Ser/Thr kinases and polyamines in the regulation of non-canonical functions of elongation factor 1A

The link between eukaryotic translation elongation factor 1A (eEF1A) and signal transduction pathways through the regulatory mechanism of phosphorylation has never been considered. In this review, we focus on the different kinases that recognize the Ser and Thr residues of the eEF1A1 and eEF1A2 isoforms and regulate their involvement in different cellular processes like cell survival and apoptosis. In this context, polyamines seem to play a role in the regulation of the translation elongation process by modulating the Ser/Thr kinases involved in the phosphorylation of translation elongation factors.     

Recent progress on the structure of Ser/Thr protein phosphatases

PP1, PP2A and PP2B, belonging to the PPP family of Ser/Thr protein phosphatases, participate in regulating many important physiological processes, such as cell cycle control, regulation of cell growth and division regulation, etc. The sequence homology between them is relatively high, and ter- tiary structure is conserved. Because of the complexity of the structure of PP2A and the diversity of its regulatory subunits, its structure is less well known than those of PP1 and PP2B. The PP2A holoen- zyme consists of a heterodimeric core enzyme, comprising a scaffolding subunit and a catalytic sub- unit, as well as a variable regulatory subunit. In this study, the subunit compositions, similarities and differences between the Ser/Thr protein phsphatases structures are summarized.     

Peptide microarrays for high-throughput studies of Ser/Thr phosphatases

Protein phosphorylation and dephosphorylation play an important role in regulation of intracellular signal transduction pathways in the biological system. A key step in the biological characterization of phosphatases and their use as drug targets is the identification of their cellular partners and suitable substrates for potential inhibitor development. Herein we describe a microarray-based protocol to map the substrate specificity of protein Ser/Thr phosphatases. This protocol uses Pro-Q dye to sensitively and quantitatively detect the amount of dephosphorylation that occurs from many putative peptide substrates in parallel, and therefore could be used to generate the so-called peptide substrate fingerprints as well as detailed kinetic information of a target phosphatase. Excluding the synthesis of the peptide substrates, the whole protocol takes a total of 11 h to complete and in future can be readily extended to the study of other classes of phosphatases, i.e., protein tyrosine phosphatases.     

Mycobacterial Ser/Thr protein kinases and phosphatases: physiological roles and therapeutic potential

Reversible protein phosphorylation is a major regulation mechanism of fundamental biological processes, not only in eukaryotes but also in bacteria. A growing body of evidence suggests that Ser/Thr phosphorylation play important roles in the physiology and virulence of Mycobacterium tuberculosis, the etiological agent of tuberculosis. This pathogen uses 'eukaryotic-like' Ser/Thr protein kinases and phosphatases not only to regulate many intracellular metabolic processes, but also to interfere with signaling pathways of the infected host cell. Disrupting such processes by means of selective inhibitors may thus provide new pharmaceutical weapons to combat the disease. Here we review the current knowledge on Ser/Thr protein kinases and phosphatases in M. tuberculosis, their regulation mechanisms and putative substrates, and we explore their therapeutic potential as possible targets for the development of new anti-mycobacterial compounds.     

Structural and binding studies of the three-metal center in two mycobacterial PPM Ser/Thr protein phosphatases

Phospho-Ser/Thr protein phosphatases (PPs) are dinuclear metalloenzymes classed into two large families, PPP and PPM, on the basis of sequence similarity and metal ion dependence. The archetype of the PPM family is the α isoform of human PP2C (PP2Cα), which folds into an α/β domain similar to those of PPP enzymes. The recent structural studies of three bacterial PPM phosphatases, Mycobacterium tuberculosis MtPstP, Mycobacterium smegmatis MspP, and Streptococcus agalactiae STP, confirmed the conservation of the overall fold and dinuclear metal center in the family, but surprisingly revealed the presence of a third conserved metal-binding site in the active site. To gain insight into the roles of the three-metal center in bacterial enzymes, we report structural and metal-binding studies of MtPstP and MspP. The structure of MtPstP in a new trigonal crystal form revealed a fully active enzyme with the canonical dinuclear metal center but without the third metal ion bound to the catalytic site. The absence of metal correlates with a partially unstructured flap segment, indicating that the third manganese ion contributes to reposition the flap, but is dispensable for catalysis. Studies of metal binding to MspP using isothermal titration calorimetry revealed that the three Mn²⁺-binding sites display distinct affinities, with dissociation constants in the nano- and micromolar range for the two catalytic metal ions and a significantly lower affinity for the third metal-binding site. In agreement, the structure of inactive MspP at acidic pH was determined at atomic resolution and shown to lack the third metal ion in the active site. Structural comparisons of all bacterial phosphatases revealed positional variations in the third metal-binding site that are correlated with the presence of bound substrate and the conformation of the flap segment, supporting a role of this metal ion in assisting enzyme-substrate interactions.     

