text:
Research Articles:
Microbial Cell, Vol. 5, No. 8, pp. 357 - 370; doi: 10.15698/mic2018.08.641
Snf1 cooperates with the CWI MAPK pathway to mediate the degradation of Med13 following oxidative stress
1 Department of Molecular Biology, Graduate School of Biomedical Sciences, Rowan University, Stratford, NJ, 08084, USA.
2 Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT 84602, USA.
3 Current address: Department of Biological Sciences, Rowan University, 201 Mullica Hill Rd, Glassboro, NJ 08028. USA.
4 Current address: Shawnee High School, Medford, New Jersey 08055, USA.
Keywords: cyclin C, Cdk8, Med13, SCFGrr1, AMPK, Snf1, ubiquitin mediated destruction, signal transduction, H2O2 stress, MAPK.
AMPK - 5' adenosine monophosphate-activated protein kinase,
CKM – cyclin C/Cdk8 kinase module,
CWI – cell wall integrity,
IDR – intrinsic disordered region,
MAPK - MAP kinase,
PCD - Programmed cell death,
ROS – reactive oxygen species,
Y2H – yeast two hybrid.
Received originally: 02/03/2018 Received in revised form: 29/05/2018
Accepted: 04/06/2018 Published: 25/06/2018
Katrina F. Cooper, Tel: (856)-566-2887, Fax: (856)-566-6366; firstname.lastname@example.com
Conflict of interest statement: The authors declare no conflict of interest.
Please cite this article as: Stephen D. Willis, David C. Stieg, Kai Li Ong, Ravina Shah, Alexandra K. Strich, Julianne H. Grose and Katrina F. Cooper (2018). Snf1 cooperates with the CWI MAPK pathway to mediate the degradation of Med13 following oxidative stress. Microbial Cell: 5(8): 357-370. doi: 10.15698/mic2018.08.641
Eukaryotic cells, when faced with unfavorable environmental conditions, mount either pro-survival or pro-death programs. The conserved cyclin C-Cdk8 kinase plays a key role in this decision. Both are members of the Cdk8 kinase module that, along with Med12 and Med13, associate with the core Mediator complex of RNA polymerase II. In Saccharomyces cerevisiae, oxidative stress triggers Med13 destruction, which releases cyclin C into the cytoplasm to promote mitochondrial fission and programmed cell death. The SCFGrr1 ubiquitin ligase mediates Med13 degradation dependent on the cell wall integrity pathway, MAPK Slt2. Here we show that the AMP kinase Snf1 activates a second SCFGrr1 responsive degron in Med13. Deletion of Snf1 resulted in nuclear retention of cyclin C and failure to induce mitochondrial fragmentation. This degron was able to confer oxidative-stress-induced destruction when fused to a heterologous protein in a Snf1 dependent manner. Although snf1∆ mutants failed to destroy Med13, deleting the degron did not prevent destruction. These results indicate that the control of Med13 degradation following H2O2 stress is complex, being controlled simultaneously by CWI and MAPK pathways.
All eukaryotic cells are continually exposed to changing environmental conditions. Consequently, they have evolved elaborate mechanisms to both sense damage and transmit this signal to the nucleus. The resulting response varies dependent upon the stress encountered but in gross terms cells have to decide whether to activate pro-survival or pro-death programs. Despite this being a critical decision point, what remains understudied are the molecular details of how cells decide their fate upon encountering unfavorable environments. Previous studies revealed that the conserved cyclin C protein plays a key role in this decision in response to increased environmental reactive oxygen species (ROS) [1][2][3][4][5][6][7][8][9]. Cyclin C, together with its kinase partner Cdk8, Med12 and Med13, form the Cdk8 Kinase Module (CKM) of the multi-subunit Mediator complex. This complex acts as an interface between DNA bound transcription factors and RNA polymerase II (RNAP-II [10][11][12]). When the CKM module is bound, it predominantly negatively regulates expression of a subset of stress response genes [1][13][14][15][16]. Following an increase in environmental ROS (induced by H2O2 treatment), this repression is relieved by the nuclear release of cyclin C to the cytoplasm [4][7][8]. Intriguingly, here cyclin C plays a second role directing stress-induced mitochondrial fission as well as promoting programmed cell death (PCD) [7][8]. Consistent with this function, cells lacking cyclin C are less able to execute stress-induced mitochondria fission and are more resistant to oxidative stress [3][7]. Taken together, these results argue that cyclin C nuclear release must be carefully controlled as it signals a commitment to PCD.
Our previous studies have revealed that a complex molecular mechanism controls cyclin C nuclear release. In response to environmental ROS, cyclin C is directly phosphorylated by Slt2, the MAP kinase of the Cell Wall Integrity (CWI) signal transduction pathway ([6] and Fig. 1A). This canonical pathway is characterized by a family of cell-surface sensors (Wsc1, Mid2 and Mtl1 [17]) that transmit the stress to a small G protein Rho1, which thereafter activates protein kinase C (Pkc1 [18][19]). Once activated, Pkc1 transmits the intracellular signal to the MAPK Slt2/Mpk1 [20] as well as to the pseudo-kinase Kdx1/Mlp1 via the MAPK module [21]. In addition to cyclin C [6], Slt2 directly phosphorylates two other transcription factors (Rlm1, Swi4/Swi6) that stimulate the expression of other stress response genes [21][22][23][24]. More recently, Slt2 has also been shown to regulate gene expression by phosphorylating tyrosine-1 of the RNAP II carboxy-terminal domain [25]. This event is associated with pause/termination processes in mammals [26]. Intriguingly, it is required for the loss of cyclin C and Cdk8 from target genes following stress in yeast [25].
FIGURE 1: (A) Cdk8 module regulation by the CWI MAPK pathway. H2O2 stimulates cell wall sensors Wsc1, Mtl1 and Mid2, leading to activation of Rho1 that in turn triggers the cell wall integrity (CWI) MAPK pathway by activating Protein Kinase C (Pkc1). Activation of this cascade triggers the MAPK, Slt2, to directly phosphorylate cyclin C, an event required for the 1st step towards its release from the nucleus. The second step requires Slt2 to directly phosphorylate Med13-degron742-844, which targets it for ubiquitin mediated degradation by SCFGrr1. Cyclin C-Cdk8 activity is needed to prime the degron before it is recognized by SCFGrr1 [9]. (B) Outline of the AMPK pathway in yeast. It remains unknown how this pathway is activated in response to H2O2 stress. The gray box represents the Snf1 kinase complex (see text for details).
In S. cerevisiae, Med13 destruction is required for cyclin C nuclear release [27]. Consistent with this, cyclin C is cytoplasmic in unstressed med13∆ and the mitochondria are predominantly fragmented [27]. More recently we identified SCFGrr1 as the E3 ligase that mediates Med13 destruction [9]. Like other SCF targets [28], recognition of this phospho-degron requires it first to be primed by one kinase (cyclin C-Cdk8) and then activated by another (Slt2). These studies also revealed that phosphorylation of cyclin C by Slt2 is required for the SCFGrr1 to recognize Med13 ([9] and Fig. 1A). Like the Slt2-degron, this domain lies within the large intrinsic disordered region (IDR) of Med13 (Fig. 2A). IDR's are defined by a continuous stretch of disordered promoting residues which can easily transition into more ordered states. These structural transitions afford IDR's great flexibility and as such these regions are known to play key roles in macro-molecular decisions especially those that influence signaling pathways [29][30].
FIGURE 2: Med13 contains two H2O2 stress responsive degrons. (A) Cartoon of the results from ProteinPredict® [31] analysis of yeast Med13. The amino and carboxyl terminal domains are structured and separated by a large intrinsic disordered region. The positions of the two degrons are indicated. (B) Yeast two hybrid analysis of degron571-650 and Grr1 derivatives. Yeast PJ69-4a cells harboring Med13-activating domain plasmid (pDS15) and either pAS2, pAS-Grr1 or pAS2-Grr1∆F∆L binding domain plasmids were grown on -LEU, -TRP drop out medium to select for both plasmids (left panel) and -TRP, -LEU, -HIS –ADE (right panel) to test for Med13-Grr1 interaction. (C) Wild-type (RSY10) or grr1∆ cells (RSY1770) harboring degron571-650 (pDS15) were treated with 0.4 mM H2O2 for the timepoints indicated and Med13571-650-HA levels analyzed by Western blot. Tub1 levels were used as loading controls. (D) Degradation kinetics of the degron571-650 constructs shown in C. Values represent averages ± SD from a total of at least two Western blots from two independent experiments.
