Proteolytic activation of protein kinase C-epsilon

Proteolysis of native protein kinase C-epsilon (PKC-epsilon) is shown to occur through tryptic attack at multiple sites within the PKC-epsilon V2/V3 domain. Following initial cleavage of PKC-epsilon with trypsin, the kinase activity using a synthetic peptide substrate was found to be lipid/phorbol-ester independent, as observed for other members of this kinase family. Interestingly, there is also an increase in the histone kinase activity, indicating that there is an influence of the regulatory domain of the enzyme on substrate specificity. This is discussed in the context of alternatively spliced PKC-epsilon mRNAs that are shown to be present in brain and lung tissues.     

TPA-induced activation of MAP kinase

Threonine and tyrosine residue phosphorylation of a 42 kDa protein identified as mitogen-activated protein kinase (MAP kinase) was stimulated in extracts from TPA-pretreated cells. It is further shown that TPA pretreatment leads to the enhancement of an activity that will induce reactivation of dephosphorylated/inactivated MAP kinase. This TPA-induced activity induces the threonine and tyrosine phosphorylation of p42 in extracts from unstimulated cells.     

Dynamin is required for the activation of mitogen-activated protein (MAP) kinase by MAP kinase kinase

Internalization of activated receptors from the plasma membrane has been implicated in the activation of mitogen-activated protein (MAP) kinase. However, the mechanism whereby membrane trafficking may regulate mitogenic signaling remains unclear. Here we report that dominant-negative dynamin (K44A), an inhibitor of endocytic vesicle formation, abrogates MAP kinase activation in response to epidermal growth factor, lysophosphatidic acid, and protein kinase C-activating phorbol ester. In contrast, dynamin-K44A does not affect the activation of Ras, Raf, and MAP kinase kinase (MEK) by either agonist. Through immunofluorescence and subcellular fractionation studies, we find that activated MEK is present both at the plasma membrane and in intracellular vesicles but not in the cytosol. Our findings suggest that dynamin-regulated endocytosis of activated MEK, rather than activated receptors, is a critical event in the MAP kinase activation cascade.     

Regulation of Drosophila p38 activation by specific MAP2 kinase and MAP3 kinase in response to different stimuli

The p38 mitogen-activated protein kinase (MAPK) signaling pathway plays an important role in cellular responses to inflammatory stimuli and environmental stress. Activation of p38 is mediated through phosphorylation by upstream MAPKK, which in turn is activated by MAPKKK. However, the mechanism of how different upstream MAP2Ks and MAP3Ks specifically contribute to p38 activation in response to different stimuli is still not clearly understood. By using double-stranded RNA-mediated interference (RNAi) in Drosophila cells, we demonstrate that D-MKK3 is a major MAP2K responsible for D-p38 activation by UV, heat shock, NaCl or peptiodglycan (PGN). Stimulation of UV and PGN activates D-p38 through D-MEKK1, heat shock-induced activation of D-p38 signals through both D-MEKK1 and D-ASK1. On the other hand, maximal activation of D-p38 by NaCl requires the expression of four MAP3Ks.     

Phosphatidylinositol 3-kinase contributes to Erk1/Erk2 MAP kinase activation associated with hepatocyte growth factor-induced cell scattering

MAP kinase cascade-dependent responses were investigated during scattering of HepG2 human hepatoma cells stimulated by HGF or phorbol ester. Inhibition of phosphatidylinositol 3-kinase with LY294002 prevented completely the dissociation of cells. Inhibition of MAP kinase kinase (MEK) with PD98059 prevented the development of characteristic morphological changes associated with cell migration. EGF, which failed to induce cell scattering, caused a short-term increase in the phosphorylation of Erk1/Erk2 MAP kinases. On the contrary, HGF or phorbol ester stimulated the phosphorylation of MAP kinases for a long time. Experiments performed with LY294002 indicated that phosphatidylinositol 3-kinase contributed to the HGF-stimulated phosphorylation of Erk1/Erk2. This finding was confirmed by the demonstration that the MAP kinase cascade-dependent expression of a high-Mr (>300 kDa) protein pair appearing in the course of cell scattering was inhibited by LY294002 in HGF-induced cells but was not inhibited in phorbol ester-treated cells.     