Overexpression of budding yeast protein phosphatase Ppz1 impairs translation

The Ser/Thr protein phosphatase Ppz1 from Saccharomyces cerevisiae is the best characterized member of a family of enzymes only found in fungi. Ppz1 is regulated in vivo by two inhibitory subunits, Hal3 and Vhs3, which are moonlighting proteins also involved in the decarboxylation of the 4-phosphopantothenoylcysteine (PPC) intermediate required for coenzyme A biosynthesis. It has been reported that, when overexpressed, Ppz1 is the most toxic protein in yeast. However, the reasons for such toxicity have not been elucidated. Here we show that the detrimental effect of excessive Ppz1 expression is due to an increase in its phosphatase activity and not to a plausible down-titration of the PPC decarboxylase components. We have identified several genes encoding ribosomal proteins and ribosome assembly factors as mild high-copy suppressors of the toxic Ppz1 effect. Ppz1 binds to ribosomes engaged in translation and copurifies with diverse ribosomal proteins and translation factors. Ppz1 overexpression results in Gcn2-dependent increased phosphorylation of eIF2α at Ser-51. Consistently, deletion of GCN2 partially suppresses the growth defect of a Ppz1 overexpressing strain. We propose that the deleterious effects of Ppz1 overexpression are in part due to alteration in normal protein synthesis.     

Global analysis of phosphoproteome regulation by the Ser/Thr phosphatase Ppt1 in Saccharomyces cerevisiae

Even though protein phosphatases are key regulators of signal transduction, their cellular mechanisms of action are poorly understood. Here, we undertook a large-scale proteomics survey to identify cellular protein targets of a serine/threonine phosphatase. We used SILAC-based quantitative MS to measure differences in protein expression and phosphorylation upon ablation of the serine/threonine phosphatase Ppt1 in Saccharomyces cerevisiae. Phosphopeptide fractionation by strong cation exchange chromatography combined with immobilized metal affinity chromatography (IMAC) enrichment enabled quantification of more than 8000 distinct phosphorylation sites in Ppt1 wild-type versus Ppt1-deficient yeast cells. We further quantified the relative expression of 1897 yeast proteins and detected no major protein changes accompanying Ppt1 deficiency. Notably, we found 33 phosphorylation sites to be significantly and reproducibly up-regulated while no phosphorylation events were repressed in cells lacking Ppt1. Ppt1 acted on its cellular target proteins in a sequence- and site-specific fashion. Several of the regulated phosphoproteins were involved in the response to heat stress in agreement with known Ppt1 functions. Additionally, biosynthetic enzymes were particularly prominent among Ppt1-regulated phosphoproteins, pointing to unappreciated roles of Ppt1 in the control of various metabolic functions. These results demonstrate the utility of large-scale and quantitative phosphoproteomics to identify cellular sites of serine/threonine phosphatase action in an unbiased manner.     

The eukaryotic translation elongation Factor 1Bgamma has a non-guanine nucleotide exchange factor role in protein metabolism

The turnover of damaged proteins is critical to cell survival during stressful conditions such as heat shock or oxidative stress. The accumulation of misfolded proteins in the endoplasmic reticulum (ER) is toxic to cells. Therefore these proteins must be efficiently exported from the ER and degraded by the proteasome or the vacuole. Previously it was shown that the loss of eukaryotic elongation factor 1Bγ (eEF1Bγ) from the yeast Saccharomyces cerevisiae results in resistance to oxidative stress. Strains lacking eEF1Bγ show severe defects in protein turnover during conditions of oxidative stress. Furthermore, these strains accumulate a greater amount of oxidized proteins, which correlates with changes in heat shock chaperones. These strains show severe defects in vacuole morphology and defects related to the maturation of carboxypeptidase Y that is not dependent on the catalytic subunit of the eEF1B complex as a guanine nucleotide exchange factor. Finally, eEF1Bγ co-immunoprecipitates with an essential component of ER-Golgi transport vesicles. Taken together, these results support a broader protein metabolism role for eEF1Bγ.     

