Novel compounds, ‘1,3-selenazine derivatives’ as specific inhibitors of eukaryotic elongation factor-2 kinase

The inhibitory activities of 5,6-dihydro-4H-1,3-selenazine derivatives on protein kinases were investigated. In a multiple protein kinase assay using a postnuclear fraction of v-src-transformed NIH3T3 cells, 4-ethyl-4-hydroxy-2-p-tolyl-5,6-dihydro-4H-1,3-selenazine (TS-2) and 4-hydroxy-6-isopropyl-4-methyl-2-p-tolyl-5,6-dihydro-4H-1,3-selenazine (TS-4) exhibited selective inhibitory activity against eukaryotic elongation factor-2 kinase (eEF-2K) over protein kinase A (PKA), protein kinase C (PKC) and protein tyrosine kinase (PTK). In further experiments using purified kinases, TS-2 (IC₅₀=0.36 μM) and TS-4 (IC₅₀=0.31 μM) inhibited eEF-2K about 25-fold more effectively than calmodulin-dependent protein kinase-I (CaMK-I), and about 6-fold (TS-2) or 33-fold (TS-4) more effectively than calmodulin-dependent protein kinase-II (CaMK-II), respectively. TS-2 and TS-4 showed much weaker inhibitory activity toward PKA and PKC, while TS-4, but not TS-2, moderately inhibited immunoprecipitated v-src kinase. TS-2 (10.7-fold) and TS-4 (12.5-fold) demonstrated more potent and more specific eEF-2K inhibitory activity than rottlerin, a previously identified eEF-2K inhibitor. TS-2 inhibited ATP or eEF-2 binding to eEF-2K in a competitive or non-competitive manner, respectively. In cultured v-src-transformed NIH3T3 cells, TS-2 also decreased phospho-eEF-2 protein level (IC₅₀=4.7 μM) without changing the total eEF-2 protein level. Taken together, these results suggest that TS-2 and TS-4 are the first identified selective eEF-2K inhibitors and should be useful tools for studying the function of eEF-2K.     

Regulation of elongation factor-2 kinase by pH

Elongation factor-2 kinase (eEF-2K) is a Ca(2+)/calmodulin-dependent protein kinase that phosphorylates and inactivates eEF-2 and that can regulate the rate of protein synthesis at the elongation stage. Here we report that a slight decrease in pH, within the range observed in vivo, leads to a dramatic activation of eEF-2K. The activity of eEF-2K in mouse liver extracts, as well as the activity of purified recombinant human eEF-2K, is low at pH 7.2-7.4 and is increased by severalfold when the pH drops to 6.6-6.8. eEF-2K requires calmodulin for activity at neutral as well as acidic pH. Kinetic studies demonstrate that the pH does not affect the K(M) for ATP or eEF-2 and activation of eEF-2K at acidic pH is due to an increase in V(max). To analyze the potential role of eEF-2K in regulating protein synthesis by pH, we constructed a mouse fibroblast cell line that expresses eEF-2K in a tetracycline-regulated manner. Overexpression of eEF-2K led to a decreased rate of protein synthesis at acidic pH, but not at neutral pH. Our results suggest that pH-dependent activation of eEF-2K may play a role in the global inhibition of protein synthesis during tissue acidosis, which accompanies such processes as hypoxia and ischemia.     

Analysis of the domain structure of elongation factor-2 kinase by mutagenesis.