In addition to Slt2, the highly conserved 5' adenosine monophosphate-activated protein kinase (AMPK), which plays a major role in the utilization of alternative carbon sources after glucose depletion [31], is also activated in response to various environmental stresses including oxidative stress [32]. In S. cerevisiae, the catalytic subunit of the heterotrimeric AMPK complex is encoded by SNF1. Other members of the complex (outlined in Fig. 1B) include two regulatory subunits, the γ subunit Snf4 and one of the three alternative β subunits, Sip1, Sip2, or Gal83 [31]. The three β isoforms determine the respective cellular addresses after activation of Snf1, with the Snf1-Gal83 isoform being enriched in the nucleus [33][34][35]. The catalytic activity of Snf1 is regulated by phosphorylation at Thr-210, which is located in the activation loop of its kinase domain [36]. This is executed by one of three upstream kinases, Sak1, Tos3, or Elm1 [32][37][38]. These, in turn, are activated by an unknown mechanism in response to a variety of stresses, which lends specificity to the system [32].
In this report we show that Snf1, Sak1 and at least one β isoform are required for the H2O2 induced degradation of Med13. Using yeast two-hybrid analysis, the Snf1-interacting domain on Med13 was identified. This domain lies in the large IDR of Med13 and is recognized by SCFGrr1 after Snf1 directed phosphorylation. Consistent with this, Snf1 is required for efficient cyclin C nuclear release following H2O2 stress. Taken together, this reveals that Med13 degradation is regulated by two SCFGrr1 degrons that are regulated by three different classes of kinases, a Cdk, a MAPK and an AMPK. As all three kinases are required for Med13 degradation, this complex molecular mechanism ensures that cyclin C nuclear release is tightly controlled and prevents its untimely release into the cytoplasm.
Med13 contains two SCFGrr1 phospho-degrons
We have previously shown that SCFGrr1 is the E3 ligase responsible for mediating Med13 degradation following H2O2 stress [9]. This degron (amino acids 742-844, Fig. 2A,) is primed by cyclin C-Cdk8 and activated by Slt2. In these studies we also observed that another Med13 domain (amino acids 571-650) can also bind to Grr1 using the Gal4 yeast two hybrid (Y2H) assay [39]. These results were repeated using two baits, wild-type Grr1 and a grr1∆F∆L mutant, which can neither bind to the SCF or recognize substrates [40][41]. As anticipated, the Gal4 activating domain Med13571-650 fusion protein (hereon out referred to as degron571-650) can associate with wild-type Grr1 as an interaction is detected selecting for the dual HIS3 and ADE2 reporter genes (Fig. 2B). The vector control and mutant bait were unable to grow on this media suggesting that the interaction is specific.
To confirm that degron571-650 is responsive to SCFGrr1 we examined its degradation, via Western blot analysis, following 0.4 mM H2O2 treatment in wild-type and grr1∆ cells. This concentration of H2O2 induces mitochondrial outer membrane permeabilization (MOMP) dependent regulated cell death response in yeast [42][43][44] and triggers cyclin C nuclear release [4][6][7][27]. The results show that although less degron571-650 is present in unstressed grr1∆ cells, the protein is more stable following H2O2 stress (Fig. 2C and quantified in 2D). Taken together with the Y2H data, these results argue that degron571-650 is a SCFGrr1 responsive degron. They are also consistent with our previous published work demonstrating that Grr1 is required for H2O2 induced degradation of Med13 [9].
Snf1 is required for stress-induced degradation of Med13
We next addressed if the H2O2 mediated destruction of degron571-650 required Cdk8 kinase activity. To execute this, the degradation of degron571-650 was monitored as described above, in cells deleted for cyclin C harboring either functional myc-tagged cyclin C or a vector control. The results revealed that degron571-650 is degraded with the same kinetics in the presence or absence of cyclin C (Fig. S1A and quantified in Fig. S1B). These results suggest that another kinase phosphorylates degron571-650. One candidate is the PAS kinase. This kinase is required for glucose homeostasis, and is encoded by two orthologs Psk1 and Psk2 [45]. Intriguingly, previous Y2H studies have revealed that a domain of Med13 (amino acids 505-703), that encompasses degron571-650, interacts with Psk1 [46]. The Y2H assay was repeated using Psk1 as bait and the smaller degron571-650 as prey and again an interaction was observed (Fig. S2A). We next tested if the PAS kinase plays a role in the degradation of full length Med13. Wild-type and psk1∆ psk2∆ cells harboring a functional HA-tagged Med13 plasmid [9] were treated with H2O2 and Med13 degradation was monitored by Western blot analysis. The results show that Med13 is degraded with similar kinetics in the mutant and wild-type cells (Fig. S2B and quantified in Fig. S2C). These results indicate that although the PAS kinase can interact with degron571-650, it is not required for Med13 degradation following H2O2 stress.
PAS kinase activation by carbon sources is dependent on the Snf1 complex [47][48]. Since Snf1 has many targets [49], we next tested if Snf1 mediates Med13 degradation following H2O2 stress as just described. The results show that Med13 is significantly more stable in snf1∆ cells (Fig. 3A and quantified in Fig. 3C). Similar results were obtained when the Snf1 kinase dead mutant (K84R, [50]) was the only source of Snf1 (Fig. 3B and quantified in Fig. 3D). Taken together, these results indicate that Snf1 activity is required for Med13 degradation following H2O2 stress.
Sak1 has been identified as the AMPK kinase that is activated in response to oxidative stress [32]. Therefore, we next addressed if Sak1 is required for H2O2 induced Med13 degradation. The degradation assays described above were repeated in sak1∆ cells and results revealed that Med13 was again significantly more stable in sak1∆ than wild-type cells (Fig. 3A and quantified in Fig. 3C). This result supports the previously proposed model that Sak1 is the AMPKK that activates Snf1 in response to oxidative stress [32]. We next addressed if the nuclear enriched isoform, Snf1-Gal83, [33][34][35][51], is required for Med13 degradation under similar circumstances. Unexpectedly, the results show that Med13 is still degraded in gal83∆ cells (Fig. S3A). Likewise, similar results were obtained when Med13 degradation was monitored in a sip1∆ sip2∆ strain (Fig. S3A). However, deletion of all three β subunits significantly inhibited the degradation of Med13 to a similar extend as observed in snf1∆ (Fig. 3A and quantified in Fig. 3C). Taken together, this suggests at least one of the β subunits of the Snf1 complex is required for Med13 degradation following H2O2 stress.
Snf1 activation alone is not sufficient to mediate Med13 degradation
We next addressed if Snf1 activation was sufficient to mediate Med13 degradation in the absence of H2O2 stress. To execute this Med13 levels were examined in wild-type cells after they had been switched from 2% to 0.05% glucose which, is sufficient to activate Snf1 (Fig. 4A and [32]). No differences in Med13 levels were observed following glucose deprivation (Fig. 4B). Taken with the results presented in Fig. 3, this suggests that Snf1 activation is necessary but not sufficient to mediate the degradation of Med13 following H2O2 treatment.
The CWI pathway sensors proteins are not required for Snf1 activation
It is known that both the CWI and AMPK pathways are activated in response to oxidative stress but how these two pathways communicate is not well understood [52]. Importantly, although it is known how the signal is transmitted to the CWI pathway, it is unclear how the AMPK pathway is activated (Fig. 1B). One possibility is that the pathways crosstalk through the cell wall sensor proteins. To test this, we examined Snf1 phosphorylation status in a strain lacking the three cell wall sensors (Wsc1, Mid2 and Mtl1, [17]). Consistent with previously published work [32], Snf1 is phosphorylated following H2O2 treatment in a Sak1 dependent manner (Fig. 4C). Likewise the cell wall sensor triple mutant is also able to activate Snf1 following H2O2 treatment (Fig. 4C). These results suggest that the activation of the CWI pathway is not required to transmit the stress signal to the AMPK pathway. Others have demonstrated that Snf1 is not required for activation of Slt2 [53]. Consistent with this, we observed that the Med13 degron571-650 is still degraded following H2O2 stress when a mutant of cyclin C, that cannot be phosphorylated by Slt2 (S266A, [6]), is used as the sole source of cyclin C (Fig. S1C and D). Taken together, these results argue that the AMPK and MAPK signaling pathways act independently of each other. The extension of this conclusion is that both pathways contribute independently to regulating Med13 destruction following H2O2 stress.
