Tamer S. Kaoud

Targeted silencing of elongation factor 2 kinase suppresses growth and sensitizes tumors to doxorubicin in an orthotopic model of breast cancer.

Eukaryotic elongation factor 2 kinase (eEF-2K), through its phosphorylation of elongation factor 2 (eEF2), provides a mechanism by which cells can control the rate of the elongation phase of protein synthesis. The activity of eEF-2K is increased in rapidly proliferating malignant cells, is inhibited during mitosis, and may contribute to the promotion of autophagy in response to anti-cancer therapies. The purpose of this study was to examine the therapeutic potential of targeting eEF-2K in breast cancer tumors. Through the systemic administration of liposomal eEF-2K siRNA (twice a week, i.v. 150 µg/kg), the expression of eEF-2K was down-regulated in vivo in an orthotopic xenograft mouse model of a highly aggressive triple negative MDA-MB-231 tumor. This targeting resulted in a substantial decrease in eEF2 phosphorylation in the tumors, and led to the inhibition of tumor growth, the induction of apoptosis and the sensitization of tumors to the chemotherapy agent doxorubicin. eEF-2K down-modulation in vitro resulted in a decrease in the expression of c-Myc and cyclin D1 with a concomitant increase in the expression of p27Kip1. A decrease in the basal activity of c-Src (phospho-Tyr-416), focal adhesion kinase (phospho-Tyr-397), and Akt (phospho-Ser-473) was also detected following eEF-2K down-regulation in MDA-MB-231 cells, as determined by Western blotting. Where tested, similar results were seen in ER-positive MCF-7 cells. These effects were also accompanied by a decrease in the observed invasive phenotype of the MDA-MB-231 cells. These data support the notion that the disruption of eEF-2K expression in breast cancer cells results in the down-regulation of signaling pathways affecting growth, survival and resistance and has potential as a therapeutic approach for the treatment of breast cancer.

193‐nm photodissociation of singly and multiply charged peptide anions for acidic proteome characterization

193‐nm ultraviolet photodissociation (UVPD) was implemented to sequence singly and multiply charged peptide anions. Upon dissociation by this method, a‐/x‐type, followed by d and w side‐chain loss ions, were the most prolific and abundant sequence ions, often yielding 100% sequence coverage. The dissociation behavior of singly and multiply charged anions was significantly different with higher charged precursors yielding more sequence ions; however, all charge states investigated (1− through 3−) produced rich diagnostic information. UVPD at 193 nm was also shown to successfully differentiate and pinpoint labile phosphorylation modifications. The sequence ions were produced with high abundances, requiring limited averaging for satisfactory spectral quality. The intact, charge‐reduced radical products generated by UV photoexcitation were also subjected to collision‐induced dissociation (termed, activated‐electron photodetachment dissociation (a‐EPD)), but UVPD alone yielded more predictable and higher abundance sequence ions. With the use of a basic (pH∼11.5), piperidine‐modified mobile phase, LC‐MS/UVPD was implemented and resulted in the successful analysis of mitogen‐activated pathway kinases (MAPKs) using ultrafast activation times (5 ns).

Calcium/calmodulin stimulates the autophosphorylation of elongation factor 2 kinase on Thr-348 and Ser-500 to regulate its activity and calcium dependence

Eukaryotic elongation factor 2 kinase (eEF-2K) is an atypical protein kinase regulated by Ca(2+) and calmodulin (CaM). Its only known substrate is eukaryotic elongation factor 2 (eEF-2), whose phosphorylation by eEF-2K impedes global protein synthesis. To date, the mechanism of eEF-2K autophosphorylation has not been fully elucidated. To investigate the mechanism of autophosphorylation, human eEF-2K was coexpressed with λ-phosphatase and purified from bacteria in a three-step protocol using a CaM affinity column. Purified eEF-2K was induced to autophosphorylate by incubation with Ca(2+)/CaM in the presence of MgATP. Analyzing tryptic or chymotryptic peptides by mass spectrometry monitored the autophosphorylation over 0-180 min. The following five major autophosphorylation sites were identified: Thr-348, Thr-353, Ser-445, Ser-474, and Ser-500. In the presence of Ca(2+)/CaM, robust phosphorylation of Thr-348 occurs within seconds of addition of MgATP. Mutagenesis studies suggest that phosphorylation of Thr-348 is required for substrate (eEF-2 or a peptide substrate) phosphorylation, but not self-phosphorylation. Phosphorylation of Ser-500 lags behind the phosphorylation of Thr-348 and is associated with the Ca(2+)-independent activity of eEF-2K. Mutation of Ser-500 to Asp, but not Ala, renders eEF-2K Ca(2+)-independent. Surprisingly, this Ca(2+)-independent activity requires the presence of CaM.