Proteolytic signaling by TNFalpha: caspase activation and IkappaB degradation

Following binding its death receptor on the plasma membrane, tumor necrosis factor (TNF) induces the receptor trimerization and recruits a number of death domain-containing molecules to form the receptor complex. The complex promotes activation of downstream caspase cascade and induces degradation of IkappaBalpha. Caspases are activated using mechanisms of oligomeration and 'self-controlled proteolysis'. According to their structures and functions, apoptosis related caspases can be divided into upstream and downstream caspases. In general, upstream caspases cleave and activate downstream caspases by proteolysis of the Asp-X site. Activated caspases then cleaved target substrates. To date, more than 70 proteins have been identified to be substrates of caspases in mammalian cells. Caspases can alter the function of their target proteins by destroying structural components of the cytoskeleton and nuclear scaffold or by removing their regulatory domains. Activation of NF-kappaB is dependent on the degradation of IkappaBalpha. IkappaB kinase (IKK) phosphorylates IkappaBalpha at the residues 32 and 36 followed by polyubiquitination at lysine 21 and 22 and subsequent degradation of the molecules by 26S proteasome. There is extensive crosstalk between the apoptotic and NF-kappaB signaling pathways that emanate from TNF-R1. On the one hand, activation of NF-kappaB can inactivate caspases; on the other hand, activated caspases can inhibit the activation of NF-kappaB. Both processes involve in proteolysis. This crosstalk may be important for maintaining the balance between the two pathways and for determining whether a cell should live or die.     

Conserved docking site is essential for activation of mammalian MAP kinase kinases by specific MAP kinase kinase kinases

Mammalian mitogen-activated protein kinase (MAPK) cascades control various cellular events, ranging from cell growth to apoptosis, in response to external stimuli. A conserved docking site, termed DVD, is found in the mammalian MAP kinase kinases (MAPKKs) belonging to the three major subfamilies, namely MEK1, MKK4/7, and MKK3/6. The DVD sites bind to their specific upstream MAP kinase kinase kinases (MAPKKKs), including MTK1 (MEKK4), ASK1, TAK1, TAO2, MEKK1, and Raf-1. DVD site is a stretch of about 20 amino acids immediately on the C-terminal side of the MAPKK catalytic domain. Mutations in the DVD site strongly inhibited MAPKKs from binding to, and being activated by, their specific MAPKKKs, both in vitro and in vivo. DVD site mutants could not be activated by various external stimuli in vivo. Synthetic DVD oligopeptides inhibited specific MAPKK activation, both in vitro and in vivo, demonstrating the critical importance of the DVD docking in MAPK signaling.     

MAP kinase activation in cells exposed to a 60 Hz electromagnetic field

This research provides evidence that mitogen-activated protein kinase or extracellular signal-regulated kinase (MAPK/ERK) is activated in HL-60 human leukemia cells, MCF-7 human breast cancer cells, and rat fibroblast 3Y1 cells exposed to a 60 Hertz (Hz), 1 Gauss (G) electromagnetic field (EMF). The effects of EMF exposure were compared to those observed using 12-O-tetradecanoylphorbal-13-acetate (TPA) treatment. The level of MAPK activation in cells exposed to EMF was approximately equivalent to that in cells treated with 0.1-0.5 ng/ml of TPA. A role for protein kinase C (PKC) in the process leading to MAPK activation in EMF exposed cells is also suggested by the results. MAPK activation is negated by an inhibitor to PKCalpha, but not PKCdelta inhibitors, in cells subjected to EMF exposure or TPA treatment. Thus, similarities between the effects of EMF exposure and TPA treatment are supported by this investigation. This provides a possible method for revealing other participants in EMF-cell interaction, since the TPA induction pathway is well documented.     

Resistance to signal activation governs design features of the MAP kinase signaling module

Given its broad influence over numerous cell functions, redesigning the mitogen-activated protein (MAP) kinase signaling module would offer a powerful means to engineer cell behavior. Early challenges include identifying quantitative module features most relevant to biological function and developing simple design rules to predictably modify these features. This computational study delineates how features such as signal amplification, input potency, and dynamic range of output may be tuned by manipulating chief module components. Importantly, the model construction identifies a metric of resistance to signal activation that quantitatively predicts module features and design trade-offs for broad perturbations in kinase and phosphatase expression. Its predictive utility extends to dynamic properties such as signal lifetime, which often dictates MAP kinase effect on cell function. Taken together, we propose that predictably altering MAP kinase signaling by tuning resistance is not only a feasible engineering strategy, but also one exploited by natural systems to allow each MAP kinase to exert pleiotropic effects in a context-dependent manner. External stimuli not only activate kinases, but also alter phosphatase expression and activity, thereby reconfiguring a single module for quantitatively distinct modes of signaling such as transient vs. sustained dynamics, each with unique effects on cell function.     