The Snf1 degron lies within the IDR of Med13
Snf1 has previously been shown to phosphorylate a SCFGrr1 phospho-degron in Pfk27, a key regulator of glycolysis [54]. Taking this into account, we asked if the Gal4 activating domain Med13571-650 fusion protein (degron571-650) is a Snf1 responsive degron by monitoring its H2O2 mediated degradation snf1∆ cells. The results show that degron571-650 is more stable in snf1∆ cells compared to wild type following H2O2 stress (Fig. 5A and quantified in Fig. 5B). This suggests that degron571-650 is a Snf1 responsive degron. To further support this conclusion, we examined the degradation kinetics of a degron mutated for either of the potential Snf1 phosphorylation sites previously identified in proteomic screens (S587 [55] and S636 [56], Fig.6A), although none of these sites perfectly fit the Snf1 consensus sequence [57][58]. The studies revealed that both mutants were degraded following H2O2 treatment with kinetics similar to wild type (Fig. S3B). We also noted that serine 634 could potentially be phosphorylated by Snf1 (see Fig. 6A). As it lies very close to serine 636, we made a double mutant (S634A, S636A) and repeated the assay. Again this mutant degron was degraded following H2O2 treatment (Fig. S3B). However, the triple mutant (S587A, S634A, S636A) exhibited enhanced stability following H2O2 stress (Fig. 5C and quantitated in Fig. 5B). Taken together, these results suggest that Snf1 phosphorylates Med13 following H2O2 stress targeting S587, S634 and S636. Consistent with this model, we (Fig. 5D and S3C) and others [59] have shown that Snf1 can co-immunoprecipitate with Cdk8 both before and after H2O2 stress. As cyclin C directly binds to the adjacent degron742-844 (Fig. 2A and [9]), this places Snf1 in close proximity to degron571-650. In addition, these data also support the notion suggested by others, that a subpopulation of Snf1 is nuclear in unstressed cells [33][51][59].
To address if Snf1 directly phosphorylates Med13, kinase assays were performed with wild-type and kinase dead Snf1 (K84R). The activated kinase was immunoprecipitated from yeast extracts and incubated with GST-Med13561-650, S608A purified from E. coli. Serine 608 was mutated to alanine as it potentially is contaminating PAS kinase site (Fig. 5E and [48]). In addition, although this site has also been identified as target of PKA mediated phosphorylation [60], we have previously shown that this site does not play a role in Med13 degradation in response to oxidative stress [9]. Lastly, it is documented that Snf1 does not phosphorylate GST, so this control was not included here [61]. The results (Fig. 5F) show that Snf1 is able to directly phosphorylate degron571-650. However, despite taking the precautions listed above, kinase activity was also observed using the kinase dead version. This suggests that another kinase that immunoprecipitates with Snf1 is able to phosphorylate this degron. One strong possibility is Cdk8, which is a proline directed kinase that can phosphorylate the minimal consensus sequence S/T-P [62]. Degron571-650 contains one such site (Fig. 5E). Therefore the kinase assays were repeated using Snf1 extracts isolated from a cdk8∆ strain. The results show that degron571-650 was phosphorylated by Snf1 and this activity was reduced in the kinase dead control (Fig. 5F). Taken together, these results are suggestive that Snf1 directly phosphorylates Med13571-650. However, as some phosphorylation of degron571-650 was observed when Snf1K84R was used, we cannot rule out the possibility that an intermediary kinase may be playing a role.
Other potential Snf1 sites on Med13 are not needed for its degradation following H2O2 stress
The Snf1 proteomic screen mentioned above also identified five additional potential Snf1 sites in Med13 [56]. Intriguingly, these sites all lie within the large IDR of Med13 (Fig. 6A). As IDR's provide ideal environments for post-translational modifications, which effect signaling events [63], we asked if these additional sites also play a role in Med13 degradation. To address this question, Med13-HA fragments were fused to the SV40 nuclear localization sequence (NLS) and assayed for H2O2 mediated destruction. The results revealed that the construct that contains both degrons as well as all the potential Snf1 sites (amino acids 306-906) is degraded. However, the Med13306-570 construct, that contains the remaining potential Snf1 sites but not the two SCFGrr1 responsive degrons, was significantly more stable (Fig. 6B). This suggests that these other potential Snf1 sites do not play a role in the H2O2-stress mediated degradation of Med13. To address if the head and tail regions of Med13 contain unidentified Snf1 sites, Med13 constructs spanning these regions (amino acids 1-306 and 907-1420) were also tested, as described above, for stability following H2O2 stress. The results (Fig. 6C) demonstrate that these constructs are not destroyed under these conditions. This supports our conclusion that the Snf1 degron lies within amino acids 571-650. Degron742-844 also contains a potential Snf1 target site (Fig. 6A). However, this degron, when fused to the Gal4 activating domain, is destroyed following H2O2 stress in wild-type and snf1∆ cells alike (Fig. 6D). Taken together, these results support the above conclusions that the Snf1 phosphorylation sites lie within degron571-650.
FIGURE 6: Other potential Snf1 sites in Med13 are not required for its degradation following H2O2 stress. (A) Map of Med13 outlying the positions of the two Med13 degrons, the consensus Snf1 target site [58] and potential Snf1 sites, identified by published proteomic screens. (B) and (C) Wild-type (RSY10) cultures harboring the NLS-Med13-HA constructs shown were grown to mid-log phase (0 h) then treated with 0.4 mM H2O2 for the indicated times. Med13-HA levels were determined by Western blot analysis. Tub1 levels were used as a loading control. (D) Mid-log wild type or snf1∆ cultures (RSY202) harboring HA tagged Med13 degron742-844 (pDS32) were subjected to an H2O2 timecourse experiment and protein extracts analyzed by Western blot. Tub1 levels were used as loading controls.
Snf1 is necessary for cyclin C nuclear release and stress-induced mitochondrial fission
As Med13 degradation is required for cyclin C nuclear release [9][27], we next tested if Snf1 was also required for this event. To address this, the location of a functionalcyclin C-YFP reporter protein [5] before and after stress in wild-type and snf1∆ cells was examined (Fig. 7A and quantified in 7B). The results show that a majority of cyclin C-YFP is cytoplasmic in wild-type cells, whereas it remains significantly more nuclear in snf1∆ cells (Fig. 7A and quantified in 7B). These data indicate that Snf1 is required for the oxidative stress induced nuclear release of cyclin C. As cyclin C nuclear release initiates stress-induced mitochondrial fission [4][7][8], mitochondrial morphology in snf1∆ cells was examined. As previously reported, unstressed wild-type cells exhibited mainly reticular mitochondrial morphology (Fig. 7C) that switched to a predominantly fragmented phenotype after 3 hours of 0.4 mM H2O2 treatment. In snf1∆ cells, significantly less fragmented mitochondria are seen after H2O2 treatment. Taken together, these results indicate that activation of Snf1 is required for stress-induced mitochondrial fragmentation through cyclin C nuclear release.
Med13-degron571-650 is not required for the degradation of Med13 following H2O2 stress
We next addressed if Snf1 mediated phosphorylation of Med13 is required for Med13 degradation. Degron571-650 was deleted from full length Med13 and the degradation kinetics of this construct (Med13deg∆571-650) was examined in med13∆ cells. The results show that Med13deg∆571-650 was degraded with kinetics similar to wild type (Fig. 8A upper two panels, quantified in Fig. 8B). We also observed that this construct retained cyclin C in the nucleus in unstressed cells and released it into the cytoplasm following H2O2 stress (Fig. S5). These results were unexpected as deletion or inactivation of Snf1 results in Med13 stabilization following H2O2 stress (Fig. 3). Similarly, deletion of the Slt2 responsive degron (amino acids 742-844) only partially rescued the deletion (Fig. 8A, bottom two panels) whereas Med13 is significantly stabilized in slt2∆ cells following H2O2 stress [9]. These results suggest a complex model in which the function of either Snf1 or Slt2 is only required when their respective degron is present. If correct such a model would predict that the degron impeaches degradation when not phosphorylated. Moreover, these studies predict independent roles for these two signaling pathways that ultimately direct in the same outcome of Med13 degradation.
FIGURE 8: Either Med13 degron is sufficient for Med13 degradation. (A) med13∆ (RSY1701) cells harboring either Med13571-650deg∆-HA (pKC805, upper panels) or Med13742-844deg∆-HA plasmids (pKC814, lower panels) were treated with 0.4 mM H2O2 for the timepoints indicated and Med13deg∆-HA levels analyzed by Western blot. Tub1 levels were used as a loading control. (B) Degradation kinetics of the results shown in (A). Values represent averages ± SD from a total of at least two Western blots from independent experiments. For clarity, the degradation kinetics of wild-type Med13-HA from previous experiments was included. (C) Model depicting how two SCFGrr1 phospho-degrons mediate the destruction of Med13 following H2O2 stress. In unstressed cells cyclin C-Cdk8 phosphorylates degron742-844 [9] but both degrons are protected by an unknown mechanism from Snf1 and Slt2 kinase activity (depicted by the red circle). Following H2O2 stress Snf1 and Slt2 are activated and permitted access to the now exposed degrons. This results in SCFGrr1 mediated degradation of Med13 and cyclin C nuclear release (not shown).