Investigating the kinetic mechanism of inhibition of elongation factor 2 kinase by NH125: Evidence of a common in vitro artifact

Evidence that elongation factor 2 kinase (eEF-2K) has potential as a target for anticancer therapy and possibly for the treatment of depression is emerging. Here the steady-state kinetic mechanism of eEF-2K is presented using a peptide substrate and is shown to conform to an ordered sequential mechanism with ATP binding first. Substrate inhibition by the peptide was observed and revealed to be competitive with ATP, explaining the observed ordered mechanism. Several small molecules are reported to inhibit eEF-2K activity with the most notable being the histidine kinase inhibitor NH125, which has been used in a number of studies to characterize eEF-2K activity in cells. While NH125 was previously reported to inhibit eEF-2K in vitro with an IC(50) of 60 nM, its mechanism of action was not established. Using the same kinetic assay, the ability of an authentic sample of NH125 to inhibit eEF-2K was assessed over a range of substrate and inhibitor concentrations. A typical dose-response curve for the inhibition of eEF-2K by NH125 is best fit to an IC(50) of 18 ± 0.25 μM and a Hill coefficient of 3.7 ± 0.14, suggesting that NH125 is a weak inhibitor of eEF-2K under the experimental conditions of a standard in vitro kinase assay. To test the possibility that NH125 is a potent inhibitor of eEF2 phosphorylation, we assessed its ability to inhibit the phosphorylation of eEF2. Under standard kinase assay conditions, NH125 exhibits a similar weak ability to inhibit the phosphorylation of eEF2 by eEF-2K. Notably, the activity of NH125 is severely abrogated by the addition of 0.1% Triton to the kinase assay through a process that can be reversed upon dilution. These studies suggest that NH125 is a nonspecific colloidal aggregator in vitro, a notion further supported by the observation that NH125 inhibits other protein kinases, such as ERK2 and TRPM7 in a manner similar to that of eEF-2K. As NH125 is reported to inhibit eEF-2K in a cellular environment, its ability to inhibit eEF2 phosphorylation was assessed in MDA-MB-231 breast cancer, A549 lung cancer, and HEK-293T cell lines using a Western blot approach. No sign of a decrease in the level of eEF2 phosphorylation was observed up to 12 h following addition of NH125 to the media. Furthermore, contrary to the previously reported literatures, NH125 induced the phosphorylation of eEF-2.

In-Situ Generation of Differential Sensors that Fingerprint Kinases and the Cellular Response to Their Expression

Mitogen-activated protein (MAP) kinases are responsible for many cellular functions, and their malfunction manifests itself in several human diseases. Usually, monitoring the phosphorylation states of MAP kinases in vitro requires the preparation and purification of the proteins or Western blotting. Herein, we report an array sensing approach for the differentiation of MAP kinases and their phosphorylated counterparts in vitro. This technique utilizes a library of differential receptors created in situ containing peptides known for affinity to MAP kinases, and a Zn(II)-dipicolylamine complex that binds phosphate groups on proteins. An indicator-displacement assay signals the binding of the individual receptors to the kinases, while chemometrics is used to create a fingerprint for the kinases and their state of activity. For example, linear discriminant analysis correctly identified kinase activity with a classification accuracy of 97.5% in vitro, while the cellular response to kinase expression was classified with 100% accuracy.

Arrestin-3 Binds c-Jun N-terminal Kinase 1 (JNK1) and JNK2 and Facilitates the Activation of These Ubiquitous JNK Isoforms in Cells via Scaffolding