A number of elongation factor-2 kinase (eEF-2K) mutants were constructed to investigate features of this kinase that may be important in its activity. Typical protein kinases possess a highly conserved lysine residue in subdomain II which follows the GXGXXG motif of subdomain I. Mutation of two lysine residues, K340 and K346, which follow the GXGXXG motif in eEF-2K had no effect on activity, showing that such a lysine residue is not important in eEF-2K activity. Mutation of a conserved pair of cysteine residues C-terminal to the GXGXXG sequence, however, completely inactivated eEF-2K. The eEF-2K CaM binding domain was localised to residues 77-99 which reside N-terminal to the catalytic domain. Tryptophan 84 is an important residue within this domain as mutation of this residue completely abolishes CaM binding and eEF-2K activity. Removal of approximately 130 residues from the C-terminus of eEF-2K completely abolished autokinase activity; however, removal of only 19 residues inhibited eEF-2 kinase activity but not autokinase activity, suggesting that a short region at the C-terminal end may be important in interacting with eEF-2. Likewise, removal of between 75 and 100 residues from the N-terminal end completely abolished eEF-2K activity.     

Elongation factor-2 kinase and its newly discovered relatives

Phosphorylation of elongation factor-2 (eEF-2) by the highly specific eEF-2 kinase results in eEF-2 inactivation and, therefore, may regulate the global rate of protein synthesis in animal cells. Cloning and sequencing of eEF-2 kinase led to the discovery of a new family of protein kinases, named alpha-kinases, whose catalytic domains display no sequence homology to conventional eukaryotic protein kinases. Several mammalian alpha-kinases have recently been cloned. Two of these alpha-kinases, named channel-kinases 1 and 2 (ChaK1 and ChaK2) represent a new type of signaling molecules that are protein kinases fused to ion channels.     

Geraniol inhibits prostate cancer growth by targeting cell cycle and apoptosis pathways

Elongation factor-2 kinase (eEF-2 kinase, also known as calmodulin-dependent protein kinase III), is a unique calcium/calmodulin-dependent enzyme that inhibits protein synthesis by phosphorylating and inactivating elongation factor-2 (eEF-2). We previously reported that expression/activity of eEF-2 kinase was up-regulated in several types of malignancies including Gliomas, and was associated with response of tumor cells to certain therapeutic stress. In the current study, we sought to determine whether eEF-2 kinase expression affected sensitivity of glioma cells to treatment with tumor the necrosis factor-related apoptosis-inducing ligand (TRAIL), a targeted therapy able to induce apoptosis in cancer cells but causes no toxicity in most normal cells. We found that inhibition of eEF-2 kinase by RNA interference (RNAi) or by a pharmacological inhibitor (NH125) enhanced TRAIL-induced apoptosis in the human glioma cells, as evidenced by an increase in apoptosis in the tumor cells treated with eEF-2 kinase siRNA or the eEF-2 kinase inhibitor. We further demonstrated that sensitization of tumor cells to TRAIL was accompanied by a down-regulation of the anti-apoptotic protein, Bcl-xL, and that overexpression of Bcl-xL could abrogate the sensitizing effect of inhibiting eEF-2 kinase on TRAIL. The results of this study may help devise a new therapeutic strategy for enhancing the efficacy of TRAIL against malignant glioma by targeting eEF-2 kinase.     

Purification and characterization of tagless recombinant human elongation factor 2 kinase (eEF-2K) expressed in Escherichia coli

The eukaryotic elongation factor 2 kinase (eEF-2K) modulates the rate of protein synthesis by impeding the elongation phase of translation by inactivating the eukaryotic elongation factor 2 (eEF-2) via phosphorylation. eEF-2K is known to be activated by calcium and calmodulin, whereas the mTOR and MAPK pathways are suggested to negatively regulate kinase activity. Despite its pivotal role in translation regulation and potential role in tumor survival, the structure, function, and regulation of eEF-2K have not been described in detail. This deficiency may result from the difficulty of obtaining the recombinant kinase in a form suitable for biochemical analysis. Here we report the purification and characterization of recombinant human eEF-2K expressed in the Escherichia coli strain Rosetta-gami 2(DE3). Successive chromatography steps utilizing Ni-NTA affinity, anion-exchange, and gel filtration columns accomplished purification. Cleavage of the thioredoxin-His(6)-tag from the N-terminus of the expressed kinase with TEV protease yielded 9 mg of recombinant (G-D-I)-eEF-2K per liter of culture. Light scattering shows that eEF-2K is a monomer of ～85 kDa. In vitro kinetic analysis confirmed that recombinant human eEF-2K is able to phosphorylate wheat germ eEF-2 with kinetic parameters comparable to the mammalian enzyme.     

