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.     

AMP-activated protein kinase and autophagy

Autophagy is inhibited by TOR-dependent signaling. Interruption of signalling by rapamycin is known to stimulate autophagy, both in mammalian cells and in yeast. However, inactivation of TOR by AMPK has yielded controversial results in the literature with regard to its effect on autophagy: activation of autophagy in yeast but inhibition in hepatocytes. In a recent study, carried out with hepatocytes, HT-29 cells, and HeLa cells, the possible role of AMPK in the control of mammalian autophagy was reexamined. The data suggest that in mammalian cells, as in yeast, AMPK is required for autophagy.     

Death-associated protein kinase 2: Regulator of apoptosis,autophagy and inflammation

Death-associated protein kinase 2 (DAPK2/DRP-1) belongs to a family of five related serine/threonine kinases that mediate a range of cellular processes, including membrane blebbing, apoptosis, and autophagy, and possess tumour suppressive functions. The three most conserved family members DAPK1/DAPK, DAPK2 and DAPK3/ZIPK share a high degree of homology in their catalytic domain, but differ significantly in their extra-catalytic structures and tissue-expression profiles. Hence, each orthologue binds to various unique interaction partners, localizes to different subcellular regions and controls some dissimilar cellular functions. In recent years, mechanistic studies have broadened our knowledge of the molecular mechanisms that activate DAPK2 and that execute DAPK2-mediated apoptosis, autophagy and inflammation. In this “molecules in focus” review on DAPK2, the structure, modes of regulation and various cellular functions of DAPK2 will be summarized and discussed.     

A dual role for receptor-interacting protein kinase 2 (RIP2) kinase activity in nucleotide-binding oligomerization domain 2 (NOD2)-dependent autophagy

Autophagy is triggered by the intracellular bacterial sensor NOD2 (nucleotide-binding, oligomerization domain 2) as an anti-bacterial response. Defects in autophagy have been implicated in Crohn's disease susceptibility. The molecular mechanisms of activation and regulation of this process by NOD2 are not well understood, with recent studies reporting conflicting requirements for RIP2 (receptor-interacting protein kinase 2) in autophagy induction. We examined the requirement of NOD2 signaling mediated by RIP2 for anti-bacterial autophagy induction and clearance of Salmonella typhimurium in the intestinal epithelial cell line HCT116. Our data demonstrate that NOD2 stimulates autophagy in a process dependent on RIP2 tyrosine kinase activity. Autophagy induction requires the activity of the mitogen-activated protein kinases MEKK4 and p38 but is independent of NFκB signaling. Activation of autophagy was inhibited by a PP2A phosphatase complex, which interacts with both NOD2 and RIP2. PP2A phosphatase activity inhibited NOD2-dependent autophagy but not activation of NFκB or p38. Upon stimulation of NOD2, the phosphatase activity of the PP2A complex is inhibited through tyrosine phosphorylation of the catalytic subunit in a process dependent on RIP2 activity. These findings demonstrate that RIP2 tyrosine kinase activity is not only required for NOD2-dependent autophagy but plays a dual role in this process. RIP2 both sends a positive autophagy signal through activation of p38 MAPK and relieves repression of autophagy mediated by the phosphatase PP2A.     

Elongation factor-2: a useful gene for arthropod phylogenetics.

Robust resolution of controversial higher-level groupings within Arthropoda requires additional sources of characters. Toward this end, elongation factor-2 sequences (1899 nucleotides) were generated from 17 arthropod taxa (5 chelicerates, 6 crustaceans, 3 hexapods, 3 myriapods) plus an onychophoran and a tardigrade as outgroups. Likelihood and parsimony analyses of nucleotide and amino acid data sets consistently recovered Myriapoda and major chelicerate groups with high bootstrap support. Crustacea + Hexapoda (= Pancrustacea) was recovered with moderate support, whereas the conflicting group Myriapoda + Hexapoda (= Atelocerata) was never recovered and bootstrap values were always <5%. With additional nonarthropod sequences included, one indel supports monophyly of Tardigrada, Onychophora, and Arthropoda relative to molluscan, annelidan, and mammalian outgroups. New and previously published sequences from RNA polymerase II (1038 nucleotides) and elongation factor-1alpha (1092 nucleotides) were analyzed for the same taxa. A comparison of bootstrap values from the three genes analyzed separately revealed widely varying values for some clades, although there was never strong support for conflicting groups. In combined analyses, there was strong bootstrap support for the generally accepted clades Arachnida, Arthropoda, Euchelicerata, Hexapoda, and Pycnogonida, and for Chelicerata, Myriapoda, and Pancrustacea, whose monophyly is more controversial. Recovery of some additional groups was fairly robust to method of analysis but bootstrap values were not high; these included Pancrustacea + Chelicerata, Hexapoda + Cephalocarida + Remipedia, Cephalocarida + Remipedia, and Malaocostraca + Cirripedia. Atelocerata (= Myriapoda + Hexapoda) was never recovered. Elongation factor-2 is now the second protein-encoding, nuclear gene (in addition to RNA polymerase II) to support Pancrustacea over Atelocerata. Atelocerata is widely cited in morphology-based analyses, and the discrepancy between results derived from molecular and morphological data deserves greater attention.     

