Roles of yeast eIF2α and eIF2β subunits in the binding of the initiator methionyl-tRNA

Heterotrimeric eukaryotic/archaeal translation initiation factor 2 (e/aIF2) binds initiator methionyl-tRNA and plays a key role in the selection of the start codon on messenger RNA. tRNA binding was extensively studied in the archaeal system. The γ subunit is able to bind tRNA, but the α subunit is required to reach high affinity whereas the β subunit has only a minor role. In Saccharomyces cerevisiae however, the available data suggest an opposite scenario with β having the most important contribution to tRNA-binding affinity. In order to overcome difficulties with purification of the yeast eIF2γ subunit, we designed chimeric eIF2 by assembling yeast α and β subunits to archaeal γ subunit. We show that the β subunit of yeast has indeed an important role, with the eukaryote-specific N- and C-terminal domains being necessary to obtain full tRNA-binding affinity. The α subunit apparently has a modest contribution. However, the positive effect of α on tRNA binding can be progressively increased upon shortening the acidic C-terminal extension. These results, together with small angle X-ray scattering experiments, support the idea that in yeast eIF2, the tRNA molecule is bound by the α subunit in a manner similar to that observed in the archaeal aIF2–GDPNP–tRNA complex.     

Methylglyoxal activates Gcn2 to phosphorylate eIF2α independently of the TOR pathway in Saccharomyces cerevisiae

Methylglyoxal is a ubiquitous 2-oxoaldehyde derived from glycolysis. Previously, we have reported that methylglyoxal attenuates the rate of overall protein synthesis in Saccharomyces cerevisiae through phosphorylation of the α subunit of translation initiation factor 2 (eIF2α) in a Gcn2-dependent manner. Phosphorylation of eIF2α impedes the formation of a translation initiation complex, and subsequently, overall protein synthesis is reduced. Uncharged tRNA plays an important role in the activation of Gcn2, although we found that MG treatment did not elevate the levels of uncharged tRNA. Rapamycin, a potent inhibitor of TOR kinase, is known to induce phosphorylation of eIF2α without affecting the levels of uncharged tRNA. We determined the correlation between methylglyoxal and TOR kinase activity and found that phosphorylation of eIF2α by methylglyoxal occurred independently of the target of rapamycin (TOR) pathway.     

A truncated Danio rerio PKZ isoform functionally interacts with eIF2α and inhibits protein synthesis

A protein kinase containing Z-DNA binding domains (PKZ), which resembles protein kinase R (PKR) in domain organization, was recently discovered to be a member of the eIF2α kinase family in fish. PKR has roles in antiviral immunity through inhibiting protein synthesis and activating NF-κB; therefore, it is thought that PKZ may have a similar role in fish antiviral immunity. In the present study, the roles of two Danio rerio PKZ isoforms (DrPKZ-A and DrPKZ-B) in eIF2α phosphorylation and protein synthesis regulation were explored. DrPKZ-A and DrPKZ-B possess N-terminal Z-DNA binding domains and a conserved eIF2α kinase domain; however, they have domains of differing lengths inserted between kinase subdomains IV and V. DrPKZ-A has an insert domain of 73 amino acids (aa), whereas DrPKZ-B has an insert sequence of only 10 aa, suggesting that DrPKZ-B could be a dysfunctional isoform or could interact with different substrates. Our results show that both DrPKZ-A and DrPKZ-B functionally interact with eIF2α and inhibit protein synthesis, although DrPKZ-B possesses attenuated kinase activity. Our results also show that deletion of the insert in either isoform results in the complete abrogation of kinase activity, suggesting that the insert is critical for PKZ kinase activity. Kinase activity appears to be independent of insert length but may depend on the presence of specific amino acids within the insert domain. Furthermore, the effects of the N-terminal regulatory domain on kinase activity were analyzed. Deletion of the N-terminus results in reduced kinase activity of these isoforms relative to the wild-type forms, indicating that the isolated kinase domain is sufficient for eIF2α phosphorylation and that DrPKZ-A and DrPKZ-B may be regulated in a similar manner. Overall, our results show that DrPKZ-B is a functional kinase in zebrafish and contribute to our understanding of the function of PKZ in fish.     