Novel compounds, '1,3-selenazine derivatives' as specific inhibitors of eukaryotic elongation factor-2 kinase

The inhibitory activities of 5,6-dihydro-4H-1,3-selenazine derivatives on protein kinases were investigated. In a multiple protein kinase assay using a postnuclear fraction of v-src-transformed NIH3T3 cells, 4-ethyl-4-hydroxy-2-p-tolyl-5, 6-dihydro-4H-1,3-selenazine (TS-2) and 4-hydroxy-6-isopropyl-4-methyl-2-p-tolyl-5,6-dihydro-4H-1, 3-selenazine (TS-4) exhibited selective inhibitory activity against eukaryotic elongation factor-2 kinase (eEF-2K) over protein kinase A (PKA), protein kinase C (PKC) and protein tyrosine kinase (PTK). In further experiments using purified kinases, TS-2 (IC(50)=0.36 microM) and TS-4 (IC(50)=0.31 microM) inhibited eEF-2K about 25-fold more effectively than calmodulin-dependent protein kinase-I (CaMK-I), and about 6-fold (TS-2) or 33-fold (TS-4) more effectively than calmodulin-dependent protein kinase-II (CaMK-II), respectively. TS-2 and TS-4 showed much weaker inhibitory activity toward PKA and PKC, while TS-4, but not TS-2, moderately inhibited immunoprecipitated v-src kinase. TS-2 (10.7-fold) and TS-4 (12.5-fold) demonstrated more potent and more specific eEF-2K inhibitory activity than rottlerin, a previously identified eEF-2K inhibitor. TS-2 inhibited ATP or eEF-2 binding to eEF-2K in a competitive or non-competitive manner, respectively. In cultured v-src-transformed NIH3T3 cells, TS-2 also decreased phospho-eEF-2 protein level (IC(50)=4.7 microM) without changing the total eEF-2 protein level. Taken together, these results suggest that TS-2 and TS-4 are the first identified selective eEF-2K inhibitors and should be useful tools for studying the function of eEF-2K.     

Protein kinase CK2 phosphorylates and activates the SR protein-specific kinase 1

The serine/arginine subfamily of protein kinases has been conserved throughout evolution and its members are thought to play important roles in the regulation of multiple cellular processes. Mammalian SRPK1 has been considered as a constitutively active kinase that is predominantly expressed in testis. In the present study, recombinant GST-SRPK1 was used as substrate to identify potential protein kinase(s) in testis extracts, involved in phosphorylating and thereby regulating the activity of this enzyme. Using a panel of chromatography media, inhibition by heparin, immunoblot analysis, and phosphopeptide mapping, CK2 was determined to be the major kinase that phosphorylates SRPK1. Phosphorylation of SRPK1 by CK2 occurred mainly at Ser(51) and Ser(555) in vitro, and resulted in approximately 6-fold activation of the enzyme. These findings suggest that SRPK1 may be an important cellular target for CK2 action.     

Downregulation of the translation elongation factor 2 kinase in Xenopus laevis oocytes at the final stages of oogenesis

Phosphorylation of translation elongation factor 2(eEF-2) by a specific Ca2+/calmodulin-dependent eEF-2 kinase plays an important role in the regulation of protein synthesis in mammalian cells. We show here that an eEF-2 kinase similar to the mammalian enzyme is present in tissues of the amphibian Xenopus laevis. We investigated changes in the activity of eEF-2 kinase in extracts of Xenopus oocytes at different stages of oogenesis. The eEF-2 kinase activity was constant from stage I to stage IV of oogenesis, but dramatically decreased after stage IV. Extracts of fully grown stage-VI oocytes showed no eEF-2 kinase activity. However, when extracts were analyzed by two-dimensional gel electrophoresis, eEF-2 was found to be present mostly, if not exclusively, in the dephosphorylated form throughout oogenesis. It is suggested that eEF-2 kinase disappears late in oogenesis to make protein synthesis insensitive to changes in intracellular Ca2+ concentration. This may be important for the induction of meiotic maturation.     

