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MAP kinases regulate essential cellular events, including cell growth, differentiation and inflammation. The solution structure of a complete MAPK–MAPK-regulatory protein complex, p38α–HePTP, was determined, enabling a comprehensive investigation of the molecular basis of specificity and fidelity in MAPK regulation. Structure determination was achieved by combining NMR spectroscopy and small-angle X-ray scattering data with a new ensemble calculation–refinement procedure. We identified 25 residues outside of the HePTP kinase interaction motif necessary for p38α recognition. The complex adopts an extended conformation in solution and rarely samples the conformation necessary for kinase deactivation. Complex formation also does not affect the N-terminal lobe, the activation loop of p38α or the catalytic domain of HePTP. Together, these results show how the downstream tyrosine phosphatase HePTP regulates p38α and provide for fundamentally new insights into MAPK regulation and specificity.
Mitogen-activated protein kinases (MAPKs; ERK, p38, JNK) are serine/threonine kinases that mediate the cellular response to a variety of extracellular stimuli ranging from growth factors to inflammatory cytokines. The activity of MAPKs is finely tuned in a cell type–specific and temporal manner by the concerted effort of multiple regulatory proteins, including activating kinases (such as MEK1, MKK3 and MKK6), deactivating phosphatases (such as KIM-PTPs and MKPs) and scaffolding proteins (such as KSRs)1. Critically, disruptions in the tight regulation of the MAPK signaling pathways are directly correlated with numerous diseases, including Alzheimer's disease, rheumatoid arthritis and cancer2.
The MAPK pathways consist of three components: a MAP kinase kinase kinase (MAP3K), a MAP kinase kinase (MAP2K) and a MAPK3. Stimulation of the pathway ultimately results in MAPK activation by the dual phosphorylation of a threonine and a tyrosine residue (Thr-Xaa-Tyr) located in the MAPK activation loop. Though the kinase cascades that direct MAPK activation seem stereotypical, the guidance and fine-tuning that direct cell- and situation-specific responses are governed by MAPK-specific phosphatases. In particular, MAPKs are dephosphorylated by the kinase interaction motif protein tyrosine phosphatases (KIM-PTPs), 11 'typical' MAP kinase phosphatases (MKPs) and several 'atypical' dual-specific phosphatases as well as serine/threonine phosphatases4. The reason for this abundance of phosphatases is most likely related to the many roles that MAPKs play in the cell, which is profoundly affected by the duration of MAPK activation and the subcellular localization of active MAPKs. A comprehensive understanding of how the activity of MAPKs is finely controlled by MAPK regulatory proteins can only be obtained by understanding how these proteins interact at a molecular level.
MAPKs have a two-lobed architecture with a five-stranded β-sheet in the N-terminal lobe and six α helices in the C-terminal lobe5. The activation loop, which contains the Thr-Xaa-Tyr motif, is at the top of the C-terminal domain immediately adjacent to the N-terminal domain. In 1994, it was discovered that MAPKs contain a docking site in the C-terminal lobe6. This docking site is located on the face of the C-terminal lobe opposite to the activation loop and binds a conserved 13- to 16-amino-acid sequence known as the kinase interaction motif (KIM) or the D motif. This motif has since been identified in nearly every MAPK regulatory and substrate protein. The KIM is characterized by a group of basic residues (two to three lysines or arginines) and two hydrophobic residues (ΦA-Xaa-ΦB) that mediate the interaction with the KIM binding site of MAPKs. Structures of MAPKs bound to KIM peptides from activating kinases, deactivating phosphatases and substrates have revealed new insights into the molecular determinants that direct these interactions7–13. However, though the KIM is required for binding14,15, it has recently become clear that residues outside the KIM are necessary to confer specificity16–18.
The KIM-PTPs are a small family of phosphatases that include hematopoietic tyrosine phosphatase (HePTP; immune system–specific)19, striatum-enriched phosphatase (STEP; brain-specific)20 and STEP-like PTP (PTPRR or PTP-SL; brain-specific)21. Each phosphatase possesses a C-terminal catalytic domain (the PTP domain) and an N-terminal unstructured extension that contains the 15-amino-acid KIM. KIM-PTPs regulate the activity of p38 and ERK by both protein localization (by retaining the MAPKs in the cytosol) and dephosphorylation of their active sites22. Despite a wealth of crystallographic data for p38 (refs. 5,7,8,23,24), little is known about the atomic-level detail of its interaction with regulatory proteins outside the KIM binding pocket and the role that these residues have in distinguishing between MAPK regulatory proteins to ensure pathway specificity. Though current structures provide clues to the conformational variability in p38 in the free and KIM peptide–bound forms, additional structures are necessary to obtain comprehensive structural models that describe the molecular interactions between MAPKs and the full-length regulatory proteins that define MAPK pathway specificity and fidelity.
Here, we combine biomolecular NMR spectroscopy, small-angle X-ray scattering (SAXS) and isothermal titration calorimetry (ITC) to determine how p38α, the p38 isoform that is highly and ubiquitously expressed in most cell types, is recognized by its KIM-PTP regulator HePTP under functional conditions. We show that, although the KIM peptide is essential for binding, p38α residues outside the KIM binding pocket play a central role in HePTP recognition. We also show that the interaction between p38α and HePTP is largely confined to the unstructured HePTP N terminus and the KIM-PTP catalytic-domain extension. Finally, using SAXS and a new ensemble-calculation routine that incorporates chemical-shift information derived from NMR spectroscopy, we show that the complex adopts an extended conformation in solution and provide an atomic model of the p38α–HePTP complex. Collectively, these results provide new insights into the molecular interactions that regulate the strength and duration of MAPK signaling and, in turn, provide unique avenues for therapeutic interventions of MAPK-related diseases.
NMR spectroscopy is a powerful technique for defining the interactions of proteins at atomic resolution. However, a precise definition of these interactions requires a highly complete sequence-specific backbone assignment of the protein (or proteins) under investigation. Because of their large size (~40 kDa) and complex dynamic characteristics, serine/threonine kinases are notoriously difficult targets for NMR spectroscopy, and only a few of them, such as the serine/threonine kinase PKA25,26 and the tyrosine kinase Abl27, have been investigated in detail using this technique. This is especially true for the MAPKs, for which only two sequence-specific backbone assignments have been reported28,29. However, unlike the assignments for PKA (80% complete)30 or Abl (96% complete)31, the assignment of p38α is only partially complete (64% complete)29. Moreover, many functionally important residues are unassigned.
To further our understanding of these previously unassigned regions of p38α, we improved the sequence-specific backbone assignment of p38α to 82% (Fig. 1a–d). Of the 334 expected peaks for nonproline residues, 297 peaks were observed in the two-dimensional 1H,15N transverse relaxation-optimized spectroscopy (TROSY) spectrum of p38α. The 37 peaks not observed could be missing because of fast solvent (H2O)-amide exchange on the NMR timescale; lack of D2O back exchange after protein growth in D2O-based expression medium, especially for amino acids in the hydrophobic core; or intermediate conformational exchange, which broadens the peaks beyond detection. To minimize solvent-amide exchange, all experiments were performed at pH 6.8 using water suppression by gradient-tailored excitation (WATERGATE)–based NMR experiments. In addition, analysis of unassigned residues suggested that only a small fraction of residues are sufficiently buried in the hydrophobic core to be missing as a result of slow D2O back exchange. Thus, the majority of missing peaks are probably in a microsecond-millisecond conformational exchange regime that broadens the peaks beyond detection.
Most newly assigned residues are located in functionally critical regions of p38α (Fig. 1b). The current assignment now includes 88% of the N-terminal lobe, including all of the GxGxxG ATP binding motif; 83% of the activation loop, which contains the Thr-Gly-Tyr phosphorylation site; 100% of the Thr-Gly-Tyr motif; 74% of the MAPK insert (residues 243–273), which includes helices α1L14 and α2L14 and forms a C-terminal domain binding pocket that is currently being exploited for the development of new p38-specific inhibitors32; 100% of the αEF-αF loop, which forms a platform on which the activation loop folds; 100% of helix αH; and 93% of the KIM-peptide binding pocket. Taken together, this is the most complete sequence-specific backbone assignment of any MAPK reported to date.