A novel transmembrane Ser/Thr kinase complexes with protein phosphatase-1 and inhibitor-2

Protein kinases and protein phosphatases exert coordinated control over many essential cellular processes. Here, we describe the cloning and characterization of a novel human transmembrane protein KPI-2 (Kinase/Phosphatase/Inhibitor-2) that was identified by yeast two-hybrid using protein phosphatase inhibitor-2 (Inh2) as bait. KPI-2 mRNA was predominantly expressed in skeletal muscle. KPI-2 is a 1503-residue protein with two predicted transmembrane helices at the N terminus, a kinase domain, followed by a C-terminal domain. The transmembrane helices were sufficient for targeting proteins to the membrane. KPI-2 kinase domain has about 60% identity with its closest relative, a tyrosine kinase. However, it only exhibited serine/threonine kinase activity in autophosphorylation reactions or with added substrates. KPI-2 kinase domain phosphorylated protein phosphatase-1 (PP1C) at Thr(320), which attenuated PP1C activity. KPI-2 C-terminal domain directly associated with PP1C, and this required a VTF motif. Inh2 associated with KPI-2 C-terminal domain with and without PP1C. Thus, KPI-2 is a kinase with sites to associate with PP1C and Inh2 to form a regulatory complex that is localized to membranes.     

Extended conformation of mammalian translation elongation factor 1A in solution

The conformation of mammalian elongation factor eEF1A in solution was examined by the small angle neutron scattering and scanning microcalorimetry. We have found that in contrast to the bacterial analogue the eEF1A molecule has no fixed rigid structure in solution. The radius of gyration of the eEF1A molecule (5.2 nm) is much greater than that of prokaryotic EF1A. The specific heat of denaturation is considerably lower for eEF1A than for EF1A, suggesting that the eEF1A conformation is significantly more disordered. Despite its flexible conformation, eEF1A is found to be highly active in different functional tests. According to the neutron scattering data, eEF1A becomes much more compact in the complex with uncharged tRNA. The absence of a rigid structure and the possibility of large conformational change upon interaction with a partner molecule could be important for eEF1A functioning in channeled protein synthesis and/or for the well-known capability of the protein to interact with different ligands besides the translational components.     

丝/苏氨酸蛋白激酶Plk1研究进展

Plk1是一类从酵母到人类都高度保守的丝氨酸/苏氨酸蛋白激酶。Plk1与不同的细胞周期检查点的精密调控有关,从而确保了细胞周期事件按照严格的时间和顺序正常进行。Plk1在增殖活跃的细胞中呈高水平表达,Plk1的高度表达和肿瘤患者的低存活率之间具有显著的统计相关性。Plk1可能是非常有效的抗癌药物设计的靶点。     

The role of protein elongation factor eEF1A2 in ovarian cancer

Frequent gains of chromosome 20q12-13 in ovarian tumors indicate that at least one important oncogene is found at that locus. One of the genes there is EEF1A2, which maps to 20q13.3 and encodes protein elongation factor eEF1A2. This review will focus on recent evidence indicating that EEF1A2 is an important ovarian oncogene and that the protein elongation network can activate tumorigenesis and inhibit apoptosis.     

The eukaryotic translation elongation factor 1A regulation of actin stress fibers is important for infectious RSV production