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.     

Targeting elongation factor-2 kinase (eEF-2K) induces apoptosis in human pancreatic cancer cells

Pancreatic cancer (PaCa) is one of the most aggressive, apoptosis-resistant and currently incurable cancers with a poor survival rate. Eukaryotic elongation factor-2 kinase (eEF-2K) is an atypical kinase, whose role in PaCa survival is not yet known. Here, we show that eEF-2K is overexpressed in PaCa cells and its down-regulation induces apoptotic cell death. Rottlerin (ROT), a polyphenolic compound initially identified as a PKC-δ inhibitor, induces apoptosis and autophagy in a variety of cancer cells including PaCa cells. We demonstrated that ROT induces intrinsic apoptosis, with dissipation of mitochondrial membrane potential (ΔΨm), and stimulates extrinsic apoptosis with concomitant induction of TNF-related apoptosis inducing ligand (TRAIL) receptors, DR4 and DR5, with caspase-8 activation, in PANC-1 and MIAPaCa-2 cells. Notably, while none of these effects were dependent on PKC-δ inhibition, ROT down-regulates eEF-2K at mRNA level, and induce eEF-2K protein degradation through ubiquitin–proteasome pathway. Down-regulation of eEF-2K recapitulates the events observed after ROT treatment, while its over-expression suppressed the ROT-induced apoptosis. Furthermore, eEF-2K regulates the expression of tissue transglutaminase (TG2), an enzyme previously implicated in proliferation, drug resistance and survival of cancer cells. Inhibition of eEF-2K/TG2 axis leads to caspase-independent apoptosis which is associated with induction of apoptosis-inducing factor (AIF). Collectively, these results indicate, for the first time, that the down-regulation of eEF-2K leads to induction of intrinsic, extrinsic as well as AIF-dependent apoptosis in PaCa cells, suggesting that eEF-2K may represent an attractive therapeutic target for the future anticancer agents in PaCa.     

Mapping the functional domains of elongation factor-2 kinase

A new class of eukaryotic protein kinases that are not homologous to members of the serine/threonine/tyrosine protein kinase superfamily was recently identified [Futey, L. M., et al. (1995) J. Biol. Chem. 270, 523-529; Ryazanov, A. G., et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 4884-4889]. This class includes eukaryotic elongation factor-2 kinase, Dictyostelium myosin heavy chain kinases A, B, and C, and several mammalian putative protein kinases that are not yet fully characterized [Ryazanov, A. G., et al. (1999) Curr. Biol. 9, R43-R45]. eEF-2 kinase is a ubiquitous protein kinase that phosphorylates and inactivates eukaryotic translational elongation factor-2, and thus can modulate the rate of polypeptide chain elongation during translation. eEF-2 was the only known substrate for eEF-2 kinase. We demonstrate here that eEF-2 kinase can efficiently phosphorylate a 16-amino acid peptide, MH-1, corresponding to the myosin heavy chain kinase A phosphorylation site in Dictyostelium myosin heavy chains. This enabled us to develop a rapid assay for eEF-2 kinase activity. To localize the functional domains of eEF-2 kinase, we expressed human eEF-2 kinase in Escherichia coli as a GST-tagged fusion protein, and then performed systematic in vitro deletion mutagenesis. We analyzed eEF-2 kinase deletion mutants for the ability to autophosphorylate, and to phosphorylate eEF-2 as well as a peptide substrate, MH-1. Mutants with deletions between amino acids 51 and 335 were unable to autophosphorylate, and were also unable to phosphorylate eEF-2 and MH-1. Mutants with deletions between amino acids 521 and 725 were unable to phosphorylate eEF-2, but were still able to autophosphorylate and to phosphorylate MH-1. The kinases with deletions between amino acids 2 and 50 and 336 and 520 were able to catalyze all three reactions. In addition, the C-terminal domain expressed alone (amino acids 336-725) binds eEF-2 in a coprecipitation assay. These results suggest that eEF-2 kinase consists of two domains connected by a linker region. The amino-terminal domain contains the catalytic domain, while the carboxyl-terminal domain contains the eEF-2 targeting domain. The calmodulin-binding region is located between amino acids 51 and 96. The amino acid sequence of the carboxyl-terminal domain of eEF-2 kinase displays similarity to several proteins, all of which contain repeats of a 36-amino acid motif that we named "motif 36".     