Zinc inhibits protein synthesis in neurons. Potential role of phosphorylation of translation initiation factor-2alpha.

In the central nervous system, Zn(2+) is concentrated in the cerebral cortex and hippocampus and has been found to be toxic to neurons. In this study, we show that exposure of cultured cortical neurons from mouse to increasing concentrations of Zn(2+) (10-300 microM) induces a progressive decrease in global protein synthesis. The potency of Zn(2+) was increased by about 2 orders of magnitude in the presence of Na(+)-pyrithione, a Zn(2+) ionophore. The basal rate of protein synthesis was restored 3 h after Zn(2+) removal. Zn(2+) induced a sustained increase in phosphorylation of the alpha subunit of the translation eukaryotic initiation factor-2 (eIF-2alpha), whereas it triggered a transient increase in phosphorylation of eukaryotic elongation factor-2 (eEF-2). Protein synthesis was still depressed 60 min after the onset of Zn(2+) exposure while the state of eEF-2 phosphorylation had already returned to its basal level. Moreover, Zn(2+) was less effective than glutamate to increase eEF-2 phosphorylation, whereas it induced a more profound inhibition of protein synthesis. These results suggest that Zn(2+)-induced inhibition of protein synthesis mainly correlates with the increase in eIF-2alpha phosphorylation. Supporting further that Zn(2+) acts at the initiation step of protein synthesis, it strongly decreased the amount of polyribosomes.     

Regulation of autophagy by sphingosine kinase 1 and its role in cell survival during nutrient starvation

The sphingolipid ceramide induces macroautophagy (here called autophagy) and cell death with autophagic features in cancer cells. Here we show that overexpression of sphingosine kinase 1 (SK1), an enzyme responsible for the production of sphingosine 1-phosphate (S1P), in MCF-7 cells stimulates autophagy by increasing the formation of LC3-positive autophagosomes and the rate of proteolysis sensitive to the autophagy inhibitor 3-methyladenine. Autophagy was blocked in the presence of dimethylsphingosine, an inhibitor of SK activity, and in cells expressing a catalytically inactive form of SK1. In SK1(wt)-overexpressing cells, however, autophagy was not sensitive to fumonisin B1, an inhibitor of ceramide synthase. In contrast to ceramide-induced autophagy, SK1(S1P)-induced autophagy is characterized by (i) the inhibition of mammalian target of rapamycin signaling independently of the Akt/protein kinase B signaling arm and (ii) the lack of robust accumulation of the autophagy protein Beclin 1. In addition, nutrient starvation induced both the stimulation of autophagy and SK activity. Knocking down the expression of the autophagy protein Atg7 or that of SK1 by siRNA abolished starvation-induced autophagy and increased cell death with apoptotic hallmarks. In conclusion, these results show that SK1(S1P)-induced autophagy protects cells from death with apoptotic features during nutrient starvation.     

Role of AMP-activated protein kinase in autophagy and proteasome function

In this work, we have examined the possible role of AMP-activated protein kinase (a key energy sensor) in regulating intracellular protein degradation. We have found that AICAR, a known activator of AMPK, has a dual effect. On one hand, it inhibits autophagy by a mechanism independent of AMPK activity; AICAR decreases class III PI3-kinase binding to beclin-1 and this effect counteracts and reverses the known positive effect of AMPK activity on autophagy. On the other hand, AICAR inhibits the proteasomal degradation of proteins by an AMPK-dependent mechanism. This is a novel function of AMPK that allows the regulation of proteasomal activity under conditions of energy demand.     

