One property of Hsp90 is its ability to undergo autophosphorylation. The N-terminal domain of Hsp90 has been shown to contain an unusual ATP-binding site. A well-known inhibitor of Hsp90 function is geldanamycin binding to the N-terminal ATP-binding site with high affinity. Recently it was shown that Hsp90 possesses a second ATP-binding site in the C-terminal region, which can be competed with novobiocin. Autophosphorylation of Hsp90 was analysed by incubation with gamma(32)P-ATP. Addition of geldanamycin did not interfere with the capability for autophosphorylation, while novobiocin indeed did. These results suggest that the C-terminal ATP-binding site is required for autophosphorylation of Hsp90.     

The C-terminus of PRK2/PKNgamma is required for optimal activation by RhoA in a GTP-dependent manner

PRK2/PKNγ is a Rho effector and a member of the protein kinase C superfamily of serine/threonine kinases. Here, we explore the structure-function relationship between various motifs in the C-terminal half of PRK2 and its kinase activity and regulation. We report that two threonine residues at conserved phosphoacceptor position in the activation loop and the turn motif are essential for the catalytic activity of PRK2, but the phosphomimetic Asp-978 at hydrophobic motif is dispensable for kinase catalytic competence. Moreover, the PRK2-Δ958 mutant with the turn motif truncated still interacts with 3-phosphoinositide-dependent kinase-1 (PDK-1). Thus, both the intact hydrophobic motif and the turn motif in PRK2 are dispensable for the binding of PDK-1. We also found that while the last seven amino acid residues at the C-terminus of PRK2 are not required for the activation of the kinase by RhoA in vitro, however, the extreme C-terminal segment is critical for the full activation of PRK2 by RhoA in cells in a GTP-dependent manner. Our data suggest that the extreme C-terminus of PRK2 may represent a potential drug target for effector-specific pharmacological intervention of Rho-medicated biological processes.