Protein phosphatase-1α interacts with and dephosphorylates polycystin-1

Polycystin signaling is likely to be regulated by phosphorylation. While a number of potential protein kinases and their target phosphorylation sites on polycystin-1 have been identified, the corresponding phosphatases have not been extensively studied. We have now determined that polycystin-1 is a regulatory subunit for protein phosphatase-1α (PP1α). Sequence analysis has revealed the presence of a highly conserved PP1-interaction motif in the cytosolic, C-terminal tail of polycystin-1; and we have shown that transfected PP1α specifically co-immunoprecipitates with a polycystin-1 C-tail construct. To determine whether PP1α dephosphorylates polycystin-1, a PKA-phosphorylated GST-polycystin-1 fusion protein was shown to be dephosphorylated by PP1α but not by PP2B (calcineurin). Mutations within the PP1-binding motif of polycystin-1, including an autosomal dominant polycystic kidney disease (ADPKD)-associated mutation, significantly reduced PP1α-mediated dephosphorylation of polycystin-1. The results suggest that polycystin-1 forms a holoenzyme complex with PP1α via a conserved PP1-binding motif within the polycystin-1 C-tail, and that PKA-phosphorylated polycystin-1 serves as a substrate for the holoenzyme.     

The SH2-domain of SHIP1 interacts with the SHIP1 C-terminus: impact on SHIP1/Ig-α interaction

The SH2-containing inositol 5'-phosphatase, SHIP1, negatively regulates signal transduction from the B cell antigen receptor (BCR). The mode of coupling between SHIP1 and the BCR has not been elucidated so far. In comparison to wild-type cells, B cells expressing a mutant IgD- or IgM-BCR containing a C-terminally truncated Ig-α respond to pervanadate stimulation with markedly reduced tyrosine phosphorylation of SHIP1 and augmented activation of protein kinase B. This indicates that SHIP1 is capable of interacting with the C-terminus of Ig-α. Employing a system of fluorescence resonance energy transfer in S2 cells, we can clearly demonstrate interaction between the SH2-domain of SHIP1 and Ig-α. Furthermore, a fluorescently labeled SH2-domain of SHIP1 translocates to the plasma membrane in an Ig-α-dependent manner. Interestingly, whereas the SHIP1 SH2-domain can be pulled-down with phospho-peptides corresponding to the immunoreceptor tyrosine-based activation motif (ITAM) of Ig-α from detergent lysates, no interaction between full-length SHIP1 and the phosphorylated Ig-α ITAM can be observed. Further studies show that the SH2-domain of SHIP1 can bind to the C-terminus of the SHIP1 molecule, most probably by inter- as well as intra-molecular means, and that this interaction regulates the association between different forms of SHIP1 and Ig-α.     

Enhanced Interaction between Pseudokinase and Kinase Domains in Gcn2 stimulates eIF2α Phosphorylation in Starved Cells

The stress-activated protein kinase Gcn2 regulates protein synthesis by phosphorylation of translation initiation factor eIF2α, from yeast to mammals. The Gcn2 kinase domain (KD) is inherently inactive and requires allosteric stimulation by adjoining regulatory domains. Gcn2 contains a pseudokinase domain (YKD) required for high-level eIF2α phosphorylation in amino acid starved yeast cells; however, the role of the YKD in KD activation was unknown. We isolated substitutions of evolutionarily conserved YKD amino acids that impair Gcn2 activation without reducing binding of the activating ligand, uncharged tRNA, to the histidyl-tRNA synthetase-related domain of Gcn2. Several such Gcn⁻ substitutions cluster in predicted helices E and I (αE and αI) of the YKD. We also identified Gcd⁻ substitutions, evoking constitutive activation of Gcn2, mapping in αI of the YKD. Interestingly, αI Gcd⁻ substitutions enhance YKD-KD interactions in vitro, whereas Gcn⁻ substitutions in αE and αI suppress both this effect and the constitutive activation of Gcn2 conferred by YKD Gcd⁻ substitutions. These findings indicate that the YKD interacts directly with the KD for activation of kinase function and identify likely sites of direct YKD-KD contact. We propose that tRNA binding to the HisRS domain evokes a conformational change that increases access of the YKD to sites of allosteric activation in the adjoining KD.     

Androgen receptor coregulator ARA267-α interacts with death receptor-6 revealed by the yeast two-hybrid

ARA267-α is a newly identified androgen receptor coactivator. In order to further elucidate its precise role in cells, using the ARA267-α fragment containing four PHD and one SET conserved domains as bait we revealed an ARA267-α-PHD-SET-interacting protein, death receptor-6 (DR6), in the yeast two-hybrid screening. DR6 is the member of TNF receptor family and has a death domain in its intracellular cytoplasmic portion (DR6cp) to mediate the cell apoptosis. The interaction between ARA267-α-PHD-SET and DR6cp was confirmed in vitro and in vivo. Our finding implied that androgen signaling pathway might cross talk with apoptosis signaling pathway through the interaction between ARA267-α and DR6.     