AMP-activated protein kinase modulates cardiac autophagy in diabetic cardiomyopathy

We have recently shown that in diabetic OVE26 mice (type I diabetes), the AMP-activated protein kinase (AMPK) is reduced along with cardiac dysfunction and decreased cardiac autophagy. Genetic inhibition of AMPK in cardiomyocytes attenuates cardiac autophagy, exacerbates cardiac dysfunction and increases mortality in diabetic mice. More importantly, we have found chronic AMPK activation with metformin, one of the most used antidiabetes drugs and a well-characterized AMPK activator, significantly enhances autophagic activity, preserves cardiac function and prevents most of the primary characteristics of diabetic cardiomyopathy in OVE26 mice, but not in dominant negative-AMPK diabetic mice. We conclude that AMPK activation protects cardiac structure and function by increasing cardiac autophagy in the diabetic heart.     

AMP-activated protein kinase modulates cardiac autophagy in diabetic cardiomyopathy

We have recently shown that in diabetic OVE26 mice (type I diabetes), the AMP-activated protein kinase (AMPK) is reduced along with cardiac dysfunction and decreased cardiac autophagy. Genetic inhibition of AMPK in cardiomyocytes attenuates cardiac autophagy, exacerbates cardiac dysfunction and increases mortality in diabetic mice. More importantly, we have found chronic AMPK activation with metformin, one of the most used antidiabetes drugs and a well-characterized AMPK activator, significantly enhances autophagic activity, preserves cardiac function and prevents most of the primary characteristics of diabetic cardiomyopathy in OVE26 mice, but not in dominant negative-AMPK diabetic mice. We conclude that AMPK activation protects cardiac structure and function by increasing cardiac autophagy in the diabetic heart.     

Inorganic lead stimulates DNA synthesis in human astrocytoma cells: role of protein kinase Calpha

As lead has been shown to activate protein kinase C (PKC), and gliomas are reported to be highly dependent on PKC for their proliferation, this study was undertaken to investigate whether lead may act as a mitogen in human astrocytoma cells, and to determine the role of PKC in this effect. Lead acetate (from 100 nM to 100 microM) induced a concentration- and time-dependent increase in DNA synthesis, as measured by incorporation of [methyl-3H]thymidine into cell DNA, without causing any cytotoxicity. Flow cytometric analysis showed that lead was able to stimulate the cell cycle transition from the G0/G1 phase to the S/G2 phase, resulting in increased percentage of cells in the latter phase. Western blot analyses showed that lead induced translocation of PKCalpha, but not of PKCepsilon or PKCzeta, from the cytosolic to the particulate fraction, with a concomitant increase in PKC enzyme activity. Prolonged exposure to lead caused down-regulation of PKCalpha, but not of PKCepsilon. The effect of lead on DNA synthesis was mediated through PKC as evidenced by the finding that two PKC inhibitors, GF 109203X and staurosporine, as well as down-regulation of PKC through prolonged treatment with 12-O-tetradecanoylphorbol 13-acetate, blocked lead-induced DNA synthesis. Further experiments using a pseudosubstrate peptide targeting classical PKCs and selective down-regulation of specific PKC isoforms indicated that the effect of lead on DNA synthesis was mediated by PKCalpha. Altogether, these results suggest that lead stimulates DNA synthesis in human astrocytoma cells by a mechanism that involves activation of PKCalpha.     

RNA localization and its role in the spatially restricted protein synthesis

RNA localization is an evolutionarily conserved phenomenon that occurs in uni- and multi-cellular animal and plant species. Localized RNA plays a role in the establishment of cell polarity and/or the determination of cell fate. In recent years, it became evident that the major function of RNA localization is the creation of a high concentration of proteins in specific cellular compartments. The movement of RNA involves interactions between targeting signals within the RNA molecule, cytoskeleton, and molecular motors. Translocating RNA must be translationally silent, and on-site translation at the destination site requires a de-repression mechanism. This is probably achieved by sequestering RNA and the regulators of translation within the multiprotein RNP complexes that co-translocate all the components to the ultimate destination within the cell.     