Background: The ability of arrestin-3 to facilitate activation of JNK1 and JNK2 has never been reported. Results: Arrestin-3 binds JNK1α1 and JNK2α2 and promotes their phosphorylation by MKK4 and MKK7 in vitro and in intact cells. Conclusion: Arrestin-3 promotes the activation of ubiquitous JNK1 and JNK2 isoforms. Significance: Arrestin-3 scaffolds MKK4/7-JNK1/2/3 signaling modules and facilitates activation of ubiquitous JNK isoforms. Non-visual arrestins scaffold mitogen-activated protein kinase (MAPK) cascades. The c-Jun N-terminal kinases (JNKs) are members of MAPK family. Arrestin-3 has been shown to enhance the activation of JNK3, which is expressed mainly in neurons, heart, and testes, in contrast to ubiquitous JNK1 and JNK2. Although all JNKs are activated by MKK4 and MKK7, both of which bind arrestin-3, the ability of arrestin-3 to facilitate the activation of JNK1 and JNK2 has never been reported. Using purified proteins we found that arrestin-3 directly binds JNK1α1 and JNK2α2, interacting with the latter comparably to JNK3α2. Phosphorylation of purified JNK1α1 and JNK2α2 by MKK4 or MKK7 is increased by arrestin-3. Endogenous arrestin-3 interacted with endogenous JNK1/2 in different cell types. Arrestin-3 also enhanced phosphorylation of endogenous JNK1/2 in intact cells upon expression of upstream kinases ASK1, MKK4, or MKK7. We observed a biphasic effect of arrestin-3 concentrations on phosphorylation of JNK1α1 and JNK2α2 both in vitro and in vivo. Thus, arrestin-3 acts as a scaffold, facilitating JNK1α1 and JNK2α2 phosphorylation by MKK4 and MKK7 via bringing JNKs and their activators together. The data suggest that arrestin-3 modulates the activity of ubiquitous JNK1 and JNK2 in non-neuronal cells, impacting the signaling pathway that regulates their proliferation and survival.

Understanding the Specificity of a Docking Interaction between JNK1 and the Scaffolding Protein JIP1

The up-regulation of JNK activity is associated with a number of disease states. The JNK-JIP1 interaction represents an attractive target for the inhibition of JNK-mediated signaling. In this study, molecular dynamics simulations have been performed on the apo-JNK1 and the JNK1•L-pepJIP1 and JNK1•D-pepJIP1 complexes to investigate the interaction between the JIP1 peptides and JNK1. Dynamic domain studies based on essential dynamics (ED) analysis of apo-JNK1 and the JNK1•L-pepJIP1 complex have been performed to analyze and compare details of conformational changes, hinge axes, and hinge bending regions in both structures. The activation loop, the αC helix, and the G loop are found to be highly flexible and to exhibit significant changes in dynamics upon L-pepJIP1 binding. The conformation of the activation loop for the apo state is similar to that of inactive apo-ERK2, while the activation loop in JNK1•L-pepJIP1 complex resembles that of the inactive ERK2 bound with pepHePTP. ED analysis shows that, after the binding of l-pepJIP1, the N- and C-terminal domains of JNK1 display both a closure and a twisting motion centered around the activation loop, which functions as a hinge. In contrast, no domain motion is detected for the apo state for which an open conformation is favored. The present study suggests that L-pepJIP1 regulates the interdomain motions of JNK1 and potentially the active site via an allosteric mechanism. The binding free energies of L-pepJIP1 and D-pepJIP1 to JNK1 are estimated using the molecular mechanics Poisson-Boltzmann and generalized-Born surface area (MM-PB/GBSA) methods. The contribution of each residue at the interaction interface to the binding affinity of L-pepJIP1 with JNK1 has been analyzed by means of computational alanine-scanning mutagenesis and free energy decomposition. Several critical interactions for binding (e.g., Arg156/L-pepJIP1 and Glu329/JNK1) have been identified. The binding free energy calculation indicates that the electrostatic interaction contributes critically to specificity, rather than to binding affinity between the peptide and JNK1. Notably, the binding free energy calculations predict that D-pepJIP1 binding to JNK1 is significantly weaker than the L form, contradicting the previous suggestion that D-pepJIP1 acts as an inhibitor toward JNK1. We have performed experiments using purified JNK1 to confirm that, indeed, D-pepJIP1 does not inhibit the ability of JNK1 to phosphorylate c-Jun in vitro.

Development of JNK2-selective peptide inhibitors that inhibit breast cancer cell migration