Our previous work has shown that nuclear release of the yeast and mammalian cyclin C predisposes cells to initiate PCD following stress [7][8]. In S. cerevisiae, this nuclear release requires the destruction of Med13 [27], mediated by the E3 ligase complex SCFGrr1, which requires Slt2 and Cdk8 activity [9]. In this current work, we provide evidence that Sak1 activated Snf1 is also required for H2O2 induced Med13 degradation and cyclin C nuclear release. In the absence of this kinase, Med13 degradation following H2O2 stress is inhibited and cyclin C remains predominantly nuclear. Consistent with this model, we showed that Snf1 is required for the degradation of the degron571-650. Paradoxically, deletion of this degron did not prevent H2O2 induced destruction of Med13. Instead, this mutant exhibited H2O2 induced Med13 degradation and cyclin C nuclear release. Intriguingly, deletion of the Slt2-resonsive degron (Med13742-844) also did not protect Med13 from H2O2 induced degradation, although the protein was slightly more stable than wild type. Taken together, this study indicates that Slt2 and Snf1 pathways cooperate to trigger Med13 destruction. The use of two required pathways may help insure the proper signals are in place to target Med13.
To accommodate these two results, the following model that best fits the data is proposed (outlined in Fig. 8C). In unstressed cells the IDR of Med13, which encompasses both degrons, is protected from SCFGrr1 activity (depicted by red circle in Fig. 8C) by an unknown mechanism. Following H2O2 stress two events happen, the hierarchy of which is unknown. The protection is lost (depicted by green circle in Fig. 8C) and activated Snf1 and Slt2 phosphorylate their respective degrons, triggering SCFGrr1 mediated degradation of Med13. Although we currently favor the hypothesis that Snf1 directly phosphorylates Med13, we could not definitively exclude the possibility that Snf1 may promote Med13 phosphorylation indirectly via an intermediary kinase. That being said, the model presented in Fig. 8C could accommodate this possibility. More importantly however, the model supports the observation that both the AMPK and MAPK pathways act independently of each other (Fig. 4), and are required for Med13 degradation. However, in the absence of either the Snf1 or Slt2 degron, this protection is lost, allowing SCFGrr1 to recognize either activated degron. This model also accounts for the observation that when either degron is expressed in isolation, it requires its respective kinase to render it recognizable by SCFGrr1.
If this model is correct, then how could this region be protected from AMPK and MAPK activity in unstressedcells? One strong possibility could be connected to the fact that both degrons lie within the very large IDR of Med13 (Fig 2A). IDR's are known to endow proteins with highly malleable structures that undergo disorder-to-order transitions [64]. It has also been proposed that IDR's can have different binding partners that transiently associate [65]. In some cases, this can lead to proteins binding the same region that possess unrelated, or even opposite functions [66]. Thus, proteins that contain IDR's are frequently involved in signaling as they can easily change their conformational state in response to changing environmental conditions. Taken together, it is feasible to propose that in unstressed cells, the IDR of Med13 is in one conformational state that associates with an unknown protein, conferring protection to this region. Upon stress, the confirmation changes and the degron becomes exposed. This model is also consistent with the observation that IDR's are notorious for being regulated by multiple kinases [63][67]. This has led to the idea that IDR's and multiple phosphorylation events together provide structural variability [68], resulting in ultra-sensitive molecular switches that are triggered at a threshold level of phosphorylation. Thus, our results suggest a model in which Med13 degradation is regulated by three types of different kinases, a cyclin dependent kinase, a MAPK and an AMPK.
Lastly, here we show that Snf1 phosphorylates Med13, either directly or by an intermediary kinase, as well as associating with the CKM before stress (Fig. 5D). These results are consistent with a growing number of papers that have shown that a sub-population of the Snf1-Gal3 isoform is present in the nucleus under normal conditions [51][59][69], as well as enriched in the nucleus upon glucose starvation [33][34][35]. In addition, Sak1 is needed for Snf1 nuclear localization [70]. In support of this model, Snf1, Gal83 and Sak1 localize to the SUC2 promotor under non-starvation conditions [51]. This promotor is also negatively regulated by the CKM [71]. Repression is relieved by Snf1 mediated phosphorylation of two proteins known to repress SUC2 expression, the DNA binding protein Mig1 [72] and the glucose kinase Hxk2 [51][73], causing them to be released into the cytoplasm [74][75]. Likewise, carbon starvation results in the degradation of cyclin C [2], one event that occurs in the cytoplasm [4]. Surprisingly, we found that although Sak1 is needed for Med13 destruction following H2O2 stress, deletion of Gal83 has no effect (Fig. 3A and S2A). However, deletion of all three β-subunits does inhibit Med13 degradation. This would suggest that in the absence of Gal83, either Sip1 or Sip2 are able to activate nuclear Snf1. However, the Snf1-Sip1 and Snf1-Sip2 isoforms have not been reported to be nuclear, dispersing either to the vacuolar membrane (Sip1) or remaining cytoplasmic following carbon deprivation (Sip2) [33][34][35][76]. Further studies need to be executed to address if Sip1 or Sip2 can translocate into the nucleus in the absence of Gal83. Intriguingly, just recently the Mitochondrial Voltage-Dependent Anion Channel Protein Por1 (yVDAC1) has been shown to enhance Snf1 nuclear enrichment by promoting the nuclear enrichment of Gal83 [69]. This unexpected finding serves to emphasize that there is still much to learn about the role Snf1 kinase plays in response to changing environmental conditions.
Yeast strains and plasmids
All strains used in this study are listed in Table S1. Most experiments were performed in the S. cerevisiae W303 strain [77] and are listed in Table S1. Exceptions to this are the sak1∆ (YPDahl17, a gift from S. Hohmann [78]), the sip1∆ sip2∆ (MML1445, a gift from E. Herrero) and psk1∆ psk2∆ strains which are W303-1A strains. The sip1∆ sip2∆ gal83∆ strain (MSY557, a gift from M. Schmidt [79]) is an S288c strain. Finally, the Y2H assays were performed in PJ69-4a [39] that was obtained from the Yeast Resource center, a gift from S. Fields. In accordance with the Mediator nomenclature unification effort [80], we use CNC1 and CDK8 gene designations for cyclin C (SSN8/UME3/SRB11) and Cdk8 (SSN3/UME5/SRB10) respectively. The snf1∆ and gal83∆ strains (RSY1949 and RSY2080 respectively) were constructed using gene replacement methodology as described [81]. The sak1∆ (RSY1976) was a gift from S. Hohmann [78]. All cells were grown at 30°C.
All plasmids used in this study are listed in Table S2. The wild-type epitope tagged plasmids pKC801, pKC803 (MED13-HA), pBK38 (ADH1Pro–CNC1-YFP), Snf1-Myc and ADH1Pro–CNC1-MYC (pKC337) are functional and have been previously described [1][4][5][9][82][83]. All other plasmids were constructed using PCR cloning techniques and details are available upon request. In short, all constructs were amplified from plasmid DNA using Phusion Taq (Thermo), digested using Thermo fast digest restriction enzymes and ligated using Thermo fast ligase into their respective vectors. Site directed mutagenesis (New England Bio-Labs Q5) was used to create plasmids harboring amino acid mutations and the change was confirmed by sequencing (Eurofins Genomics). The MED13 Y2H plasmids were constructed by PCR cloning regions of Med13 in frame with the Gal4 activating domain of pACT2. The NLS-Med13 fusion constructs were made by first creating a backbone vector (pNLS-HA) that contains the SV40 nuclear localization sequence (NLS) in frame with a single HA epitope tag under the control of the ADH1 promotor. PCR-cloning was then used to place fragments of Med13 in frame with NLS. All in frame fusion proteins were verified by sequence analysis. Other plasmids that were used in this study that have been previously described.