Cellular protein eukaryotic translation elongation factor 1A (eEF1A) is an actin binding protein that plays a role in the formation of filamentous actin (F-actin) bundles. F-Actin regulates multiple stages of respiratory syncytial virus (RSV) replication including assembly and budding. Our previous study demonstrated that eEF1A knock-down significantly reduced RSV replication. Here we investigated if the eEF1A function in actin bundle formation was important for RSV replication and release. To investigate this, eEF1A function was impaired in HEp-2 cells by either knock-down of eEF1A with siRNA, or treatment with an eEF1A inhibitor, didemnin B (Did B). Cell staining and confocal microscopy analysis showed that both eEF1A knock-down and treatment with Did B resulted in disruption of cellular stress fiber formation and elevated accumulation of F-actin near the plasma membrane. When treated cells were then infected with RSV, there was also reduced formation of virus-induced cellular filopodia. Did B treatment, similarly to eEF1A knock-down, reduced the release of infectious RSV, but unlike eEF1A knock-down, did not significantly affect RSV genome replication. The lower infectious virus production in Did B treated cells also reduced RSV-induced cell death. In conclusion, the cellular factor eEF1A plays an important role in the regulation of F-actin stress fiber formation required for RSV assembly and release.     

Analysis of the role of Ser1/Ser2/Thr9 phosphorylation on myosin II assembly and function in live cells

Background

The therapeutic efficacy of human mesenchymal stem cells (hMSCs) for the treatment of hypoxic-ischemic diseases is closely related to level of hypoxia in the damaged tissues. To elucidate the potential therapeutic applications and limitations of hMSCs derived from human umbilical cords, the effects of hypoxia on the morphology and proliferation of hMSCs were analyzed.

Results

After treatment with DFO and CoCl₂, hMSCs were elongated, and adjacent cells were no longer in close contact. In addition, vacuole-like structures were observed within the cytoplasm; the rough endoplasmic reticulum expanded, and expanded ridges were observed in mitochondria. In addition, DFO and CoCl₂ treatments for 48 h significantly inhibited hMSCs proliferation in a concentration-dependent manner (P < 0.05). This treatment also increased the number of cells in G0/G1 phase and decreased those in G2/S/M phase.

Conclusions

The hypoxia-mimetic agents, DFO and CoCl₂, alter umbilical cord-derived hMSCs morphology and inhibit their proliferation through influencing the cell cycle.     

Structural basis for nucleotide exchange and competition with tRNA in the yeast elongation factor complex eEF1A:eEF1Balpha

The crystal structure of a complex between the protein biosynthesis elongation factor eEF1A (formerly EF-1alpha) and the catalytic C terminus of its exchange factor, eEF1Balpha (formerly EF-1beta), was determined to 1.67 A resolution. One end of the nucleotide exchange factor is buried between the switch 1 and 2 regions of eEF1A and destroys the binding site for the Mg(2+) ion associated with the nucleotide. The second end of eEF1Balpha interacts with domain 2 of eEF1A in the region hypothesized to be involved in the binding of the CCA-aminoacyl end of the tRNA. The competition between eEF1Balpha and aminoacylated tRNA may be a central element in channeling the reactants in eukaryotic protein synthesis. The recognition of eEF1A by eEF1Balpha is very different from that observed in the prokaryotic EF-Tu:EF-Ts complex. Recognition of the switch 2 region in nucleotide exchange is, however, common to the elongation factor complexes and those of Ras:Sos and Arf1:Sec7.     

Dynamics of protein phosphorylation on Ser/Thr/Tyr in Bacillus subtilis

The Ser/Thr/Tyr phosphoproteome of Bacillus subtilis was analyzed by a 2-D gel-based approach combining Pro-Q Diamond staining and [(33)P]-labeling. In exponentially growing B. subtilis cells 27 proteins could be identified after staining with Pro-Q Diamond and/or [(33)P]-labeling and one additional protein was labeled solely by [(33)P] resulting in a total of 28 potentially phosphorylated proteins. These proteins are mainly involved in enzymatic reactions of basic carbon metabolism and the regulation of the alternative sigma factor sigma(B). We also found significant changes of the phosphoproteome including increased phosphorylation and dephosphorylation rates of some proteins as well as the detection of four newly phosphorylated proteins in response to stress or starvation. For nine proteins, phosphorylation sites at serine or threonine residues were determined by MS. These include the known phosphorylation sites of Crh, PtsH, and RsbV. Additionally, we were able to identify novel phosphorylation sites of AroA, Pyk, and YbbT. Interestingly, the phosphorylation of RsbRA, B, C, and D, four proteins of a multicomponent protein complex involved in environmental stress signaling, was found during exponential growth. For RsbRA, B, and D, phosphorylation of one of the conserved threonine residues in their C-termini were verified by MS (T171, T186, T181, respectively).