The assignment of residues surrounding the ATP binding pocket were confirmed by titration of both 2′,3′-(2,2,5,5-tetramethyl-3-pyrroline-1-oxyl-3-carboxylic acid ester) adenosine triphosphate (SL-ATP)33,34, a spin-labeled ATP analog, and the prototypical p38α inhibitor SB203580 (ref. 35), a competitive inhibitor of ATP that binds at the ATP binding site, into [2H,15N]p38α (two- to four-fold excess of titrant) using two-dimensional 1H,15N-TROSY spectroscopy. As expected, only residues surrounding the ATP binding pocket showed either reduced intensities (SL-ATP; Supplementary Results, Supplementary Fig. 1) or large chemical shift changes (SB203580; Fig. 1c). In addition, titration with SB203580 did not change the number of observed peaks (296 peaks), demonstrating that the microsecond-millisecond intermediate timescale dynamics of p38α are unchanged when it is bound to SB203580.
Notably, the largest contiguous patch of unassigned p38α residues connects the N- and the C-terminal lobes of p38α (Supplementary Fig. 2a,b). This connection is mediated by the DFG loop and stretches into the MAPK insert in the C-terminal lobe. Titration with BIRB796 (Fig. 1d), a prototypical DFG-out inhibitor that binds and stabilizes the DFG loop in the inactive 'out' conformation36, resulted not only in chemical shift perturbations of residues near the BIRB796 binding site but also in altered microsecond-millisecond intermediate timescale p38α dynamics, as a total of 15 additional peaks were observed in the spectrum. Thus, BIRB796 changes the conformational equilibrium of all DFG loop residues, including Phe169 (ref. 36), as well as additional residues in this region (Supplementary Fig. 2c). This is notable as this finding correlates well with a recently introduced model for the catalytic reactions of kinases that is based on hydrophobic spines that coordinate internal kinase dynamics37. The hydrophobic spine, the R-spine (Phe169, unassigned; Leu75, unassigned; Leu86, assigned; His148, assigned), is centered on the DFG loop. Our data show that stabilization of Phe169 (for example, by an inhibitor such as BIRB796) and, in turn, other spine residues such as Leu86 influence kinase dynamics.
We used NMR spectroscopy to define the complete p38α–HePTP resting-state complex interface at atomic resolution (Fig. 2a–e). In this experiment, [2H,15N]p38α was incubated with unlabeled HePTP (constructs used in this study are illustrated in Fig. 2a), the 78-kDa p38α–HePTP complex was purified by size-exclusion chromatography and a two-dimensional 1H,15N-TROSY spectrum of the complex was recorded. The complex interacts tightly (Kd = 2.5 ± 0.6 μM; Table 1 and Supplementary Fig. 3) and is stable for days at 23 °C. The spectrum is of excellent quality for a complex of this size (78 kDa), allowing the chemical shift perturbations in p38α caused by HePTP binding to be readily detected (Fig. 2b).
Direct comparison of the two-dimensional 1H,15N-TROSY spectra of free and HePTP-bound p38α revealed chemical shift perturbations in 45 peaks, 34 of which were more than 4 s.d. (σ) above the mean (Fig. 3a,b). As expected, a subset of the chemical shift perturbations were localized to the KIM binding pocket, including helix αD, the αD-αE loop, the β7-β8 reverse turn, the ED site and the CD site (the latter two sites are often collectively referred to as the acidic binding pocket). However, perturbation of residues that are distal to the KIM binding pocket were also detected, including multiple residues from the αC-β4 loop, the αE-β7 loop, helix αF and the αF-αG loop. To our knowledge, this demonstrates for the first time that residues outside the p38α KIM binding pocket contribute to HePTP interaction and thus are important for binding.
The structure of the HePTPKIM peptide (HePTP residues 16–31) in complex with ERK2 (ref. 13), as well as three structures of KIM peptides (one with MEF2a and two with MKK3b) bound to p38α (ref. 7,8), have been reported. In the p38α–KIM peptide complexes, no interaction of the peptides with the CD domain (Asp313 and Asp316) was observed. In contrast, the HePTPKIM peptide makes multiple contacts with the CD domain of ERK2. Our chemical shift perturbation data with the p38α–HePTP complex unequivocally confirm the interaction of HePTP with the p38α CD and ED domains in solution, which involves CD domain residues Gln310–Asp313, Asp315–Glu317 (Pro314 is undetectable in the two-dimensional 1H,15N-TROSY spectrum) and ED residues Glu160 and Asp161. Thus, HePTP clearly interacts with both the ED and the CD sites in the p38α–HePTP complex and, based on the observed chemical shift changes, most likely adopts a conformation similar to that observed in the ERK2–HePTPKIM peptide complex. This demonstrates a critical point for MAPK selectivity: the sequence of the KIM, and not the identity of the MAPK, defines how KIM peptides from different MAPK effector proteins bind and interact with MAPKs.
To identify the residues of HePTP interacting with p38α, we isolated the individual domains of HePTP that most likely contribute to p38α binding, namely the KIM peptide (HePTPKIM31; Fig. 2a) and the HePTP catalytic domain (HePTPCAT; Fig. 2a). We then used NMR chemical shift perturbation mapping experiments to determine which of these domains participate in binding. First, we probed the interaction of the HePTPKIM31 peptide with p38α at four different protein-peptide ratios (Supplementary Fig. 4). Upon addition of the HePTPKIM31 peptide to [2H,15N]p38α, 35 large chemical shift changes corresponding to residues in the p38α C lobe and Leu108 in the hinge region were observed. Consistent with the p38α–HePTP complex chemical shift perturbation experiment, no substantial chemical shift differences were observed for peaks corresponding to residues in the p38α N-lobe (Figs. 2c and 3a,b). Remarkably, the total number of perturbed peaks was lower in the titration of p38α with HePTPKIM31 than in the p38α–HePTP complex. Whereas complex formation between p38α and HePTP resulted in large chemical shift changes in multiple residues from helix αD, the αD-αE loop, the β7-β8 reverse turn, the ED site and the CD site, titration with HePTPKIM31 resulted in fewer shifts in these regions (Fig. 3a,b). Moreover, titration with HePTP resulted in multiple shifts outside the KIM that were not observed with HePTPKIM31 alone. This shows that, though HePTPKIM31 binds and interacts with p38α, it does not recapitulate the shifts observed with HePTP, demonstrating that surfaces outside the KIM binding pocket are essential for the formation of the p38α–HePTP complex.
We also investigated the role of the HePTP residue Val31. Sequence alignments with other MAPK KIMs predicted that the HePTP ΦA-X-ΦB residues correspond to Leu27-Xaa-Leu29. However, the crystal structure of the ERK2–HePTPKIMpeptide complex13 revealed a register shift in the ΦA-Xaa-ΦB sequence in which Leu27 binds in a third hydrophobic pocket, ΦA-2, and Leu29 actually binds in the ΦA pocket. Similar ΦA-2 and ΦA interactions have been seen for the MEF2a peptide with p38α (ref. 8). Thus, Val31 putatively binds in the ΦB pocket. However, the absence of electron density observed for Val31 indicates flexibility and thus potential dispensability for binding. This is consistent with the observation that ERK2-bound KIM peptide conformations are heterogeneous, with only weak overlap between corresponding ΦA and ΦB residues. In contrast, the KIM peptides bound to p38α adopt highly similar conformations, and the corresponding ΦA and ΦB residues overlap nearly perfectly (Fig. 4a,b and Supplementary Fig. 5).
Because the ΦB interactions between KIM peptides and p38α are so highly conserved, we hypothesized that Val31 is not dispensable but instead has an important role in binding to p38α, which we examined again using NMR chemical shift mapping with the peptide HePTPKIM30, which lacks Val31, at four different protein-peptide ratios (Supplementary Fig. 4). The addition of Val31 resulted in a considerable increase in the number of residues that experienced chemical shift perturbations: from 23 (HePTPKIM30) to 35 (HePTPKIM31) (Fig. 3a,b). The localization of the majority of additional shifts to the ΦB binding pocket indicates that this is the binding site for Val31 (Fig. 4b). Remarkably, the addition of Val31 also resulted in a chemical shift perturbation of Leu108 in the hinge region, which connects the N and C lobes of p38α. This suggests that the domain closure mediated by the hinge in MAPKs upon KIM-peptide binding most likely requires occupation of the ΦB binding pocket. Unexpectedly, increases in the number of chemical shift perturbations for residues that contribute to the ΦA-2 and ΦA binding pockets, as well as the acidic binding pocket, were also observed for HePTPKIM31. Taken together, these data suggest that HePTPKIM31 interacts more tightly with p38α than HePTPKIM30. This was confirmed using ITC, which showed that the HePTPKIM31 binds p38α with a Kd of 5.2 ± 1.3 μM (Table 1 and Supplementary Fig. 3), whereas the Kd for the HePTPKIM30-p38α interaction was sufficiently weak that it could not be accurately determined. These results demonstrate that HePTPKIM31 occupies all three hydrophobic binding pockets (ΦA-2, ΦA and ΦB) in p38α and provide additional evidence that the mechanism of MAPK binding is determined by the KIM sequence and not the MAPK.