Despite their lack of selectivity toward c-Jun N-terminal kinase (JNK) isoforms, peptides derived from the JIP (JNK Interacting Protein) scaffolds linked to the cell-penetrating peptide TAT are widely used to investigate JNK-mediated signaling events. To engineer an isoform-selective peptide inhibitor, several JIP-based peptide sequences were designed and tested. A JIP sequence connected through a flexible linker to either the N-terminus of an inverted TAT sequence (JIP(10)-Δ-TAT(i)) or to a poly arginine sequence (JIP(10)-Δ-R(9)) enabled the potent inhibition of JNK2 (IC(50) ≈ 90 nM) and exhibited 10-fold selectivity for JNK2 over JNK1 and JNK3. Examination of both peptides in HEK293 cells revealed a potent ability to inhibit the induction of both JNK activation and c-Jun phosphorylation in cells treated with anisomycin. Notably, Western blot analysis indicates that only a fraction of total JNK must be activated to elicit robust c-Jun phosphorylation. To examine the potential of each peptide to selectively modulate JNK2 signaling in vivo, their ability to inhibit the migration of Polyoma Middle-T Antigen Mammary Tumor (PyVMT) cells was assessed. PyVMTjnk2-/- cells exhibit a lower migration potential compared to PyVMTjnk2+/+ cells, and this migration potential is restored through the overexpression of GFP-JNK2α. Both JIP(10)-Δ-TAT(i) and JIP(10)-Δ-R(9) inhibit the migration of PyVMTjnk2+/+ cells and PyVMTjnk2-/- cells expressing GFP-JNK2α. However, neither peptide inhibits the migration of PyVMTjnk2-/- cells. A control form of JIP(10)-Δ-TAT(i) containing a single leucine to arginine mutation lacks ability to inhibit JNK2 in vitro cell-free and cell-based assays and does not inhibit the migration of PyVMTjnk2+/+ cells. Together, these data suggest that JIP(10)-Δ-TAT(i) and JIP(10)-Δ-R(9) inhibit the migration of PyVMT cells through the selective inhibition of JNK2. Finally, the mechanism of inhibition of a D-retro-inverso JIP peptide, previously reported to inhibit JNK, was examined and found to inhibit p38MAPKα in an in vitro cell-free assay with little propensity to inhibit JNK isoforms.

JNK3 enzyme binding to arrestin-3 differentially affects the recruitment of upstream mitogen-activated protein (MAP) kinase kinases.

Background: An interaction between arrestin-3 and MKK7 has never been elucidated. Results: Arrestin-3 directly binds MKK7 and promotes JNK3α2 phosphorylation by MKK7 in vitro and in intact cells. Conclusion: Arrestin-3 recruits JNK3α2 and both upstream MKKs. Significance: Arrestin-3 promotes full JNK3α2 activation; MKK binding is regulated by JNK3α2. Arrestin-3 was previously shown to bind JNK3α2, MKK4, and ASK1. However, full JNK3α2 activation requires phosphorylation by both MKK4 and MKK7. Using purified proteins we show that arrestin-3 directly interacts with MKK7 and promotes JNK3α2 phosphorylation by both MKK4 and MKK7 in vitro as well as in intact cells. The binding of JNK3α2 promotes an arrestin-3 interaction with MKK4 while reducing its binding to MKK7. Interestingly, the arrestin-3 concentration optimal for scaffolding the MKK7-JNK3α2 module is ∼10-fold higher than for the MKK4-JNK3α2 module. The data provide a mechanistic basis for arrestin-3-dependent activation of JNK3α2. The opposite effects of JNK3α2 on arrestin-3 interactions with MKK4 and MKK7 is the first demonstration that the kinase components in mammalian MAPK cascades regulate each others interactions with a scaffold protein. The results show how signaling outcomes can be affected by the relative expression of scaffolding proteins and components of signaling cascades that they assemble.

Solution NMR insights into docking interactions involving inactive ERK2.

The mitogen-activated protein (MAP) kinase ERK2 contains recruitment sites that engage canonical and noncanonical motifs found in a variety of upstream kinases, regulating phosphatases and downstream targets. Interactions involving two of these sites, the D-recruitment site (DRS) and the F-recruitment site (FRS), have been shown to play a key role in signal transduction by ERK/MAP kinases. The dynamic nature of these recruitment events makes NMR uniquely suited to provide significant insight into these interactions. While NMR studies of kinases in general have been greatly hindered by their large size and complex dynamic behavior leading to the suboptimal performance of standard methodologies, we have overcome these difficulties for inactive full-length ERK2 and obtained an acceptable level of backbone resonance assignments. This allowed a detailed investigation of the structural perturbations that accompany interactions involving both canonical and noncanonical recruitment events. No crystallographic information exists for the latter. We found that the chemical shift perturbations in inactive ERK2, indicative of structural changes in the presence of canonical and noncanonical motifs, are not restricted to the recruitment sites but also involve the linker that connects the N- and C-lobes and, in most cases, a gatekeeper residue that is thought to exert allosteric control over catalytic activity. We also found that, even though the canonical motifs interact with the DRS utilizing both charge-charge and hydrophobic interactions, the noncanonical interactions primarily involve the latter. These results demonstrate the feasibility of solution NMR techniques for a comprehensive analysis of docking interactions in a full-length ERK/MAP kinase.