Cell growth
Yeast cells were grown in either rich, non-selective medium (YPDA) or synthetic minimal medium (SC) allowing plasmid selection as previously described [1]. For all experiments, the cells were grown to mid-log phase (~ 6×106 cells/ml) before treatment with low concentrations of 0.4 mM H2O2 as previously described [5]. 25 ml of cells were collected per timepoint, washed in water, then the pellet flash frozen in liquid nitrogen. Yeast two hybrid experiments were executed as described [40]. E. coli cells were grown in LB medium with selective antibiotics. For the kinase assays cells were initially grown in SD-Ura (1.7 g Yeast Nitrogen Base, 5 g Ammonium Sulfate, 20 g Dextrose per liter), overnight, diluted 1:100-fold into 500 ml of SD-Ura, and grown for 10–12 h. Thereafter the cells were pelleted, and resuspended in 500 ml SGal-Ura (1.7 g Yeast Nitrogen Base, 5 g Ammonium Sulfate, 20 g Galactose per liter)
Western blot analysis and co-immunoprecipitation
Tagged full length Med13-HA constructs were detected by using NaOH lysis of cell pellets exactly as described in [9]. To detect Med13-HA, 1 in 5000 dilutions of anti-HA antibodies (Abcam) were used. The co-immunoprecipitation analysis was performed essentially as described [84]. Anti-alpha tubulin antibodies (12G10) were obtained from the Developmental Studies Hybridoma Bank, University of Iowa. Western blot signals were detected using either goat anti-mouse or goat anti-rabbit secondary antibodies conjugated to alkaline phosphatase (Sigma) and the CDP-Star chemiluminescence kit (Thermo). Signals were quantitated by CCD camera imaging (Kodak) and standardized to the loading control. All degradation assays were performed two or three times. SEM's were generated for each point (error bars are indicated on the graphs) and the data analyzed using linear regression analysis using GraphPad Prism 7 program.
Snf1 activation and kinase assays
The Snf1 activation assays were executed as follows. Cells were grown to mid-log phase and treated with 0.4 mM H2O2 for the times indicated They were immediately boiled for 3 minutes and pellets frozen using liquid nitrogen. Protein extracts were made using the NaOH method described in [9] and analyzed by SDS-PAGE and Western blotting. Anti-phospho-Thr172–AMPK (Cell Signaling Technology; Cat. No. 2531) was used to detect phosphorylated Snf1 and polyhistidine antibody H1029 (Sigma-Aldrich) to detect Snf1. This is possible as Snf1 ORF contains 13 contiguous histidine residues at its N terminus.
The in vitro kinase assays were executed as follows. Wild-type or cdk8∆ cells harboring either myc-tagged Snf1 or Snf1 kinase dead (K84R) under the control of the GAL1-10 promotor, were first grown in SD-Ura followed by SGal-Ura as described above and then resuspended in lysis buffer (20 mM HEPES, 10 mM KCl, 1 mM EDTA, 1 mM ethylene glycol tetra-acetic acid (EGTA), 50 mM NaCl, 10% glycerol, 1 mM β-mercaptoethanol, Pierce™ Protease Inhibitor Tablets, pH 7.4 with phosphatase inhibitors). Resuspended cultures were lysed using a Microfluidics M-110P homogenizer (Microfluidics) and cell debris removed by centrifugation at 12,000 x g for 30 min. Supernatants were incubated with 5–10 µl of Myc-conjugated magnetic beads (Cell Signaling) for 2–3 h at 4°C. Beads were separated using magnetic force and washed four times with 1 ml of lysis buffer lacking protease inhibitors. Beads containing Myc-tagged proteins were used directly for in vitro kinase assays without eluting. GST-Med13571-650S608A was purified from E. coli BL21 DE3 strain as previously described [9] and incubated with the Snf1 immunoprecipitates in 30 μl of reaction buffer containing 1X Snf1 kinase buffer (50 mM Tris-HCl, 10 mM MgCl2, 1 mM dithiothreitol (DTT), pH 7.5, 10 μM ATP, and 7.5 μCi of γ32P-ATP, and reactions were incubated for 35 min at 30°C. Kinase assays were stopped by the addition of SDS–PAGE sample buffer, analyzed by SDS-PAGE, stained with coommassie (30 min), destained for 2 h, dried and exposed to film.
Fluorescence Microscopy
Cyclin C-YFP subcellular localization and mitochondrial morphology were monitored as described previously [4][7]. For all experiments, the cells were grown to mid-log (6×106 cells/ml), treated with 0.4 mM H2O2 for the timepoints indicated, then analyzed by fluorescence microscopy. Cyclin C-YFP release analysis was performed on cells stained with DAPI in mounting medium (10 mg/ml p-phenylenediamine, 50 ng/ml 4′,6-diamidino-2-phenylindole, Molecular Probes) to visualize nuclei and prevent photo bleaching. Images were obtained using a Nikon microscope (model E800) with a 100X objective with 1.2X camera magnification (Plan Fluor Oil, NA 1.3) and a CCD camera (Hamamatsu model C4742). Data were collected using NIS software and processed using Image Pro software. All images of individual cells were optically sectioned (0.2 µM slices at 0.3 µM spacing) deconvolved and the slices were collapsed to visualize the entire fluorescent signal within the cell. Cyclin C-YFP foci were scored as being cytoplasmic when 3 or more foci were observed outside of the nucleus. Mitochondrial fission assays were performed on live cells as described [7]. In brief, mitochondrial fission was scored positive if no reticular mitochondria were observed that transversed half the cell diameter. Fusion was scored when cells exhibited one or more reticular mitochondria the diameter of the cell. Fission and fusion was scored for 200 cells from three independent isolates. Statistical analysis was performed using the Student's T-test.
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Download Supplemental Information
We thank. S. Fields, S. Hohmann, E. Herrero, J. Nunnari, M. Schmidt and M. Solomon for strains and plasmids. We thank R. Strich for critical reading of this manuscript. This work was supported by grants from the National Institutes of Health awarded to K.F.C. (GM113196), and J.G (GM100376). The yeast two-hybrid strain was made by work supported by NIH grant P41 RR11823 awarded to T. N. Davis.
© 2018
Snf1 cooperates with the CWI MAPK pathway to mediate the degradation of Med13 following oxidative stress by Willis et al. is licensed under a Creative Commons Attribution 4.0 International License.


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subset: commoncrawl


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Score one for me (Bartl's blog)
Score one for me.
Players rarely admit to reading blogs by the media guys, but my blog last week about famous Condors trades actually got a tweet: from Evan Stoflet, who, needless to say, considered his deal a "blockbuster."
I also received a bunch of feedback from the fans, and I think I disappointed our current head coach for not including any of his deals. In all fairness, I think one or two of them would make the list, but I will wait until the season ends to judge. But it's hard not to look at the Helfrich and Dee deals and not feel very good about them. So just wait your turn, Odie…
* * * * *
Ryan Holt is on the road with the team and he took along with him a dusty copy of They Don't Play Hockey In Heaven that was sitting on a display shelf in the office, so he could read it on this trip. From the texts I received during the team's layover it's clear that he's enjoying it.
There are multiple ways to read that book and judge its contents. One is from the perspective of working or playing for a minor league hockey team, imagining the behind the scenes things happening. Which probably brings you a few chuckles. Another is from a dedicated fans' perspective, perhaps bristling at some of the not-so-flattering things said about the town and the players that you have admired. One is from the angle of a general person unfamiliar with the names, teams and the city, just reading it for the intriguing story line. And yet another is from the perspective of having been there along the way and knowing where and when some liberties to story angles were taken. And with that, one would also know there was a lot that he left OUT of the book, thank God (some things need to stay in the locker room).
Many people from inside that team were not happy with the finished product that Ken Baker published, but there's no denying that it was a compelling tale, with quirky characters, odd events and situations, and the drama and excitement of his one game in the pro ranks. I remember much about that game, and probably always will, because at the time, calling it was one of the biggest highlights of my career. It IS a very good read, with a remarkable and unlikely ending. And I still recommend it, 10 years later.
* * * * *
Evan Trupp's goal last weekend has garnered over 28,000 views on our YouTube page. "The Brawl" video has been watched over 170,000 times on our page alone, and that's not counting the 10's of thousands of views it has from other assorted websites that lifted it. Think about that. A goal, scored in a Condors game, has been watched online 28,000 times. That's truly amazing.
Other stats from the brawl video are noteworthy as well. The top websites that brought in the most "views" of that fight were (in order): BarstoolSports.com, DeadSpin.com, Yahoo!, BarstoolU.com and aktuality.sk. Besides the fact that Barstool Sports was responsible for a combined 26,000 views between their sites, the '.sk' website made Slovakia the third highest ranked COUNTRY for people watching that video. That's right, it goes: the United States, Canada, Slovakia.
I guess I don't know where else it would end up being so popular, but I would not have predicted Slovakia would be third. Ukraine was next. Probably Denis Shiryaev and Vlad Serov's friends and family, still following along with the Condors.