Crystallographic studies of MAPK–KIM peptide complexes have also shown that docking interactions alter the structure of the activation loop, resulting in a more 'active-like' conformation of the MAPK7–13,18. To determine whether the same changes occur in solution, we examined the chemical shifts of the p38α activation loop residues (172–183) in p38α alone and bound to either the HePTPKIM31 peptide or the HePTP protein. Remarkably, no chemical shift perturbations were observed for any of the peaks corresponding to activation-loop residues (10 of the 12 residues are assigned, including the Thr-Gly-Tyr motif). This demonstrates that, in solution, binding of HePTPKIM31 or HePTP to p38α does not change the local environment of activation-loop residues.
It is well known that the HePTPKIM is necessary for the formation of a stable p38α–HePTP complex15. However, it is unknown whether weak NMR-detectable interactions (mM) occur between p38α and HePTPCAT. Thus, we probed the interaction of p38α with HePTPCAT, which lacks residues 1–43 but includes HePTP α0, a secondary structural element that is unique to the KIM-PTP family among the classical PTPs38. Surprisingly, we detected absolutely no interactions between p38α and HePTPCAT (Figs. 2d and and3b).3b). This was unexpected because activated p38α is dephosphorylated by HePTP15, and thus, at some point during catalysis, the phosphorylated tyrosine of the p38α activation loop must bind the HePTP active site. Thus, all chemical shift perturbations observed for the p38α–HePTP complex are mediated by residues N-terminal to HePTPCAT, namely, the HePTPKIM31, and the residues immediately C-terminal to the HePTPKIM31 known as the kinase specificity sequence (KIS), namely, the HePTPKIS (ref. 16).
To confirm this hypothesis, we performed reverse NMR titration experiments with [2H,15N]HePTP and unlabeled p38α. The two-dimensional 1H,15N-TROSY spectrum of HePTP overlaps nearly perfectly with that of HePTPCAT, demonstrating that there is no interaction between the residues N-terminal to the catalytic domain (residues 15–43) and HePTPCAT (Supplementary Fig. 6a). This allowed us to transfer the previously published assignment of HePTPCAT (ref. 39) to that of HePTP. Additionally, the new peaks in the spectrum, owing to the additional 29 residues at the N terminus, were clustered at ~8.0 p.p.m., a chemical shift range typical for unstructured residues. We also detected chemical shift perturbations for residues 44–56 corresponding to helix α0, which indicate that the flexibility of residues 15–43 alters the chemical environment of residues 44–56.
To test the effect of p38α binding, the complex between [2H,15N]HePTP and unlabeled p38α was formed, and a two-dimensional 1H,15N-TROSY spectrum of the 78-kDa complex was collected (Supplementary Fig. 6b). All crosspeaks that clustered at ~8.0 p.p.m. in the free HePTP spectrum were perturbed in the spectrum of the p38α–HePTP complex, demonstrating that the flexible residues in HePTP, 15–56, bind strongly to p38α and adopt an ordered conformation. Thus, HePTP residues that mediate strong and specific p38α binding include not only the HePTPKIM31 peptide but also the HePTPKIS, which includes helix α0 (residues 31–56) and thus HePTP residues 15–56 (Supplementary Fig. 6c).
To validate these results, we generated the HePTPKIMKIS peptide, which corresponds to HePTP residues 15–56. The HePTPKIMKIS peptide was directly titrated into [2H,15N]p38α (it also copurifies with p38α using size-exclusion chromatography). As predicted by our earlier studies, the chemical shift perturbation mapping of the HePTPKIMKIS peptide bound to p38α almost completely recapitulated that observed in the p38α–HePTP complex, demonstrating that all of the interactions between p38α and HePTP are mediated by the HePTPKIMKIS peptide (Figs. 2e and 3a,b). We also performed a titration with 15N-labeled HePTPKIMKIS peptide and unlabeled p38α as an additional experiment to show that all residues of the HePTPKIMKIS peptide bind to p38α (Supplementary Fig. 7). Notably, the HePTPKIMKIS peptide interaction with p38α showed a few additional chemical shift perturbations. Correspondingly, it had an approximately three-fold increase in binding affinity over HePTP, as measured by ITC (Table 1 and Supplementary Fig. 3). The additional interaction is most likely due to nonpolar contacts between HePTPKIS hydrophobic residues (Ile40, Val43, Pro46 and Val49) and p38α residues (Leu217, Thr218, His228, Asn269, Asn272, Phe274, Ile275 and Ala277). These p38α residues showed larger chemical shift perturbations when bound to the HePTPKIMKIS peptide than when in complex with HePTP. Thus, both NMR and ITC data show that the presence of HePTPCAT slightly weakens the interactions observed for p38α residues that preferentially interact with the HePTPKIMKIS peptide.
SAXS provides information on the global size and shape of a protein in solution. The experimental scattering curve represents the average scattering of all conformations weighted by the number of molecules in each conformational state. We therefore used SAXS to determine the size and shape of the individual p38α and HePTP proteins and the p38α–HePTP complex in solution. SAXS profiles (I(q) versus q, with intensity I and scattering vector q) and pair- distribution functions are shown in Figure 5a,b (p38α–HePTP complex) and Supplementary Figure 8 (p38α and HePTP alone). The I0-to-concentration ratio was consistent for measurements of at least two concentrations, demonstrating no detectable aggregation and thus confirming the high quality of the samples. The experimental radius of gyration (Rg) value of 33.2 ± 0.5 Å for the p38α–HePTP complex was determined from Guinier analysis (n = 4; Supplementary Table 1). As expected, this was considerably larger than that for either protein alone (Rg = 23.7 ± 0.4 Å for p38α and 24.2 ± 0.2 Å for HePTP). Ab initio determination of the molecular envelope also converged on an Rg of 34.1 Å for the complex. These values for Rg are, respectively, 7.2 Å and 8.1 Å larger than what is predicted for a globular protein with the same number of amino acids40. Collectively, these results demonstrate that in the resting state the p38α–HePTP complex is highly extended, with p38α and HePTP associating in a head-to-head, head-to-tail or tail-to-tail manner.
To further our understanding of the p38α–HePTP complex, we developed a new computational procedure, ensemble refinement of SAXS (EROS)-NMR, which allowed us to determine the relative orientation of p38α and HePTP that provides the best fit to the experimental SAXS data. EROS-NMR includes the key features of EROS41: a coarse-grained model for protein binding is used to simulate the molecular assembly, which generates an initial ensemble of starting protein conformations. This ensemble is subsequently refined to improve the fit to the SAXS data. In addition, EROS-NMR accounts for NMR chemical shift data by incorporating a bias in the initial p38α–HePTP simulation. Namely, a nonspecific attractive potential was applied between the HePTPKIS linker (HePTP residues 32–56) and the p38α residues (Asn114, Asn115, Lys118, Lys121, Ser154, Gly219, Arg220) that fulfill three conditions: they show large chemical shift changes, they are surface exposed and they are not in the vicinity of the p38 KIM binding pocket. The bias potential was weak so that the HePTPKIS linker could bind and unbind repeatedly from p38α during the course of the simulation. The conformational space occupied by these rigid bodies was thoroughly sampled by simulation to identify conformations that best fit the SAXS data.