Specific in vitro phosphorylation of plant eIF2α by eukaryotic eIF2α kinases

Phosphorylation of the subunit of eukaryotic initiation factor 2 (eIF2) is known to be an important translational control mechanism in all eukaryotes with the major exception of plants. Regulation of mammalian and yeast eIF2 activity is directly governed by specific phosphorylation on Ser-51. We now demonstrate that recombinant wheat wild-type (51S) but not mutant 51-Ala (51A) protein is phosphorylated by human PKR and yeast GCN2, which are defined eIF2 kinases. Further, only wheat wild-type eIF2 is a substrate for plant-encoded, double-stranded RNA-dependent kinase (pPKR) activity. Plant PKR and GCN2 phosphorylate recombinant yeast eIF2 51S but not the 51A mutant demonstrating that pPKR has recognition site capability similar to established eIF2 kinases. A truncated version of wild-type wheat eIF2 containing 51S but not the KGYID motif is not phosphorylated by either hPKR or pPKR suggesting that this putative eIF2 kinase docking domain is essential for phosphorylation. Taken together, these results demonstrate the homology among eukaryotic eIF2 species and eIF2 kinases and support the presence of a plant eIF2 phosphorylation pathway.     

Androgen receptor coregulator ARA267-α interacts with death receptor-6 revealed by the yeast two-hybrid

ARA267-α is a newly identified androgen receptor coactivator. In order to further elucidate its precise role in cells, using the ARA267-α fragment containing four PHD and one SET conserved domains as bait we revealed an ARA267-α-PHD-SET-interacting protein, death receptor-6 (DR6), in the yeast two-hybrid screening. DR6 is the member of TNF receptor family and has a death domain in its intracellular cytoplasmic portion (DR6cp) to mediate the cell apoptosis. The interaction between ARA267-α-PHD-SET and DR6cp was confirmedin vitro andin vivo. Our finding implied that androgen signaling pathway might cross talk with apoptosis signaling pathway through the interaction between ARA267-α and DR6.     

p38α MAP kinase phosphorylates RCAN1 and regulates its interaction with calcineurin

RCAN1, also known as DSCR1, is an endogenous regulator of calcineurin, a serine/threonine protein phosphatase that plays a critical role in many physiological processes. In this report, we demonstrate that p38?? MAP kinase can phosphorylate RCAN1 at multiple sites in vitro and show that phospho-RCAN1 is a good protein substrate for calcineurin. In addition, we found that unphosphorylated RCAN1 noncompetitively inhibits calcineurin protein phosphatase activity and that the phosphorylation of RCAN1 by p38?? MAP kinase decreases the binding affinity of RCAN1 for calcineurin. These findings reveal the molecular mechanism by which p38?? MAP kinase regulates the function of RCAN1/calcineurin through phosphorylation.     

The Alström syndrome protein, ALMS1, interacts with α-actinin and components of the endosome recycling pathway

Alstr?m syndrome (ALMS) is a progressive multi-systemic disorder characterized by cone-rod dystrophy, sensorineural hearing loss, childhood obesity, insulin resistance and cardiac, renal, and hepatic dysfunction. The gene responsible for Alstr?m syndrome, ALMS1, is ubiquitously expressed and has multiple splice variants. The protein encoded by this gene has been implicated in ciliary function, cell cycle control, and intracellular transport. To gain better insight into the pathways through which ALMS1 functions, we carried out a yeast two hybrid (Y2H) screen in several mouse tissue libraries to identify ALMS1 interacting partners. The majority of proteins found to interact with the murine carboxy-terminal end (19/32) of ALMS1 were α-actinin isoforms. Interestingly, several of the identified ALMS1 interacting partners (α-actinin 1, α-actinin 4, myosin Vb, rad50 interacting 1 and huntingtin associated protein1A) have been previously associated with endosome recycling and/or centrosome function. We examined dermal fibroblasts from human subjects bearing a disruption in ALMS1 for defects in the endocytic pathway. Fibroblasts from these patients had a lower uptake of transferrin and reduced clearance of transferrin compared to controls. Antibodies directed against ALMS1 N- and C-terminal epitopes label centrosomes and endosomal structures at the cleavage furrow of dividing MDCK cells, respectively, suggesting isoform-specific cellular functions. Our results suggest a role for ALMS1 variants in the recycling endosome pathway and give us new insights into the pathogenesis of a subset of clinical phenotypes associated with ALMS.     