Silencing of EEF2K (eukaryotic elongation factor-2 kinase) reveals AMPK-ULK1-dependent autophagy in colon cancer cells

EEF2K (eukaryotic elongation factor-2 kinase), also known as Ca²⁺/calmodulin-dependent protein kinase III, functions in downregulating peptide chain elongation through inactivation of EEF2 (eukaryotic translation elongation factor 2). Currently, there is a limited amount of information on the promotion of autophagic survival by EEF2K in breast and glioblastoma cell lines. However, the precise role of EEF2K in carcinogenesis as well as the underlying mechanism involved is still poorly understood. In this study, contrary to the reported autophagy-promoting activity of EEF2K in certain cancer cells, EEF2K is shown to negatively regulate autophagy in human colon cancer cells as indicated by the increase of LC3-II levels, the accumulation of LC3 dots per cell, and the promotion of autophagic flux in EEF2K knockdown cells. EEF2K negatively regulates cell viability, clonogenicity, cell proliferation, and cell size in colon cancer cells. Autophagy induced by EEF2K silencing promotes cell survival and does not potentiate the anticancer efficacy of the AKT inhibitor MK-2206. In addition, autophagy induced by silencing of EEF2K is attributed to induction of protein synthesis and activation of the AMPK-ULK1 pathway, independent of the suppression of MTOR activity and ROS generation. Knockdown of AMPK or ULK1 significantly abrogates EEF2K silencing-induced increase of LC3-II levels, accumulation of LC3 dots per cell as well as cell proliferation in colon cancer cells. In conclusion, silencing of EEF2K promotes autophagic survival via activation of the AMPK-ULK1 pathway in colon cancer cells. This finding suggests that upregulation of EEF2K activity may constitute a novel approach for the treatment of human colon cancer.     

Cofilin phosphorylation by protein kinase testicular protein kinase 1 and its role in integrin-mediated actin reorganization and focal adhesion formation

Testicular protein kinase 1 (TESK1) is a serine/threonine kinase with a structure composed of a kinase domain related to those of LIM-kinases and a unique C-terminal proline-rich domain. Like LIM-kinases, TESK1 phosphorylated cofilin specifically at Ser-3, both in vitro and in vivo. When expressed in HeLa cells, TESK1 stimulated the formation of actin stress fibers and focal adhesions. In contrast to LIM-kinases, the kinase activity of TESK1 was not enhanced by Rho-associated kinase (ROCK) or p21-activated kinase, indicating that TESK1 is not their downstream effector. Both the kinase activity of TESK1 and the level of cofilin phosphorylation increased by plating cells on fibronectin. Y-27632, a specific inhibitor of ROCK, inhibited LIM-kinase-induced cofilin phosphorylation but did not affect fibronectin-induced or TESK1-induced cofilin phosphorylation in HeLa cells. Expression of a kinase-negative TESK1 suppressed cofilin phosphorylation and formation of stress fibers and focal adhesions induced in cells plated on fibronectin. These results suggest that TESK1 functions downstream of integrins and plays a key role in integrin-mediated actin reorganization, presumably through phosphorylating and inactivating cofilin. We propose that TESK1 and LIM-kinases commonly phosphorylate cofilin but are regulated in different ways and play distinct roles in actin reorganization in living cells.     

Macrophage protein kinase C: its role in modulating membrane microviscosity and superoxide in leishmanial infection

Pretreatment of macrophages with, an agonist of PKC, showed diverse effects on degradation and survival of two virulent strains of Leishmania donovani promastigotes. Treatment of macrophages with PMA for 45 min at 37 degrees C generated significant amounts of superoxide anions and reduced the parasite burden of macrophages by up to 48 and 43% when AG83 and GE-1 strains were used for infection. Staurosporine, an inhibitor of PKC, inhibited PMA-dependent killing of the parasites, while tyrphostin AG 126, an inhibitor of protein tyrosine kinase, showed very little effect. Depletion of PKC by prolonged incubation with PMA drastically reduced the superoxide anion generation and increased the uptake and multiplication of the parasites. Finally, to understand the mechanism of higher uptake of the parasites by PKC-depleted macrophages, membrane microviscosity was measured by fluorescence depolarization. Membrane microviscosity was found to be approximately 40% lower in PKC-depleted macrophages than in normal macrophages, indicating the role of membrane fluidity in the infection process. Together, these data suggest PKC activation, superoxide generation, and membrane fluidity are essential factors in the efficient regulation of leishmanial infection.