The best fit to the experimental data (χ2 = 1.3) was obtained for an ensemble of four conformations, which includes the single-best-fit model (SBFM) with the highest relative weight (47%), as well as three additional models (relative weights 23%, 16%, 14%; Fig. 5c,d and Supplementary Fig. 9). This fit is considerably better than that observed for the SBFM alone (χ2 = 1.5), for an ensemble that includes only two models (χ2 = 1.4), or for ensembles with five or more models, as the inclusion of more models only marginally improves χ2. All models in the four-model ensemble are globally similar, with p38α and HePTP associating in an end-to-end manner such that the HePTPCAT is localized to the bottom of the p38α C-terminal domain. They only differ in the position of the HePTPCAT domain. In fact, overlaying the models using p38α suggests that in solution the HePTPCAT fluctuates in a fan-like motion below p38α about the central SBFM conformation, with the weights of the additional models steadily decreasing as the distance from the SBFM increases (Supplementary Fig. 9). These changes in the position of HePTPCAT are accommodated by changes in the flexible HePTPKIS peptide, which adopts one of two classes of conformations: it either extends back toward the ATP binding pocket before bending down toward the bottom of the p38α C-terminal domain (the dominant SBFM conformation belongs to this class), or it bends down immediately after the last HePTPKIM residue. Remarkably, in none of the four structures was the active site of HePTP (Cys270) localized near the p38α activation loop, an interaction that must occur when HePTP dephosphorylates activated p38α. To quantify this important point, we also performed a full maximum-entropy refinement of 1,000 simulation structures41. The region of conformational space spanned by the resulting ensemble is consistent with that spanned by the smaller four-model ensemble, with, at most, 1% of structures in which the Cα atoms Tyr182 (p38α) and Cys270 (HePTP) are within 18 Å of each other (Supplementary Fig. 10). Notably, the corresponding structures extend the fan-like motion previously seen for the four-conformer ensemble toward the p38α activation loop (Supplementary Fig. 9). Thus, under resting-state conditions, HePTP and p38α adopt an elongated end-to-end complex that rarely (<~1%) samples the orientation required for dephosphorylation.
It is commonly accepted that KIM peptides are only partially responsible for recognition of effector proteins by MAPKs, as multiple studies have shown that additional interactions between MAPK effectors and substrates are required to maintain the exquisite specificity observed in MAPK signaling pathways16–18. Here, we have combined the molecular detail provided by NMR spectroscopy with the overall molecular organization afforded by SAXS to determine which residues outside the HePTPKIM31 direct and stabilize the resting-state conformation of the p38α–HePTP complex. One of the central findings of this study is that the HePTPKIM31 peptide alone is not able to recapitulate the interactions observed in the p38α–HePTP complex. Instead, multiple NMR titration experiments of p38α with HePTP constructs of varying lengths and domain architectures, coupled with ITC, reveal that both the HePTPKIM31 and HePTPKIS sequences are necessary and sufficient to mediate all binding interactions with p38α. To our knowledge, this is the first time that residues outside a KIM of a MAPK effector protein have been determined at the molecular level.
Using SAXS, we have also shown that in solution the interactions mediated by the HePTPKIMKIS lead to the formation of a p38α–HePTP complex in which p38α and HePTP interact in an elongated, end-to-end manner. This was unexpected because phosphorylated p38α is dephosphorylated by HePTP, which requires the formation of a more globular complex. However, it is clear from our SAXS studies that under resting-state conditions the p38α–HePTP complex is not globular but instead is highly elongated. This result differs considerably from that of a recently published crosslinking study42 in which a resting-state PTP-SL–ERK2 complex was crosslinked and the structure of the complex was modeled using the crosslinking distances as constraints. Jointly, these results demonstrate that the overall orientation of MAPK–KIM-PTP resting-state complexes may differ depending on the MAPK, the KIM-PTP or both.
To generate a structural model for the p38α–HePTP complex in solution, we developed a new SAXS refinement procedure, EROS-NMR, which incorporates NMR chemical shift data into a recently developed SAXS refinement procedure. Incorporation of the NMR chemical shift data ensures that the models are consistent with not only the SAXS data, which define overall shape, but also the NMR chemical shift data, which define molecular interactions that direct the organization of the complex. As expected on the basis of multiple failed attempts to crystallize the resting-state complex, the SAXS refinements show that the complex is dynamic, as four models provide a better fit to the SAXS data than a single model. The models in the four-model ensemble have similar overall conformations, which differ primarily in the relative orientation of the HePTPCAT below the p38α C-terminal domain. The HePTPKIS conformation is variable, but adopts highly similar conformations as it projects downward from the p38α KIM binding pocket. The largest differences are observed in the C-terminal portion of the HePTPKIS, which adopts an extended conformation and interacts differentially with p38α in the four ensemble models of the p38α–HePTP complex. This shows that the N-terminal HePTPKIS residues interact directly with p38α, whereas the C-terminal HePTPKIS residues transiently bind p38α, allowing additional conformations to be sampled about the central dominant conformation (the single-best-fit conformation). This model is also consistent with a slight decrease in binding affinity between HePTPKIMKIS and HePTP with p38α, as measured by ITC, showing that this flexibility slightly lowers the overall strength of binding.
Taken together, these results provide fundamental new insights into the molecular basis of MAPK regulation and specificity. As additional similar studies are completed, the subset of residues outside the KIM binding pocket that are specific for different MAPK–MAPK regulatory protein complexes will be determined and, in turn, will allow for the development of new drug therapies that target these specific complexes.
The HePTPKIM31 peptide (VRLQERRGSNVALMLDV) was synthesized and HPLC purified (> 98% purity; Biosynthesis); the HePTPKIM30 peptide (RLQERRGSNVALMLD) was synthesized and HPLC purified (>98% purity; gift from J. Offer, University of Cambridge). Expression and purification of HePTP and HePTPCAT were carried out as previously described14,38 except that the final size-exclusion chromatography (SEC) step was carried out using p38α protein buffer (50 mM HEPES, pH 6.8, 150 mM NaCl, 5 mM dithiothreitol). p38α and the HePTPKIMKISC42S peptide were subcloned into a derivative of the pET28 vector that includes an N-terminal His6 tag and a TEV protease–recognition sequence43. Unlabeled proteins were expressed in BL21-(DE3) RIL Escherichia coli cells (Stratagene) in LB medium. [2H,15N]p38α and HePTP were expressed in M9 medium supplemented with 15NH4Cl (1 g l−1) in 99% D2O, and [2H,1315N]p38α was expressed in M9 medium supplemented with 15NH4Cl (1 g l−1) and [13C]d-d7-glucose (4 g l−1) in 99% D2O. p38α and the HePTPKIMKIS were purified using standard protocols (Supplementary Methods). For p38α–HePTP complex formation, p38α was incubated with HePTP (1:1 molar ratio) on ice for 45 min and purified using SEC equilibrated in p38α protein buffer. The p38α–HePTP complex elutes at the position expected for the p38α–HePTP heterodimer. The [2H,15N]p38α–HePTP complex and the p38α–[2H,15N]HePTP complex were concentrated to 0.2 mM and stored on ice (<8 h) until NMR data acquisition.
All NMR experiments were performed at 298 K on a Bruker Advance II 800 MHz spectrometer equipped with a TCI HCN z-gradient cryoprobe. NMR samples were prepared in protein buffer in 90%/10% (v/v) H2O/D2O. The NMR spectra were processed with Topspin 2.1 (Bruker) and analyzed using the CARA software package (http://www.nmr.ch/doku.php). Purified [2H,13C,15N]p38α was concentrated to 0.58 mM and stored on ice (<8 h) until data acquisition. The sequence-specific backbone assignment was obtained from TROSY versions of the following experiments: two-dimensional 1H,15N-TROSY, three-dimensional HNCA, three-dimensional HNCACB, three-dimensional HN(CO)CA, three-dimensional HN(CO)CACB and a three-dimensional 15N-edited 1H,1H-NOESY spectrum. The assignment of residues around the ATP binding pocket were confirmed by titration of 2′,3′-(2,2,5,5-tetramethyl-3-pyrroline-1-oxyl-3-carboxylic acid ester) adenosine triphosphate (SL-ATP, a generous gift from P. Vogel, Southern Methodist University)34 into [2H,13C,15N]p38α; final ratios were 0.16 mM p38α/0.32 mM SL-ATP (1:2) and 0.16 mM p38α/0.64 mM SL-ATP (1:4). Two-dimensional 1H,15N-TROSY spectra were recorded. All quantitative spin-label experiments were processed with NMRPipe44, and peak integrals were determined using CARA (http://www.nmr.ch/doku.php).