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.     

Structural Dynamics of the Activation of Elongation Factor 2 Kinase by Ca2 +-Calmodulin

Eukaryotic elongation factor 2 kinase (eEF-2K), the only known calmodulin (CaM)-activated α-kinase, phosphorylates eukaryotic elongation factor 2 (eEF-2) on a specific threonine (Thr-56) diminishing its affinity for the ribosome and reducing the rate of nascent chain elongation during translation. Despite its critical cellular role, the precise mechanisms underlying the CaM-mediated activation of eEF-2K remain poorly defined. Here, employing a minimal eEF-2K construct (TR) that exhibits activity comparable to the wild-type enzyme and is fully activated by CaM in vitro and in cells, and using a variety of complimentary biophysical techniques in combination with computational modeling, we provide a structural mechanism by which CaM activates eEF-2K. Native mass analysis reveals that CaM, with two bound Ca^2 + ions, forms a stoichiometric 1:1 complex with TR. Chemical crosslinking mass spectrometry and small-angle X-ray scattering measurements localize CaM near the N-lobe of the TR kinase domain and the spatially proximal C-terminal helical repeat. Hydrogen/deuterium exchange mass spectrometry and methyl NMR indicate that the conformational changes induced on TR by the engagement of CaM are not localized but are transmitted to remote regions that include the catalytic site and the functionally important phosphate binding pocket. The structural insights obtained from the present analyses, together with our previously published kinetics data, suggest that TR, and by inference, wild-type eEF-2K, upon engaging CaM undergoes a conformational transition resulting in a state that is primed to efficiently auto-phosphorylate on the primary activating T348 en route to full activation.     

Eukaryotic elongation factor 2 kinase confers tolerance to stress conditions in cancer cells

Eukaryotic elongation factor 2 (eEF2) is a member of the GTP-binding translation elongation factor family that is essential for protein synthesis. eEF2 kinase (eEF2K) is a structurally and functionally unique protein kinase in the calmodulin-mediated signaling pathway. eEF2K phosphorylates eEF2, thereby inhibiting eEF2 function under stressful conditions. eEF2K regulates numerous processes, such as protein synthesis, cell cycle progression, and induction of autophagy and apoptosis in cancer cells. This review will demonstrate the mechanisms underlying eEF2K activity in cancer cells under different stresses, such as nutrient deprivation, hypoxia, and DNA damage via eEF2 regulation. In vivo, in vitro, and clinical studies indicated that eEF2K may be a novel biomarker and therapeutic target for cancer.     

Protein kinase C phosphorylates and regulates UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase

UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (UDP-GlcNAc 2-epimerase) is the key enzyme in the de novo synthesis pathway of neuraminic acid, which is widely expressed as a terminal carbohydrate residue on glycoconjugates. UDP-GlcNAc 2-epimerase is a bifunctional enzyme and catalyzes the first two steps of neuraminic acid synthesis in the cytosol, the conversion of UDP-N-acetylglucosamine to ManAc and the phosphorylation to ManAc-6-phosphate. So far, regulation of this essential enzyme by posttranslational modification has not been shown. Since UDP-N-acetylglucosamine is a cytosolic protein containing eight conserved motifs for protein kinase C (PKC), we investigated whether its enzymatic activity might be regulated by phosphorylation by PKC. We showed that UDP-GlcNAc 2-epimerase interacts with several isoforms of PKC in mouse liver and is phosphorylated in vivo. Furthermore, PKC phosphorylates UDP-GlcNAc 2-epimerase and this phosphorylation results in an upregulation of the UDP-GlcNAc 2-epimerase enzyme activity.     