That's all I have for now.
Kevin Bartl is the Vice President of Communications and part of the broadcast team for the Condors, in his ninth season with the team.


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Mitsubishi EVO XI Will Be a Hybrid Confirms Company President
Rumors that the next-generation Mitsubishi EVO would go hybrid first surfaced over a year ago and now company president Osamu Masuko has reportedly confirmed as much.
Speaking with the UK's AutoCar, Masuko said the Evo XI will come with a hybrid powertrain and that it will be on sale within the next three years.
Rumors have suggested the car could be a plug-in hybrid, or that it could use a diesel-hybrid powertrain, helping it deliver substantial amounts of torque from a very small displacement engine.
Several months back came reports that the EVO would be discontinued as Mitsubishi poured substantial resources into developing future electric cars. That was eventually rebuked by Masuko has insisted the EVO would live on, but "evolve" in a different direction.
[Source: AutoCar]


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Massachusetts state Senator Joan B. Lovely was sexually abused as a 6-year-old by a maternal uncle, trauma that ultimately has led her to file comprehensive legislation to protect kids.
Lovely's bill focuses on schools and youth organizations, places where adults might recognize that child abuse is happening at home or, more seriously, be in a position to target victims for exploitation. As revelations unfold about abuse at a wide range of educational institutions, it's only sensible to begin reform efforts there. A Globe Spotlight investigation last year found unchecked sexual misconduct in at least 67 New England private schools — including 33 in Massachusetts — with more than 200 victims. Lovely's bill also targets the gray area of sexual relations between educators and students that strikes most as morally wrong but legally ambiguous. When it comes to the teacher-student relationship, sex should be legally off limits.
The proposed legislation, scheduled for a State House hearing tomorrow, cracks down on teacher-student sexual relations by closing a loophole in existing law and imposing stiffer criminal sanctions for any educator or other school or youth organization worker to engage in sexual relations with any teen 19 or younger who is under their authority and hasn't yet graduated from high school. While the age of consent in Massachusetts is 16, if the consenting teen is a student and the other person is an adult in a position of authority, the relationship is not equal, rendering the teen's consent invalid.
Lovely's legislation also requires training for all teachers, personnel, independent contractors, and volunteers at all schools — public and independent — as well as youth organizations, on how to prevent, respond, and report child sexual abuse. It also modernizes outdated regulations by expanding the list of mandated reporters of child abuse to include volunteer coaches, professional tutors, and other independent contractors at schools.
"This bill is unique in the country," says Jetta Bernier, executive director of Massachusetts Citizens for Children, or MassKids. "More than two dozen states have passed similar legislation mandating child sex abuse education, but none include private schools in the scope."
Such a law would send a clear message in education circles. "No teacher should ever have any sort of relationship with their student, because we don't send our children to school to have sex with them; teachers are there to guide them," said Brenda Demers, whose 16-year-old daughter engaged in sexual relations with her special education teacher in Upton a few years ago. She believes the teacher, then 26, would have thought twice about getting involved with her daughter had he known he would face prosecution. Instead, he went on to work at another school.
Lovely's proposal would make sure schools have strong hiring policies and screening standards in place to prevent what's known as "passing the trash," when problem educators are hired despite histories of sexual misconduct. It would also ban schools from signing agreements keeping any information about an individual's past allegations secret.
Educators generally honor their position of trust. For the small minority who may be tempted to abuse their authority, Lovely's bill would make it clear that what is morally wrong also is a violation of the law.


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Discover more from Asia Sentinel
Human Rights Groups Seek Justice for Thai Dissidents
On May 22, the one-year anniversary of the coup that brought Army general Prayuth Chan-ocha to power in Thailand, handfuls of university students in Bangkok and the northern city of Khon Kaen held peaceful demonstrations advocating community rights and democracy.
That may have been a mistake. The National Council for Peace and Order, as the junta now calls itself, has come down hard on 14 of the students, charging them with sedition, which could carry as much as seven years in prison as well as an additional six-month prison term and a fine of up to 10,000 baht (US$296) for breaching the ban on public assembly. Some of the students in Bangkok were dragged to a local police station where at least three were punched and kicked. Three were subsequently hospitalized.
And, not only is there no letup on the circumscription of rights of students and any other protesters who might rise, the junta is quite comfortable in its role. Last week, when the US State Department issued its annual human rights report, which criticized the curtailment of rights, Sansern Kaewkamnerd, the major general who acts as spokesman for the junta, instead suggested that US officials be summoned to explain the basis for the report. Thailand, he said, has placed the utmost importance on the real situation and the "restoration of peace and happiness."
The students are now held in Bangkok jails while they await trial in military courts, which have earned universal criticism from Human Rights Watch, Amnesty International and other human rights NGOs, although with no discernible effect on the military leaders who have taken over Thailand. At least 25 military courts are operating across the country.
Amnesty International has accused the Thai authorities of "using the courts to silence dissent and make an example of those who voice opposition against military rule." Civilians convicted in Thai military courts have no right of appeal to higher courts, which is contrary to international law. There is no right to habeas corpus. The military can use the pretrial period to obtain evidence before the suspect has even appeared before a tribunal.
Defendants have no right to counsel. Note-taking is banned in the courtroom. Often cases are prosecuted in secret. The courts are not accessible to relatives or rights activists. Requests for temporary release are almost never granted, with the authorities citing the political nature of the cases as the reason for the denial.
The military court is a single-tiered system. Based on cases which have already been adjudicated, according to Thai Lawyers for Human Rights, the military courts tend to impose much harsher sanctions than civilian courts.
"This forces civilian defendants to choose to plead guilty to the charges rather than to fight them," according to the Thai Lawyers group. "In addition, no effort is made to explore the background of the accused persons, which should be examined for the possible reduction of sentences. Concern about the harsh and unjust practices of the military courts has pressured people to flee and live in exile."
"Thai authorities should immediately drop all charges and release unconditionally 14 student activists who peacefully expressed opposition to military rule," Human Rights Watch said in a statement by Brad Adams, the Asia director of Human Rights Watch. "While insisting they aren't dictators, the Thai generals have used the military courts as a central feature of their crackdown against peaceful criticism and political dissent."
Since the May 2014 coup, the junta has banned political gatherings of more than five people. The authorities have arrested at least 80 people for organizing or taking part in such public gatherings. In addition hundreds of people, mostly political dissidents and critics of the NCPO, have been sent to trials in military courts since the coup although international human rights law prohibits governments from using military courts to try civilians when civilian courts are functioning.
The use of military courts in Thailand also fails to meet international fair trial standards under the International Covenant on Civil and Political Rights. The United Nations Human Rights Committee, the international expert body that monitors state compliance with the covenant, has stated on the right to a fair trial that "the trial of civilians in military or special courts may raise serious problems as far as the equitable, impartial and independent administration of justice is concerned."
This is particularly problematic in Thailand where every element of military courts functions within the Defense Ministry's chain of command, the UN Human Rights Committee said.
"With each new arrest, Thailand's path toward democracy is getting harder to find," Adams said. "Governments around the world should press the junta to end repression and respect fundamental rights."
The United Nations Human Rights Office for South East Asia also has urged the government to drop criminal charges against the students and release them from custody.
On 23 May 2014, a day after the coup d'état, the United Nations High Commissioner for Human Rights publicly expressed serious concern about the restrictions on fundamental freedoms imposed by the NCPO, adding that freedom of expression and freedom of assembly are particularly important in resolving difficult political issues through dialogue and debate. Now more than one year on, despite pledges by the Government to promptly restore the rule of law, restrictions on fundamental freedoms remain in place.


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In the early days of my walk with Christ, I was taught to believe that miracles were an everyday occurrence. The Christians I knew were generous in their definition of what constituted a miracle, as likely to call a good parking spot an act of God as someone's sudden recovery from cancer. Every situation was treated as an occasion for divine intervention. I confess that this was part of what attracted me to the Christian faith. I was not interested in a God who was merely an abstraction; I wanted to know that God was real. I was looking for a God who paid attention to me when I spoke to Him. It did not occur to me that I was the one who was supposed to do the listening.
I often prayed for God to intervene in my life. But I did not always get what I wanted. I asked Him to heal my mother when she was unexpectedly hospitalized for an illness that the doctors did not seem to be able to diagnose. She died. I asked God to deliver my father from alcoholism. He did not. I prayed to win the lottery (only once). You can guess how that turned out. I am not saying that God has never answered my prayers. Only that God refused my request often enough to know that an affirmative answer is not always a given.