Both HePTPKIM31 and HePTPKIM30 peptides were solubilized in protein buffer. Experiments were performed at ratios of 1:1, 1:3, 1:5 and 1:8 excess peptide, which were added to [2H,15N]p38α, and a two-dimensional 1H,15N-TROSY spectrum was recorded (100 μM p38α; 1:1 experiment was also recorded at 250 μM p38α). A two-fold excess of unlabeled HePTPCAT was added to [2H,15N]p38α (0.2 mM p38α/0.4 mM HePTPCAT), and a two-dimensional 1H,15N-TROSY spectrum was recorded. An equal molar ratio of unlabeled HePTPKIMKIS was added to [2H,15N]p38α (94 μM p38α/94 μM HePTPKIMKIS), and a two-dimensional 1H,15N-TROSY spectrum was recorded. The [2H,15N] p38α–unlabeled HePTP complex was purified and concentrated to 0.2 mM, and a two-dimensional 1H,15N-TROSY spectrum was recorded. p38α–HePTPKIM30 titrations in ratios of 1:1, 1:3, 1:5 and 1:8 were used to follow chemical shift changes in p38α upon binding, as nearly all peaks showed fast exchange and thus could be easily followed. This data was used for the analysis of the 1:1-, 1:3-, 1:5- and 1:8-ratio p38α–HePTPKIM31 titrations and all other interactions (HePTPKIMKIS and HePTP), allowing for high confidence of peak assignments of p38α in the bound state. All chemical shift differences (Δδ) were calculated using the following equation:
We adapted the EROS method for the refinement of SAXS data41 to incorporate NMR data. EROS was previously used to determine the structure of protein kinase C in solution45, and a related method has been used to refine SAXS data of Hck kinase46. In the simulations of the p38α–HePTP complex, a nonspecific, attractive potential was applied between the KIS linker (HePTP residues 32–56) and the p38α residues (Asn114, Asn115, Lys118, Lys121, Ser154, Gly219, Arg220) that fulfill three conditions: they show large chemical shift changes, they are surface exposed and they are not in the vicinity of the p38 KIM binding pocket. The bias potential increased the well depth of the amino acid interactions by only 1 kBT, such that the KIS linker could easily bind and unbind from p38α during the course of the simulation. The scattering intensities Ik(q) were calculated for all simulated structures k, and the ensemble intensity was defined as
where wk is the relative weight assigned to structure k in an ensemble of n structures. The difference between computed and measured intensities was quantified by
where N is the number of data points and a and b are fitting parameters obtained from conditions χ2/a = χ2/b = 0. The parameter a sets the intensity scale and the parameter b << Iexp(q=0) is a small background correction. In the SAXS profile calculations, the hydration-shell electron-density contrast ρ was also varied, and best fits were obtained for ρ = 0.005e/Å3. To fit the experimental data with a small number of simulation structures, we defined a function G = χ2 + μn, which was minimized numerically by varying the number of structures n with nonzero weights and their weights wk under the constraint Σkwk = 1. Here μ > 0 is a control parameter that captures our preference for having a small number n of structures in the ensemble, analogous to a chemical potential. As μ is decreased, n increases and the value of χ2 decreases upon minimization of G. Beyond n = 4, the quality of the fit does not improve substantially (with χ2 = 1.3, 1.4 and 1.5 for n = 4, 2 and 1, respectively). For reference, we also performed a full maximum-entropy refinement over the entire ensemble41, which produced an ensemble consistent with the region of conformation space spanned by the set of n = 4 structures.
Methodological details of additional p38α–HePTP interaction studies, isothermal titration calorimetry and SAXS are described in Supplementary Methods.
The authors thank L. Yang and M. Allaire (National Synchrotron Light Source, NSLS) for their support at NSLS beamline X9. The authors thank P. Vogel (Southern Methodist University) for providing SL-ATP for the titration studies. This research was supported by grant RSG-08-067-01-LIB from the American Cancer Society to R.P. B.R. was supported by a Marie Curie International Outgoing Fellowship within the Seventh European Community Framework Program. B.R. and G.H. were supported by the Intramural Research Program of the National Institute of Diabetes and Digestive and Kidney Diseases, US National Institutes of Health (NIH). Use of the NSLS at Brookhaven National Laboratory was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences under contract no. DE-AC02-98CH10886. 800 MHz NMR data were recorded at Brandeis University and were purchased with support from NIH S10-RR017269. Molecular simulations were performed on the Biowulf computing cluster at NIH.
Author contributions D.M.F. and D.K. cloned and purified all proteins. D.M.F. and W.P. performed and analyzed all NMR measurements. D.M.F., W.P. and R.P. performed and analyzed the SAXS measurements. D.K. performed all ITC measurements. R.P. and W.P. helped design the biochemical and NMR experiments. B.R. and G.H. performed all EROS and EROS-NMR calculations. R.P. and W.P. wrote the paper and all authors discussed the results and commented on the manuscript.
Competing financial interests The authors declare no competing financial interests.
Additional information Supplementary information is available online at http://www.nature.com/naturechemicalbiology/.
Accession codes. Protein Data Bank: the previously published structures of p38 alone, in the presence of SB203580 and in complex with BIRB796 are deposited under accession codes 1P38, 1A9U and 1KV2, respectively. Biological Magnetic Resonance Bank: chemical shifts recorded in this study are deposited under accession number 17471.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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|By The Bos
|More from The Bos
|Very Positive
|The Dai Li Arise
|Chapter information
Avatar: Guardian
|Written by
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|Release date
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The Dai Li Arise is the third chapter of Avatar: Guardian.
The Team searches the skies for signs of the Phoenix Warriors. After deciding that they are gone, they return for a nights rest. The next day, while they are interrogating a captured Phoenix Warrior, the Dai Li attack.
Toph creates a back to the invaders casing and stands them upright. More civilians arrive to attend to the wounded. Zuko orders the recently arrived imperial guard to move the invaders to the interrogation room Ozai had built. He adds not to execute them.
Aang, startled by this, exclaims "Wait, What?"
Zuko, understanding his distress, assures him "Don't worry Aang, I said not. I'm making sure the guards don't follow Ozai's policy."
The Team gets on Appa and soar into the air. They circle the capital and the route down to the Royal Plaza. They find no ships or signs of a battle. Toph exclaims "Wait! There they are!" while pointing in front of her. The entire team turns towards her then toward the place she was pointing at, then back at her, irritated. "Jeez, I thought you'd have learned by now..."
Zuko, ignoring Toph's statement, says "Obviously we can't find them. We should go back and get some rest. We can interrogate the traitors tomorrow. I have rooms prepared for all of you."
The team agrees and Aang circles Appa back to the capital.
The next day, in a dimly lit room the invader is secured in a rock sleeve covering all but his head. The invader was staring at the wall, blankly, waiting for what he expected to be a long interrogation. Zuko and Aang walked in followed by Katara and Toph. Zuko got right down to business, asking "Who is your leader?"
Ignoring everyone, the prisoner continued staring at the wall, and muttered "You will not get anything out of me. You will not get anything out of any of us. We have all taken an oath to die before being traitorous vermin... like the rest the nation."
Zuko again, asks a straight forward question "Where is my father?"
The invader never even looked up while answering "The Phoenix King is safe; far away from his scourge of a son."
Aang asked him who he was working for. This time the invader looked up at Aang, before turning his head to look at the rest of Team Avatar. "None of you are very bright, are you? I told you, I don't talk."
The lanterns begin to get brighter as Zuko's temper begins to flare. Aang calms him down as he asks the prisoner why they took Ozai. The prisoner took a liking to that question, beginning to rant "We believe that the war is not over. The Fire Nation is still superior to the other savage tribes that claim to be nations. We will burn down this world and rise from the ashes..." As he finishes the word, massive crashes are heard from outside the room.
Toph immediately identifies the crashes as walls being destroyed. Zuko, in disbelief, tells her "These walls are made of solid rock feet thick. No one in the Fire Nation could break them down."
A mysterious voice echoes throughout the room "Good thing we're not from the Fire Nation!" As the voice echoes, rock bullets fly at the group. Aang and Toph create shields of earth to block the bullets. Several Dai Li agents enter the room, either by coming from the ceiling, floor or walls. Aang, trying to solve the situation without fighting, sinks while sinking the wall to get a view of some of the agents, while asking them "What do you want?"
All the Dai Li agents reply in unison "Revenge!" before launching rocks at the team.