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 p27(Kip1). 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.     

Mutations in protein kinase subdomain X differentially affect MEKK2 and MEKK1 activity

MAPK/ERK kinase kinase 2 (MEKK2) is a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family of protein kinases. MAP3Ks are components of a three-tiered protein kinase pathway in which a MAP3K phosphorylates and activates a mitogen-activated protein kinase kinase (MAP2K), which in turn activates a mitogen-activated protein kinase (MAPK). We have previously identified residues within protein kinase subdomain X in the MAP3K, MEKK1, that are critical for its interaction with the MAP2K, MKK4, and MEKK1-induced MKK4 activation. We report here that kinase subdomain X also plays a critical role in MEKK2 activity. Select point mutations in subdomain X impair MEKK2 phosphorylation of the MAP2Ks, MKK7 and MEK5, abolish MEKK2-induced activation of the MAPKs, JNK1 and ERK5, and diminish MEKK2-dependent activation of an AP-1 reporter gene. Interestingly, the spectrum of mutations in subdomain X of MEKK2 that affects its activity is overlapping with but not identical to those that have effects on MEKK1. Thus, mutations in subdomain X differentially affect MEKK2 and MEKK1.     

Increased activity of the eEF-2 specific, Ca2+ and calmodulin dependent protein kinase III during the S-phase in Ehrlich ascites cells.

The activity of the eukaryotic elongation factor (eEF-2) specific, Ca2+ and calmodulin dependent protein kinase III (CaM PK III) was studied in homogenated Ehrlich ascites tumour cells. The eEF-2 kinase activity was determined in the presence of an excess of the substrate, i.e., non-limiting concentrations of eEF-2. The homogenates showed both kinase and phosphatase activity. The latter activity was inhibited by the protein phosphatase 2A inhibitor okadaic acid. Analysis of the kinase using cells from defined stages of the cell cycle showed that the highest activity was found in cells from the early S-phase, whereas the phosphatase activity was most pronounced during the G2+M phase.     

Frequent activation of AKT2 kinase in human pancreatic carcinomas

Activation of AKT/protein kinase B promotes a variety of biological activities important in tumorigenesis, such as cell survival and cell cycle progression. We previously demonstrated amplification and overexpression of the AKT2 gene in a subset of human pancreatic carcinomas. In this investigation, we assessed AKT2 catalytic activity in 50 frozen pancreatic tissues (37 carcinomas, four benign tumors and nine normal pancreata) by in vitro kinase assay. Twelve of 37 (32%) pancreatic carcinomas showed markedly elevated levels of AKT2 activity compared to normal pancreata and begin pancreatic tumors. To delineate mechanisms contributing to AKT2 activation in malignant pancreatic tumors, we examined the status of upstream components of the phosphatilydlinositol 3-kinase (PI3K)/AKT pathway. Western blot analysis revealed loss of PTEN protein expression in two of the 12 pancreatic carcinomas with activated AKT2. In vitro PI3K assays demonstrated high levels of PI3K activity in seven carcinoma specimens that showed AKT2 activation. Immunohistochemical staining confirmed high levels of phosphorylated (active) AKT in malignant pancreatic tumors compared to normal pancreata. Overall, these data suggest that upstream perturbations of the PI3K/AKT pathway contribute to frequent activation of AKT2 in pancreatic cancer, which may contribute to the pathogenesis of this highly aggressive form of human malignancy.