"Them that's got shall get, and them that's not shall lose," Billie Holiday sang. The whole world seems to be divided into a few privileged people who get everything they want and the majority who do not. Why not Kingdom of God too? We often wonder why God grants to others the thing He denies to us. The effect this has on our prayers is often an attitude of ambivalence. We conclude our prayers with a resigned shrug and interpret delay as denial. We secretly think that God is playing favorites. But the truth is we are the ones who suffer from bias. Our memory is selective, more inclined to dwell on God's refusals than to remember the many times He has granted our requests. Impatience distorts our sense of God's timing in His answers so that we ignore the winding and unexpected path that leads from entreaty to answer that earlier generations called providence.
Praying Like a Child
Such thinking is childish, of course. Yet there is no spiritual act that is more childlike than the act of prayer. Jesus acknowledges as much when He encourages His disciples to be persistent in their praying and asks: "Which of you, if your son asks for bread, will give him a stone? Or if he asks for a fish, will give him a snake?" (Matt. 7:9–10). When it comes to our most basic needs, God often grants them without our even having to ask. He "causes his sun to rise on the evil and the good, and sends rain on the righteous and the unrighteous" (Matt. 5:45). God "gives everyone life and breath and everything else" (Acts 17:25).
God is generous by nature. But He is no pushover. Whatever our prayers are, they are not a means by which we may manipulate God. We cannot bully God or wheedle Him into granting us the answer that we prefer. The divine right to refuse our requests is necessary if prayer to be something more than a merely mechanical or transactional event. Anthony Bloom's observation about the possibility that those who pray might still experience the absence of God's presence also applies the answers we seek in prayer. "If we could mechanically draw Him into an encounter, force Him to meet us, simply because we have chosen this moment to meet Him, there would be no relationship and no encounter," Bloom explains. "We can do that with an image, with the imagination, or with the various idols we can put in front of us instead of God; we can do nothing of the sort with the living God, any more than we can do it with a living person."
Mechanical Praying
In His teaching about prayer, Jesus described this same mechanical style as the sort of approach the pagans use when they pray, "for they think they will be heard because of their many words" (Matt. 6:7). The one who piles up words in prayer has lost sight of God. When we cease to relate to God as we would a person, He might as well be a vending machine. At best, we treat God as if He were a mere functionary, the same way we might treat someone who gives us our order at the drive-through. We are hardly aware of them. We hand over our money, take our meal and drive off. We don't know their name, and minutes after we have left the parking lot, we cannot recall their face.
Our failure to grasp this can turn prayer into an attempt at manipulation. We desperately try to gauge whether the amount of our faith is enough to trigger the desired response from God. This uncertainty, in turn, lends itself to spiritual posturing. We put on a show in the vain hope that we will somehow convince God that we have the kind of faith that warrants an answer. We fuss over our delivery, trying to sound confident and prove that we have enough faith to gain our request. Or we conclude that the weight God will give to our prayers is a function of the number of people we can persuade to take up our request. We approach prayer as if it were an oral petition drive, hoping that the sound of so many others will drown out the uncertainty of our own voice. We mount a lobbying campaign, inviting those we consider spiritual authorities to pray for us, convinced that their prayers have more influence with God than ours.
More than Answers
Miracles do not always lead to faith any more than answers to prayers do. John 6 tells how the crowd followed Jesus to the other side of the lake after He fed the multitude. "You are looking for me, not because you saw the signs I performed, but because you ate the loaves and had your fill," Jesus chided them (John 6:26). The Israelites ate bread that fell from heaven and still grumbled about the menu. The disciples saw Jesus raise people from the dead. But when the women came and told them that they had seen Jesus alive after His crucifixion, they thought they were talking nonsense (Luke 24:11). As important as the answers to our prayers are, there is more to prayer than getting. Getting the answer is certainly no small thing, but it is not the only thing. "In Gethsemane, the holiest of all petitioners prays three times that a certain cup might pass from Him. It did not," C. S. Lewis points out. "After that the idea that prayer is recommended to us as a sort of infallible gimmick may be dismissed." That one denial, combined with Jesus' resigned, "Yet not my will, but yours be done," is evidence that prayer is an exercise in trusting God's answer as much as it is the act of making our request (Luke 22:42).
When I was a boy, Superman was my favorite television show. I wanted to fly like him, so I did the natural thing. I asked my father to teach me. When he told me that he did not know how to fly, I didn't believe him. It is in a child's nature to assume not only the willingness of their parents to grant their requests but their ability to do so, no matter how unreasonable the request may be. One of the first lessons of maturity is that of learning to accept our parents' limitations in such matters. But where our prayers are concerned, the limitation is with us rather than with God. We are not always the best judge of what we need. Like a child who demands a pony for Christmas, our requests are sometimes frivolous. Others are selfish. A few of the things we ask for may even be so bad for us that God dismisses them outright. Yet, many of our requests are reasonable and even beneficial.
If the Bible reveals anything about God's power, it indicates that He is a God of miracles. All the miracles of Scripture ultimately point to the miracle of miracles, the resurrection of Jesus Christ. The resurrection is the central miracle of the Bible, the one which makes our salvation possible. Yet it is a miracle that was possible only because the Father refused the Savior's prayer in Gethsemane. We do not always understand why God withholds from us the thing we have asked of Him. But we do not need to know why to understand that His answer is good for us. The Father's refusal of the prayer of His own Son is all the proof we need that sometimes God's "no" is more loving than His "yes."
John's book, Dangerous Virtues: How to Follow Jesus When Evil Masquerades as Good is available from Moody Publishers. Check out the free small group resources by clicking on the Group Resources tab above.


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Hamilton Mayor Bob Bratina was referred to by embattled Toronto Mayor Rob Ford as "Mayor Britannia" during a media scrum this morning in Toronto.
Ford, a avid football fan, changed the topic of the scrum from his own problems to this weekend's CFL Eastern Final between the Tiger-Cats and Argos.
"I want to call Mayor Britannia in Hamilton and tell him that we're going to have to spank their little Tiger-Cats."
Bratina's Reaction
Bratina, another avid CFL fan who was the stadium announcer – "the voice of the Ticats" – for many years, was at the new Hamilton football stadium on a tour of the facility when he received news of the comment.
I was standing beside the Mayor on what will be the 55-yard line as he looked at his Blackberry. I notice he appeared to be trying to understand whatever he was reading.
Bratina turned to CFL Board of Governors Chair Jim Lawson. Lawson asked Bratina what was occurring, Bratina responded:
'Mayor Ford mentioned me in a press conference'. Bratina continued by saying the context of Ford comments was Sunday's game, and that the person who emailed him wished him "luck" now that his name had been used by Ford.
Lawson and Bratina discussed the matter, I didn't overhear the discussion. It ended with a laugh by both – some sort of joke having been made, and Bratina stated he was staying out of the Ford matter.
Official Comment from Bratina's Office
The official comment from the Mayor's office, as given by his Chief of Staff Peggy Chapman is they are not responding to Mayor Ford's incorrect pronunciation of Bratina's name.
On the bet for Sunday's game, Chapman says Ford had not connected Bratina about friendly bet. Chapman says the Eastern Final is different from the Labour Day Classic or Grey Cup and that Bratina would not be betting on the game regardless of Ford's current embattled situation.
Not Speaking on Ford Situation
Mayor Bratina has carefully avoided any comment or reference to Mayor Ford's behaviour as the Toronto scandal has grown.
No other Mayor's have commented on the Ford situation either.
Bratina carried on his day as planned with a trip to the Boys and Girls Clubs of Hamilton after the stadium where he participated in a major private sector funding announcement.


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What is Geopathic stress (GS) and how does it affect us?
Geopathic stress, wherein Geo means "Earth", Pathos means "sorrow" and stress is the trauma we experience. Geopathic Stress is due to the distorted Electromagnetic field of the Earth. The Earth resonates with an electromagnetic frequency of approximately 7.83 Hz called Schumann Waves. These waves were discovered by a German physicist W.O. Schumann in 1952. These Schuman waves oscillate at a frequency of human brainwaves. These help to regulate the functioning of the human body but are unable to penetrate the concrete or metal used to make houses.
When this natural resonance of Earth is disturbed by man- made structures like sewers, water pipes, drains, electricity, tunnels, quarries, mines, underground railways or natural mineral concentrations, underground caverns, underground streams, geological fault lines, it creates Geopathic Stress.
Sleeping or spending a lot of time in Geopathic Stress Zones can affect our body's resistance leading to ill-health and Stress. It directly affects our well-being and functioning.
How long have we known about Geopathic Stress?