Toph and Aang create more earth walls and stop the attacks. Aang sinks into the earth and appears behind an agent blasting him away with an air wave. Toph breaks her section of the wall into smaller pieces and launches them at different Dai Li agents. Some agents manage to stop the boulders but Zuko's agent dodged a fireball right into Toph's boulder.
Zuko compliments her on the timing of the attacks. Toph replies while sinking a Dai Li agent in earth "Yeah, I am awesome like that." Another agent frees him and starts firing more rock bullets at Zuko, who creates a fire wall to absorb them. A Dai Li agent jumps off of the ground toward Toph, and Zuko uses a fire whip to knock him away. The agent falls to the ground, before spring boarding into the wall and vanishing.
Aang asks "What was that about?"
Toph replies with her usual sass "Judging from what they said, revenge." Katara asks what they would want revenge for.
Zuko says he has an answer to the question "Shame. When you've lost your honor, you want it back. They probably felt shameful after a group of kids beat them. A lot. Then they were disbanded. Plus you imprisoned their leader, then I-" pauses while looking at Katara "we defeated their new leader."
Aang agreed with Zuko "We need to get the rest of the team back if we're going to be ready for what happens next." The rest of the team agrees. They leave while forgetting the prisoner who was still staring at the wall, which has massive holes in it.
The Dai Li Report
Later, on an Earth Kingdom boat, a man is standing on the deck looking at the sunset. Another few men appear and bow in respect. One speaks "Sir, we failed."
The leader coolly says in a stern, harsh voice "Since I was freed, there has been one failure after another. I will not stand for this!" He turns around in rage "Long Feng will not stand for this!"
The same agent tries to make up for what he said, "We will not fail again. The Dai Li will never betray you."
Long Feng brushes this off, bringing up their recent failures in an increasingly angry tone "You failed to kill the Fire Lord with that cave in at his capital. Then you failed facing him directly." He pauses for a moment to regain his composure "Why do you displease me?"
Another agent speaks "If I may, sir: we managed to free you."
Long Feng acknowledges this, "That you did, but you were the reason I was imprisoned to begin with! You all betrayed me!"
Yet another agent tries to claim his loyalty to his leader, but Long Feng grows too angry and orders them all to leave, before turning to look back out at the sunset.
- The Phoenix Warrior's actions were inspired by the Joker in the Dark Knight.
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Dream. Dare. Do – that is Suyati's work principle in a nutshell.
As businesses continue to face security threats, cyber security has become one of the top most priorities for business honchos and government leaders alike. In recent times, there has been a paradigm shift from channeling resources towards investing in software security mechanisms to turning to the cloud as an effective solution to counter cyber terrorism.
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Despite countless hours of evaluating and researching prospect's tendencies and talent levels, there really is no way to tell with 100 percent certainty a player will be successful at the Power 5 level. A fifth star doesn't mean an addition 200 rushing yards a year and a 2-star recruit's ceiling isn't just a special teamer.
Since I'm an eternal optimist, I went ahead and looked at some of the "overachievers" in the Leach era, starting with his first recruiting class at WSU that was put together over about eight weeks after Leach was hired.
2012: 2-star defensive end Destiny Vaeao
Vaeao was a solid contributor as a sophomore in 2013 with 24 tackles, 3 1/2 tackles for loss and 2 interceptions. He was good, but not great, as a junior with 14 tackles in 10 starts. But Vaeao soared to new heights in a senior campaign that saw him rack up 41 stops including 12 tackles for loss and 4.5 sacks along with second team All-Pac-12 honors. When it was all said and down, the 2-star recruit who never redshirted posted 84 tackles (47 solo) and got to the quarterback seven times during his Cougar career, with 19 tackles for loss. In his rookie season in the NFL this year, he's played in every game for Philadelphia with 13 tackles, 2 sacks and a forced fumble.
2013: 2-star guard Cody O'Connell, 3-star safety Isaac Dotson
O'Connell is the overachiever of this group of overachievers. "The Continent" has always had the size, he was 6-foot-8 and 350 pounds out of high school, but the 2-star recruit massively transformed his game this season at Washington State. A first-year starter in 2016, it was hoped back in August that O'Connell could do a credible job in his first season as a starter and that his expected growing pains wouldn't be overly detrimental. Instead, the Wenatchee product was of the biggest stories of year in becoming only the second unanimous All-American in WSU program history (Jason Hanson). O'Connell, along with another overachiever later in this list, helped the Cougar offensive line become one of the best in the nation this season.
Dotson was ranked the No. 85 safety prospect nationally in the 2013 class and that's nothing to sneeze at. But he became a mainstay for the Cougars in the 2016 season – at linebacker. The Cougar WIL was fourth on the team in tackles (64) and visited the backfield consistently with six tackles for loss and two quarterback hurries. Throw in two interceptions and the former high school quarterback has ably made the transition to becoming a productive member of the Wazzu defense.
2014: 2-star offensive tackle Andre Dillard (pictured above); 2-star defensive lineman Hercules Mata'afa
While O'Connell had the bulk coming into high school, the man he shares the left side of the offensive line with has added 50 pounds since joining the Cougars and cracking the starting offensive line. Dillard in 2016 was assigned the task generally considered the most important on an offensive line - protecting Luke Falk's blind side. And while it seemed early on as though the legacy Cougar would contribute in his time at Washington State, he has arrived early, exceeded expectations and helped the offensive line not miss a beat in replacing current Detroit Lion Joe Dahl and longtime starter Gunnar Eklund.
Mata'afa, a 2-star prospect out of Lahainaluna, Hawaii, went under the radar in recruiting largely because he wasn't, well, large. Entering college, he stood at 6-foot-2 and 225 pounds, minuscule compared to other defensive lineman in the conference. But Mata'afa makes it work and then some. Still undersized at 6-2, 255 pounds, his natural strength is immense and he posted 45 tackles, 12 1/2 tackles for loss and four sacks in his first season starting, leading to a first-team selection on the Pro Football Focus All-Pac-12 team.
2015: 3-star cornerback Darrien Molton
Molton was the No. 128 cornerback in the 2015 class. In just two seasons and without the benefit of a redshirt year, he was the Cougars second-leading tackler (71) behind only linebacker Peyton Pelluer. The 5-foot-10, 175-pound cornerback is generally held to be the Cougars' top corner and will be one of the Pac-12's more seasoned defensive backs in the coming years.
2016: 2-star corner back Robert Taylor (JUCO)
Taylor made big plays on both the defense and special teams in the Cougars' eight-win season. In the secondary, he was top-five in tackles with 54 and recovered three fumbles in 2016. He also didn't waste a lot of time in coming from City College of San Francisco to live up to his nickname, Run It Back Rob, in ending the inglorious streak of Wazzu not having a kick returned for a touchdown since 2003 with his 100-yard mad dash against Arizona State.
AS IT STANDS NOW, Washington State has 21 known commitments in its 2017 class. You can bet at least one, and maybe more, will overachieve and find both the field and appreciation among the Cougar faithful earlier than expected.
An impressive list: Recruiting diamonds in the rough under Mike Leach at Washington State
Despite countless hours of evaluating and researching prospect's tendencies and talent levels, there really is no way to tell with 100 percent certainty a player will be successful at the Power 5 level. A fifth star doesn't mean an addition 200 rushing yards a year and a 2-star recruit's ceiling isn't just a special teamer.


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Hyderabad, Oct. 6 : All India Majlis-e-Ittehadul Muslimeen (AIMIM) president Asaduddin Owaisi on Tuesday lashed out at Uttar Pradesh Minority Welfare and Urban Development Minister Azam Khan over his assertion that he would take up the Dadri lynching case to the United Nations, saying it is the country's internal matter.
"This matter is an internal matter of the nation… This is a fight against the Samajwadi Party and other governments who have failed to fulfil their constitutional duties. The government's constitutional duty is to protect an individual irrespective of his religion," Owaisi told the media here.
"I want to ask Mulayam Singh Yadav if he backs the statement of Azam Khan. If he backs him, then he should ask for imposing the President's rule in Uttar Pradesh," he added.
Owaisi further said the BJP-led NDA Government at the Centre and the ruling Samajwadi Party in Uttar Pradesh have failed to curb the Sangha Pariwar outfits, who are involved in such incidents.