The Ebola virus soluble glycoprotein contributes to viral pathogenesis by activating the MAP kinase signaling pathway

Ebola virus (EBOV) expresses three different glycoproteins (GPs) from its GP gene. The primary product, soluble GP (sGP), is secreted in abundance during infection. EBOV sGP has been discussed as a potential pathogenicity factor, however, little is known regarding its functional role. Here, we analyzed the role of sGP in vitro and in vivo. We show that EBOV sGP has two different functions that contribute to infectivity in tissue culture. EBOV sGP increases the uptake of virus particles into late endosomes in HEK293 cells, and it activates the mitogen-activated protein kinase (MAPK) signaling pathway leading to increased viral replication in Huh7 cells. Furthermore, we analyzed the role of EBOV sGP on pathogenicity using a well-established mouse model. We found an sGP-dependent significant titer increase of EBOV in the liver of infected animals. These results provide new mechanistic insights into EBOV pathogenicity and highlight EBOV sGP as a possible therapeutic target.     

Proteolytic activation of ETK/Bmx tyrosine kinase by caspases

Etk/Bmx is a member of the Btk/Tec family of kinases, which are characterized by having a pleckstrin homology domain at the N terminus, in addition to the Src homology 3 (SH3), SH2, and the catalytic domains, shared with the Src family kinases. Etk, or Btk kinases in general, has been implicated in the regulation of apoptosis. To test whether Etk is the substrate for caspases during apoptosis, in vitro translated [(35)S]methionine-labeled Etk was incubated with different apoptotic extracts and recombinant caspases, respectively. Results showed that Etk was proteolyzed in all conditions tested with identical cleavage patterns. Caspase-mediated cleavage of Etk generated a C-terminal fragment, containing the complete SH2 and tyrosine kinase domains, but without intact pleckstrin homology and SH3 domains. This fragment has 4-fold higher kinase activity than that of the full-length Etk. Ectopic expression of the C-terminal fragment of Etk sensitized the PC3 prostate cancer cells to apoptosis in response to apoptosis-inducing stimuli. The finding, together with an earlier report that Etk is potentially antiapoptotic, suggests that Etk may serve as an apoptotic switch, depending on the forms of Etk existing inside the cells. To our knowledge, this is the first case where the activity of a tyrosine kinase is induced by caspase cleavage.     

Overexpression of Arabidopsis MAP kinase kinase 7 leads to activation of plant basal and systemic acquired resistance

There is a growing body of evidence indicating that mitogen-activated protein kinase (MAPK) cascades are involved in plant defense responses. Analysis of the completed Arabidopsis thaliana genome sequence has revealed the existence of 20 MAPKs, 10 MAPKKs and 60 MAPKKKs, implying a high level of complexity in MAPK signaling pathways, and making the assignment of gene functions difficult. The MAP kinase kinase 7 (MKK7) gene of Arabidopsis has previously been shown to negatively regulate polar auxin transport. Here we provide evidence that MKK7 positively regulates plant basal and systemic acquired resistance (SAR). The activation-tagged bud1 mutant, in which the expression of MKK7 is increased, accumulates elevated levels of salicylic acid (SA), exhibits constitutive pathogenesis-related (PR) gene expression, and displays enhanced resistance to both Pseudomonas syringae pv. maculicola (Psm) ES4326 and Hyaloperonospora parasitica Noco2. Both PR gene expression and disease resistance of the bud1 plants depend on SA, and partially depend on NPR1. We demonstrate that the constitutive defense response in bud1 plants is a result of the increased expression of MKK7, and requires the kinase activity of the MKK7 protein. We found that expression of the MKK7 gene in wild-type plants is induced by pathogen infection. Reducing mRNA levels of MKK7 by antisense RNA expression not only compromises basal resistance, but also blocks the induction of SAR. Intriguingly, ectopic expression of MKK7 in local tissues induces PR gene expression and resistance to Psm ES4326 in systemic tissues, indicating that activation of MKK7 is sufficient for generating the mobile signal of SAR.     

Calmodulin-dependent activation of MAP kinase for ROS homeostasis in Arabidopsis

Rapid recognition and signal transduction of mechanical wounding through various signaling molecules, including calcium (Ca2+), protein phosphorylation, and reactive oxygen species (ROS), are necessary early events leading to stress resistance in plants. Here we report that an Arabidopsis mitogen-activated protein kinase 8 (MPK8) connects protein phosphorylation, Ca2+, and ROS in the wound-signaling pathway. MPK8 is activated through mechanical wounding, and this activation requires direct binding of calmodulins (CaMs) in a Ca2+-dependent manner. MPK8 is also phosphorylated and activated by a MAPKK MKK3 in the prototypic kinase cascade, and full activation of MPK8 needs both CaMs and MKK3 in planta. The MPK8 pathway negatively regulates ROS accumulation through controlling expression of the Rboh D gene. These findings suggest that two major activation modes in eukaryotes, Ca2+/CaMs and the MAP kinase phosphorylation cascade, converge at MPK8 to monitor or maintain an essential part of ROS homeostasis.     