Geopathic stress is not a recent phenomenon or discovery. People from ancient times have been known to check the ill effects of Geopathic Stress. The Romans before building a structure on a land would let the sheep graze there for a year, then cut them open to see whether the organs inside were still healthy or not. Similarly the Bavarians from Germany would make an anthill in a potential building to check whether they would stay there or not, since like dogs and some fishes, ants do not stay at a place with Geopathic Stress. The Chinese also refer to these Geopathic stress as Claws of Dragon or underground Demons in their Feng Shui. Although some of the insects like bees and wasps like Geopathic Stressed areas. Honeybees are more productive in GS areas. Also animals like cats, snakes, owls etc. fall in the same category.
How Does Geopathic Stress Affect Us?
People living in GS mostly suffer from serious long term illnesses, psychological conditions and financial problems.
- Fatigue, severe muscle pain, headaches, dizziness and inability to concentrate.
- Sleeplessness leading to a distorted decision making. They feel more comfortable and relaxed outside the house.
- Nightmares, bad dreams and insomnia.
- Suicidal Tendencies.
- Deadly diseases like Cancer, Aids, Multiple Sclerosis, Parkinson's disease and Many Other Central Nervous System, Wasting and Paralyzing Diseases
- Infertility, Miscarriages, difficult pregnancies, unhealthy New Born babies or premature births.
- Financial problems haunt the occupants like property will not sell, gets involved in legal trouble or comes under lot of debt.
- The person in the GS area will have poor workmanship, will not have good relationships with the staff or family members.
- Continuous loss in business, murders, robberies and accidents are common symptoms for people living in GS area.
Ways to detect GS lines and their Treatment
The general symptoms that all of us can recognize are:
- In your house when you wake up in the morning, instead of feeling rejuvenated you feel drained out.
- The house does not feel a welcome place, you feel more comfortable outside the house.
- The house has a history of serious illness like cancer.
- The treatment also does not have the desired effect on such patients living in GS areas.
- The walls or glass in the Geopathic Stress area tend to develop cracks and have a tendency to get mold.
- The office building has sick building like symptoms for no reason, like the employee do not feel good, fall sick very often, high rate of absenteeism, low morale, lack of concentration and high employee turnover rate.
- These Geopathic stress lines can be detected using the Universal Aura Scanner using water and IR sample.
- If you measure the aura of a person and then make him stand on a GS line, his aura will reduce considerably.
- The magnetic compass will deflect by 29 degrees more if placed at a nodal point of GS in respect to the true North of the place.
Treatment of the Geopathic Stress
- Re-arrange your living or working space if possible, so as to avoid the spending lot of time in the GS areas. If the area is very large or re-arranging is not possible, then other tools should be used to neutralize its effect.
- Applying Vastu principles helps to balance the energies of a place.
- Certain metal rods like of copper, brass or steel can be placed at specific points to neutralize the negative energies. Sometimes these rods contain specific herbs, minerals and yantras which help in tackling GS. For example Oaks and redwoods are very resilient to GS.
- Crystals, pyramids are used to resolve the energy disturbance for example Quartz or shungite crystals, Amethyst, zeolite, and tourmaline are used for treating GS.
- Reduce the electromagnetic pollution around yourself as much as possible. Stay away from satellite towers, unplug your electrical appliances when not in use.
- Harmonizers are used to neutralize the effects of GS.


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Then with spiced ginger beer and a chocolate mince tart left out for Santa, she dropped quickly to sleep.
French lavender salt and some apricot, nectarine and vanilla jam (that I will write about later).
In the morning we opened presents and had cranberry nut rolls with jarlsberg cheese for breakfast before heading down to my parents house in Geelong for more presents and food.
I loved some quirky gift tags. Sylvia got one with chocolate in it and one that was actually made out of a candy cane cookie cutter.
Sylvia had been asking for a Frozen doll all December. Yet it was the hairdresser head and the blue ukelele that she really loved.
nut roast as usual and had lots of vegies. Mum almost forgot the cauliflower cheese. I was glad she didn't! She also made a delicious sangria to drink with dinner.
Most of my siblings were off to in-laws after lunch and we relaxed. By evening we were still full and just snacked on some leftovers.
panforte, stollen, and cheesecake. I was so full I didn't get to try my mum's Christmas fruit cake after dinner.


text:
The campaign depicts the stories that are told through the collection of seemingly insignificant objects. These objects are proof of the life lived—from an overfilled passport to a race bib or even a rock picked up along the journey. The initial concepts draw inspiration from Teva's sponsored athletes and ambassadors and the first video will feature Brad Ludden, Teva professional kayaker and founder of First Descents.
Kicking off in spring, the campaign supports the new Fuse-ion, a high performance water shoe that highlights Teva's supremacy in, around and on water. The campaign, which will receive over 40 million impressions globally from February through June, will feature specific story executions speaking to wakeskating, kayaking, surf and stand-up paddleboarding.
"The campaign was born in my closet," says Lucas Martinez, Teva Associate Marketing Manager. "I was digging through a bunch of old junk I had in a shoebox, and I realized that everything reminded me of the stories in my life. I spent hours looking through that box. Then I looked down and saw a pair of Teva flips that I wore in Fiji and it brought me right back to that moment, and I realized that this is what 'Live Better Stories' is about. The things we all choose to keep are proof of our better stories."
Conceptualized by Teva's internal creative team, the campaign was brought to life with the help of freelance art director, Dustin Smith who first gained notoriety from his work with Altoids and has since worked with clients like Nintendo and Clorox's Green Works division. Complimenting Dustin's direction, the campaign was shot by Dwight Eschliman, a photographer out of San Francisco who has worked with clients such as Apple, Absolut and Audi.
Accompanying the campaign, the brand will launch a full retail program starting in February though selected retail partners which will include shop-in-shop, window displays and seasonal point-of-purchase fixtures. Also starting in February, the brand will run the "Teva House" promotion via their Facebook page for six weeks which allows individuals an opportunity to win a trip for two to Vail, Colorado for the 2012 Summer Teva Mountain Games. Two winners will be selected from North America and the remaining 11 winners will be selected from regions across Asia Pacific and Europe.
For more information on Teva or the activation around Stories are the Proof of Life, please contact Jaime Eschette at firstname.lastname@example.com or via phone at 805-967-7611 x1178.
About Teva
Teva is a global adventure brand dedicated to helping people Live Better Stories. The concept of "adventure" varies, but everyone understands an adrenaline rush and staggering sense of wonderment. These are the life-defining moments that Teva helps to provide. Born on the Colorado River in 1984, Teva's connection to water is manifested in the products it makes and its support of clean water. In partnership with the Waterkeeper Alliance and Ocean Conservancy, Teva's "A Pair for a Foot" campaign will clean a linear foot of global waterways for every pair of shoes sold. www.teva.com
About Deckers Outdoor Corporation
Deckers Outdoor Corporation is the premier lifestyle marketer that builds niche brands into global market leaders by designing and marketing innovative, functional and fashion-oriented footwear developed for both high performance outdoor activities and everyday casual lifestyle use. UGG® Australia, Teva®, Sanuk®, TSUBO®, Mozo® and Ahnu® are registered trademarks of Deckers Outdoor Corporation.
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Spinach Artichoke Pull-Apart Bread
Here's a video that shows you how to make it:
Servings: 6-8
2 sheets of puff pastry, thawed
8 ounces cream cheese, softened
14 ounce can (about 1½ cups) artichoke hearts, drained and chopped
3/4 cup spinach, cooked and drained of excess water (or frozen and thawed)
1/4 cup sour cream
1/2 cup grated parmesan cheese
1/2 cup shredded mozzarella cheese
2 teaspoons garlic powder
1 teaspoon salt
1 teaspoon pepper
½ teaspoon red pepper flakes
1 egg, beaten
1. Preheat oven to 400ºF/200ºC.
2. In a large bowl, combine cream cheese, artichoke hearts, spinach, sour cream, parmesan, mozzarella, garlic powder, salt, pepper, and red pepper flakes.
3. Place the two sheets of puff pastry on a parchment paper-lined baking tray.
4. Using a dinner plate as a guide, cut out equal size circles from both sheets.
5. Remove the top sheet.
6. On the bottom sheet, spread spinach artichoke mixture.
7. Lay the other sheet of puff pastry on top.
8. Take a glass and place it in the center of the circle.
9. Using a knife, cut away from the edge of the glass to create 12 equally spaced strips.
10. Take each strip and twist.
11. Brush with beaten egg.
12. Bake at 400°F/200°C for 30 minutes, or until golden.
13. Enjoy!