"But that doesn't mean that the Samajwadi Party should take this matter to the United Nations," he added. (ANI)


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Keywords: stuffings Yield: 10 servings Preparation Time: 15 minutes Cooking Time: 40 minutes Ingredients: 1/2 stick 1 each 2 ribs 1 clove 1/2 tsp 1/2 tsp 1 tsp 2 1/2 cups 8 cups 1 tbsp 2 tbsp 1/4 cup Butter onion ,diced Celery,diced Garlic,minced Salt freshly ground black pepper Sage,rubbed chicken broth bread cubes or croutons Fresh thyme leaves, chopped Fresh tarragon leaves -chopped Fresh parsley, chopped
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Or download a printable copy here.
The annual auction is our biggest fundraiser and is facilitated using an online catalog for collecting and viewing donations, and most importantly, bidding. Members, friends, and local businesses donate items and create events, filling the catalog with diverse and unique offerings.
Member events, such as meals, performances, and outings make this more than a fundraiser. It is also a way to connect with other members and enrich our relationships within the congregation.
Get Ready: Make sure your payment information is on file so online bidding is activated.
Get Set: Review the catalog at togetherauction.com/unitytemple to create your wish list and bidding strategy - then LOG IN to reveal bidding information and the place bid button.
Go Bid: Place your bid or buy outright with the Win It Now option. Select "Place Bid", enter Qty. and Bid Price, then SAVE.
If you are interested in helping with the 2017 auction, email us at firstname.lastname@example.com.
See Below: Frequently Asked Questions about Donating and Bidding FAQs
Email: email@example.com
Your Auction Team: Coaches Barbara Heskett and Laura Reeb, Corinne Rohr, Jerry Seeber, Tracy Welin, Jennifer Walters, Susan Huizinga, Connie Strait and Charlotte Lee.


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Free shipping To
If you think the price is too high, please click to apply for lower price
LED Light-up Rubber Slingshot Helicopter Toys for Kids - Orange (5PCS)
Free shipping
6-Sided Cube Dice Finger Toy - White + Deep Pink
Free shipping
6-Sided Cube Dice Finger Toy - Grey + Black
Free shipping
Triangular Hand Spinner Metal Finger Gyro Spiral Fingertips Toy -Black
Free shipping
Finger Peacock Raise Tail Optical Fiber Lamp Toy - Random Color (5PCS)
Free shipping
|4 piece(s) per pack
|Silver + Multicolored
|Alloy + color changing material
|4 size: diameter 16 / 17 / 18 / 19mm
|Other Features
|The ring will vary 12 different colors as your body temperature varies, and indicates different moods
|Packing List
|4 x Rings
All packages from DX.com are sent without DX logo or any information indicating DX.com. Due to package variations from suppliers, the product packaging customers receive may be different from the images displayed.
To enable volume discounts on this site, use coupon code: BULKRATE during checkout. You will see a discount applied at the bottom of the shopping cart. Competitive pricing is available. Contact us for details.
|3+ units
|5+ units
|10+ units
|US$2.93 shipped
|US$2.92 shipped
|US$2.91 shipped
What is Bulk Rate? BulkRate is a semi-wholesale system with items priced separately from retail. When you use bulk rates, a flat $1.70 registered air mail fee will automatically be added to your cart to ensure delivery of package. While BulkRate's intention is to offer cheaper prices when you buy in bulk, because it is priced separately it on occassions show a higher than retail price. That's why we ask you to enter BULKRATE as a coupon code to manually activate the rates. You can always opt not to use the bulk rates. If you are looking for wholesale prices please feel free to contact us.
Share your reviews, photos and videos here!Review Program
Comes in 4 sizes, one will probably fit your finger. When you put it in your finger, it changes color very fast. The ring is not confortable, the edges may be unconfortable after an extended use. I haven't saw colors you I... Read more
It works as advertised. It changes color... I like the glitter detail in it. I'll give a ring to each of my sisters. It doesn't have a guide or paper indicating what the colors mean. Two of the four rings are exactly the I... Read more
Comes in 4 sizes, one will probably fit your finger. When you put it in your finger, it changes color very fast. The ring is not the may... Read more
It works as advertised. It changes color... I like the glitter detail in it. I'll give a ring to each of my sisters. It doesn't have a or... Read more
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About Us
Adoption Info
Rescue Stories
We are a non-profit organization based in
Turlock, California
that saves and rescues unwanted and abandoned cats. Our main goal is to help control the animal population in our surrounding communities. We are a
rescue group. None of this could be possible if is was not for the help of our local veterinarians. Since 1996 we have found homes for over 10,000+ animals. In the year 2014 we adopted out 261 cats/kittens and 39 dogs and puppy's!
Be sure to check out our Facebook page!
Our 2016 Calendars Are Here!
The Friends of Turlock Animal Shelter is proud to announce the arrival of our 2016 fundraising calendar! Now you can purchase this calendar on our website, by clicking on the "Buy Now" button below.
All proceeds of the calendars will go to our spay and neuter assistance program for 2016. This calendar makes a great gift for that animal lover in your life.
Calendars cost $10 each.
There will be a $3 shipping fee.
Pick Up Your Shares Card!
Please pick up your Shares Card with John at Pet extreme on the weekend. When you use your card at the stores below, a percent will go to help the animals.
On behalf of Friends of Turlock Animal Shelter, we would like to thank Pedigree for their generous $1,000 operations grant. This donation has helped us with caring for our dogs! The money has helped with their vaccinations, microchiping, spaying and neutering, as well as many other things. Thank you Pedigree for helping us give better care to our dogs!
FRIENDS OF TURLOCK ANIMAL SHELTER recently celebrated their 14th year as a highly respected asset to the City of Turlock and a true friend to the animals in Stanislaus County.
Thank you for your trust and loyalty in HELPING US...TO HELP THE ANIMALS!
Friends of Turlock Animals is a 501(c)3 nonprofit corporation.
Animal lovers have been here since
November 16, 2007
Copyright © 2007 - 2016 Friends of Turlock Animal Shelter. All Rights Reserved.


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If an item you ordered from TextbookRush does not meet your expectations due to an error on our part, simply fill out a return request and then return it by mail within 30 days of ordering it for a full refund of item cost.
Learn more about our returns policy
Description: An authoritative bestseller for over fifty years, PERRINE'S LITERATURE: STRUCTURE, SOUND, AND SENSE continues to be an essential and highly effective introduction to literature for today's students. Written for students beginning a serious study of literature, the text introduces the fundamental elements of fiction, poetry, and drama in a concise and engaging way, addressing vital questions that other texts tend to ignore, such as "Is some literature better?" and "How can it be evaluated?" A remarkable selection of classic, modern, and contemporary readings serves to illustrate the elements of literature and ensure broad appeal to students of diverse backgrounds and interests. Now thoroughly updated with nearly 80 new stories, poems, and plays by some of the finest authors of any era, the eleventh edition remains true to Perrine's original vision while addressing the needs of a new generation of students.
Limited time offer:
Get the first one free! (?)
All the information you need in one place! Each Study Brief is a summary of one specific subject; facts, figures, and explanations to help you learn faster.
List price: $134.95Edition: 11thCopyright year: 2012Publisher: WadsworthPublication date: 4/18/2012Binding: HardcoverPages: 1760Size: 6.00" wide x 8.75" long x 1.75" tall Weight: 2.816Language: English
Thomas R. Arp received a B.A. in English from the University of Michigan (1954) and a Woodrow Wilson Fellowship to Stanford University. In 1955-1956, he produced educational television for the University of Michigan. He received an M.A. from Stanford University in 1960 and a Ph.D. from Stanford in 1962. He has taught at Bowdoin College, Princeton University, University of California at Berkeley, Hull University (England), and Southern Methodist University. Macmillan published his volume, THE FORM OF POETRY, in 1966, and he received a Fulbright lectureship at University of Bucharest (Romania) in 1969-1970. Arp joined Laurence Perrine in preparing revised editions of SOUND AND SENSE, STORY AND STRUCTURE, and LITERATURE: STRUCTURE, SOUND, AND SENSE beginning in 1982. He became sole author of the books in 1997, and was joined by Greg Johnson in 2002.