Opioid receptor contributes to ischemic preconditioning through protein kinase C activation in rabbits

Recent studies have reported that protection from ischemic preconditioning (PC) is blocked by the opioid receptor antagonist naloxone (NAL). We tested whether an opioid agonist could mimic PC in the rabbit heart, whether that protection involved protein kinase C (PKC) activation, and whether opioid receptors act in concert with other PKC-coupled receptors. Rabbit hearts were subjected to 30min coronary occlusions and were reperfused for either 3 (in situ) or 2 (in vitro) h. Infarct size was determined by staining with triphenyltetrazolium chloride. In untreated in situ hearts 38.5 ± 1.6% of the risk zone infarcted. PC with 5 min ischemia/10 min reperfusion significantly limited infarction to 12.7 ± 2.9% (p < 0.01). NAL infusion did not modify infarction (39.6 ± 1.6%) in non-PC hearts, but blocked the effect of one cycle of PC (34.4 ± 3.6% infarction). NAL, however, could not block cardioprotection when PC was amplified with 3 cycles of ischemia/reperfusion (9.9 ± 1.4% infarction, p < 0.01 vs. control). Morphine could also mimic ischemic preconditioning, but only at a dose much higher than would be used clinically (3 mg/kg). In isolated hearts pretreatment with morphine (0.3 M) significantly limited infarction to 9.3 ± 1.2% (p < 0.01 vs. 32.0 ± 3.1% in controls). This cardioprotective effect of morphine could be blocked by either the PKC inhibitor chelerythrine (30.4 ± 2.6% infarction) or NAL (34.0 ± 2.6% infarction). Neither chelerythrine nor NAL by itself modified infarction in non-PC hearts. NAL could not block protection from one cycle of PC in isolated hearts indicating that an intact innervation may be required for endogenous opioid production. Thus, opioid receptors, like other PKC-coupled receptors, participate in the triggering PC in the rabbit heart.     

Dissociation of MAP kinase activation and MPF activation in hormone-stimulated maturation of Xenopus oocytes.

MAP kinase activation occurs during meiotic maturation of oocytes from all animals, but the requirement for MAP kinase activation in reinitiation of meiosis appears to vary between different classes. In particular, it has become accepted that MAP kinase activation is necessary for progesterone-stimulated meiotic maturation of Xenopus oocytes, while this is clearly not the case in other systems. In this paper, we demonstrate that MAP kinase activation in Xenopus oocytes is an early response to progesterone and can be temporally dissociated from MPF activation. We show that MAP kinase activation can be suppressed by treatment with geldanamycin or by overexpression of the MAP kinase phosphatase Pyst1. A transient and low-level early activation of MAP kinase increases the efficiency of cell cycle activation later on, when MAP kinase activity is no longer essential. Many oocytes can still undergo reinitiation of meiosis in the absence of active MAP kinase. Suppression of MAP kinase activation does not affect the formation or activation of Cdc2-cyclin B complexes, but reduces the level of active Cdc2 kinase. We discuss these findings in the context of a universal mechanism for meiotic maturation in oocytes throughout the animal kingdom.     

P2X7 receptor mediated phosphorylation of p38MAP kinase in the hippocampus

This study was designed to explore the effect of P2X7 receptor (P2X7R) activation on the expression of p38 MAP kinase (p38 MAPK) enzyme in hippocampal slices of wild-type (WT) and P2X7R^−/− mice using the Western blot technique and to clarify its role in P2X7 receptor mediated [³H]glutamate release. ATP (1 mM) and the P2X7R agonist BzATP (100 μM) significantly increased p38 MAPK phosphorylation in WT mice, and these effects were absent in the hippocampal slices of P2X7R^−/− mice. Both ATP- and BzATP-induced p38 MAPK phosphorylations were sensitive to the p38 MAP kinase inhibitor, SB203580 (1 μM). ATP elicited [³H]glutamate release from hippocampal slices, which was significantly attenuated by SB203580 (1 μM) but not by the extracellular signal-regulated kinase (ERK1/2) inhibitor, PD098095 (10 μM). Consequently, we suggest that P2X7Rs and p38 MAPK are involved in the stimulatory effect of ATP on glutamate release in the hippocampal slices of WT mice.