*A minimum purchase of $35 is required. Shipping is provided via FedEx SmartPost®
and FedEx Express Saver®. Average delivery time is 1 – 5 business days, but
is not guaranteed in that timeframe. Also allow 1 - 2 days for processing. Free shipping is eligible only in the continental United States and excludes
Hawaii, Alaska and Puerto Rico. FedEx service marks used by permission."Marketplace"
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While not needed for our canvases, solid wood framing in a contemporary shadow box style is available. Our substantial frame provides a polished, designer finish at a very nice price. Each frame size is offered in either a warm white finish or deep pewter (which has a slight undertone of metallic in the brown/black color). Your chosen art canvas will arrive installed in the frame you select, ready to hang from a wire hanger on back. Unframed stretched canvases achieve a more casual, laid-back look, opt for framing your canvas if you want a more sophisticated interior.
Please keep in mind that the frame will add 1½" to the dimensions of your canvas (both width and height), and the depth of the framed piece is 2⅛".
Our frames fit the following canvas sizes:
10"x10" 14″x14″ 18″x18″ 21″x21″ 24″x18″ 24″x24″ 30″x30″ 39″x39″ 30″x24″ 40″x30″


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Mayor Eileen Swan explained that the committee made a Regional Contribution Agreement (RCA), which involves the committee transferring the units to another municipality to help fulfill its state-ordered quota. It has not yet been decided which municipality will take on the units, and Swan said Lebanon Township could not fulfill its obligation to COAH because there was no demand for them.
Through RCAs, a municipality can satisfy up to 50 pecent of its total affordable housing quota.
In other news, Township Committeeman Gottfried Pletzer mentioned that there is no speed limit on Newport Road, and that some drivers take advantage of this, noting that "sometimes it gets a little bit hairy."
He said he has been contacted by township residents who had concerns about drivers speeding on the road. The committee agreed with Gottfried's request that the police do a study on this.
County Did Mediocre Job Repairing Bridge
Also, Mayor Swan discussed an historic bridge located on Hickory Run Road that has been repaired poorly. The bridge extends over Hickory Run Creek and the issue came up at a recent Planning Board meeting, where officials and residents discussed the repairs to the keystone bridge. Township Attorney Phillip George estimated the bridge may be about 100 years old and was a replacement for another smaller bridge that was probably also 100 years old. The committee decided to let county officials know about the mediocre repairs by sending them a letter.
Proposed Benefit Concert Could Attract 500 People
Mayor Swan also said she had been approached last Wednesday by a Cokesbury Road resident wishing to hold a benefit concert to raise money for those affected by the World Trade Center attacks. Attorney Phillip George said certain provisions were necessary. A non-profit event such as this would need to have provisions for crowd control, the serving of alcoholic beverages and traffic.
Swan said the resident, whose name was not available, expected between 250 and 5 00 attendees, and added that neighbors were aware of the possibility of this con cert. The committee decided that if the concert were to be delayed to a date later in the month than Saturday, Oct. 13, as originally intended, a permit could possibly be granted, but otherwise the time frame would not allow it. The necessary provisions would be for crowd control, traffic and the serving of alcohol.


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Hampton Inn Sheridan3
980 Sibley Circle, Sheridan, 82801
Price FromView Rooms
This hotel doesn't have any reviews yet.
About the hotel
the best of the west!. . .
welcome to the Hampton Inn Sheridan. Beautiful Sheridan is one of Wyoming's treasures, where legendary Western history and seeping mountain vistas meet New West comfort and the peace of wide-open spaces. The frontier spirit is alive and well at our hotel in Sheridan. We're located just a lasso rope away from lots of fun things to do, from rodeos and museum to skiing and horseback riding. The Hampton Inn® hotel in Sheridan is central to everything in this pretty piece of the west. Buffalo Bill's Wild West is alive and well wherever you turn, so get ready to re-live the life of a cattle baron, rancher or cowboy's life. You're also just down the street from several terrific museums, great western shopping and more, so welcome to our hotel in Sheridan! services & amenities Even if you're in Sheridan to enjoy the great outdoors, we want you to enjoy our great indoors as well. That's why we offer a full range of services and amenities at our hotel to make your stay with us exceptional. Are you planning a meeting? Wedding? Family reunion? Little League game? Let us help you with our easy booking and rooming list management tools. * Meetings & Events * Local Restaurant Guide
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- Business
- Meeting Facilities, Wi-Fi Available In Public Areas
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- Disabled Access Public Areas
Useful Info Show/hide
- From 14:00 hours
- By 11:00 hours
- Up to 8 years old
- The price shown is based on the occupancy and dates you have searched for. Prices and availability are subject to change based on the arrival date, number of nights and occupancy searched for. All prices are inclusive of all local taxes.
- Pets are not allowed
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Senex and its joint venture partners have drilled 11 oil wells over the last four months, with nine wells cased and suspended for future testing and production.
Dunlop-1 exploration well (PEL 113: Senex 100%)
The Dunlop-1 exploration well flowed at 1,200 barrels of oil per day (bopd) during drill stem testing after intersecting approximately three metres of net pay in the McKinlay Member.
Dunlop-1 spudded on 1 October and reached a total depth of 1,469 metres. The well is located 4.7 kilometres southwest of the Harpoono-2 oil well in the southern Cooper-Eromanga Basin.
Senex will complete the well for production during November.
Sprigg-1 exploration well (PEL 514: Senex 80% and Operator)
Ensign Rig 754 cased and suspended the Sprigg-1 exploration well after successfully intersecting 6.3 metres interpreted net pay in the Murta Formation with gas shows in the Arrabury and Birkhead Formations. Testing of the potential pay zones will be undertaken subject to rig availability. The well reached a total depth of 2,472 metres and is located 4.5 kilometres west of the Acrasia oil field.
Senex Energy Limited
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Grey's Anatomy TV Store > Va-jay-jay - Grey's Anatomy Laundry Bag
"Stop looking at my va-jay-jay!" - Miranda Bailey, Grey's Anatomy. A t-shirt or hoodie with this quote makes a great gift for fans of Grey's Anatomy & the doctors of Seattle Grace. Seriously.
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Unveiled by Felina Bissette Demi Contour - Black / Sugar Baby
This must have bra is the epitome of elegant design with clean lines and impeccable composition. Soft and subtle, the stretch mesh covered molded cups make this piece ideal for a streamlined silhouette.
• Brand: Unveiled
• Style #: 230062
• Silhouette: Demi contour
• Strap: Adjustable back strap
- Fine mesh T-shirt bra with lightly molded, seamless cups.
- 2 way stretch contour pads for perfect fit and in between sizes
- Embellished shoulder straps are elasticized and adjustable in the back
- 1/4 wing stabilized double ply clean finish wing.
Fabric Content:
• Body Fabric: 87% nylon, 13% elastane
• Embroidery: 100% polyester
Exclusive of decoration
Size Chart


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Where It's 70 Degrees And Sunny All Year Long!
The "Peak" is a 44-by-44-by-37 foot high climbing, low crawling, slip sliding Northwoods adventure. Climb into the 30-foot tall oak tree, slide down one of the four giant tube slides or wash down the triple wave slide. Navigate across a canyon, scale the climbing wall and venture to the 30 foot lookout to spy around the Park. From the lookout, slide down the very popular and very fast new super slide. Adventure Peak also features great areas for toddlers to explore. Socks are required!
Little Peak is our Toddler Area, for children ages 4 and under, includes climbing, crawling, sliding and even bouncing in an inflatable air bounce geared just for them! The entire padded, netted and enclosed structure has more than 45 activities to keep kids challenged and entertained for hours.
The Great Hall is a 60-by-90 foot multipurpose space that you will not want to miss. This large play area can accommodate even the most energetic child! Kids will love to bounce in our large moonwalk, ride scooters and play with hula-hoops, balls and other Park-provided equipment.
Did You Know? Adventure Peak, the Toddler Area and the Moonwalks are cleaned and sanitized daily.
Don't forget your socks! Adventure Peak is a shoeless environment. Socks must be worn on the equipment.
Forget socks? No problem! They are available for purchase for $2.50/pair at the guest services desk.
Admission wristbands are required for the Playpark. If you are a frequent visitor, purchase a 10-admission pass or an annual membership to get the best value. Passes and memberships can be purcased in-person at the park or under the Fees & Hours tab.
And, it's a great place for a birthday party or group outing.
For more information, call 952-833-9540.
7700 York Ave. S.
Edina, MN 55435
952-833-9541 (fax)
Patty McGrath
General Manager
Dawn Beitel
Assistant Manager
Birthday Coordinator
© 2017 City Of Edina, Minnesota