SIRPα interacts with nephrin at the podocyte slit diaphragm

The slit diaphragm (SD) is an intercellular junction between renal glomerular epithelial cells (podocytes) that is essential for permselectivity in glomerular ultrafiltration. The SD components, nephrin and Neph1, assemble a signaling complex in a tyrosine phosphorylation dependent manner, and regulate the unique actin cytoskeleton of podocytes. Mutations in the NPHS1 gene that encodes nephrin cause congenital nephrotic syndrome (CNS), which is characterized by the loss of the SD and massive proteinuria. Recently, we have identified the expression of the transmembrane glycoprotein signal regulatory protein α (SIRPα) at the SD. In the present study, we analyzed the expression of SIRPα in developing kidneys, in kidneys from CNS patients and in proteinuric rat models. The possibility that SIRPα interacts with known SD proteins was also investigated. SIRPα was concentrated at the SD junction during the maturation of intercellular junctions. In the glomeruli of CNS patients carrying mutations in NPHS1, where SD formation is disrupted, the expression of SIRPα as well as Neph1 and nephrin was significantly decreased, indicating that SIRPα is closely associated with the nephrin complex. Indeed, SIRPα formed hetero-oligomers with nephrin in cultured cells and in glomeruli. Furthermore, the cytoplasmic domain of SIRPα was highly phosphorylated in normal glomeruli, and its phosphorylation was dramatically decreased upon podocyte injury in?vivo. Thus, SIRPα interacts with nephrin at the SD, and its phosphorylation is dynamically regulated in proteinuric states. Our data provide new molecular insights into the phosphorylation events triggered by podocyte injury. Structured digital abstract ? Sirp-alpha?physically interacts?with?Nephrin?by?anti bait coimmunoprecipitation?(View interaction) ? Sirp-alpha?physically interacts?with?Nephrin?by?anti tag coimmunoprecipitation?(View interaction).     

Doxorubicin bypasses the cytoprotective effects of eIF2α phosphorylation and promotes PKR-mediated cell death

The eukaryotic cell responds to various forms of environmental stress by adjusting the rates of mRNA translation thus facilitating adaptation to the assaulting stress. One of the major pathways that control protein synthesis involves the phosphorylation of the α-subunit of eukaryotic initiation factor eIF2 at serine 51. Different forms of DNA damage were shown to induce eIF2α phosphorylation by using PERK, GCN2 or PKR. However, the specificity of the eIF2α kinases and the biological role of eIF2α phosphorylation pathway in the DNA damage response (DDR) induced by chemotherapeutics are not known. Herein, we show that PKR is the eIF2α kinase that responds to DDR induced by doxorubicin. We show that activation of PKR integrates two signaling pathways with opposing biological outcomes. More specifically, induction of eIF2α phosphorylation has a cytoprotective role, whereas activation of c-jun N-terminal kinase (JNK) by PKR promotes cell death in response to doxorubicin. We further show that the proapoptotic effects of JNK activation prevail over the cytoprotection mediated by eIF2α phosphorylation. These findings reveal that PKR can be an important inducer of cell death in response to chemotherapies through its ability to act independently of eIF2α phosphorylation.     

A Critical Role of the mTOR/eIF2α Pathway in Hypoxia-Induced Pulmonary Hypertension

Enhanced proliferation of pulmonary arterial vascular smooth muscle cells (PASMCs) is a key pathological component of vascular remodeling in hypoxia-induced pulmonary hypertension (HPH). Mammalian targeting of rapamycin (mTOR) signaling has been shown to play a role in protein translation and participate in the progression of pulmonary hypertension. Eukaryotic translation initiation factor-2α (eIF2α) is a key factor in regulation of cell growth and cell cycle, but its role in mTOR signaling and PASMCs proliferation remains unknown. Pulmonary hypertension (PH) rat model was established by hypoxia. Rapamycin was used to treat rats as an mTOR inhibitor. Proliferation of primarily cultured rat PASMCs was induced by hypoxia, rapamycin and siRNA of mTOR and eIF2α were used in loss-of-function studies. The expression and activation of eIF2α, mTOR and c-myc were analyzed. Results showed that mTOR/eIF2α signaling was significantly activated in pulmonary arteries from hypoxia exposed rats and PASMCs cultured under hypoxia condition. Treatment with mTOR inhibitor for 21 days attenuated vascular remodeling, suppressed mTOR and eIF2α activation, inhibited c-myc expression in HPH rats. In hypoxia-induced PASMCs, rapamycin and knockdown of mTOR and eIF2α by siRNA significantly abolished proliferation and increased c-myc expression. These results suggest a critical role of the mTOR/eIF2αpathway in hypoxic vascular remodeling and PASMCs proliferation of HPH.     

The binding mechanism of eIF2β with its partner proteins, eIF5 and eIF2Bε

The eukaryotic translation initiation factor eIF2 delivers Met-tRNAiMet to the ribosomal small subunit in GTP-bound form associated with eIF1, eIF1A, eIF3 and eIF5, and dissociates together with eIF5 as eIF5-eIF2-GDP complex from the ribosomal small subunit after formation of start codon-anticodon base pairing between Met-tRNAiMet and mRNA. The inactive form eIF2-GDP is then exchanged for the active form eIF2-GTP by eIF2B for further initiation cycle. Previous studies showed that the C-terminal domains of eIF5 (eIF5-CTD) and eIF2Bε (eIF2Bε-CTD) have a common eIF2β-binding site for interacting with an N-terminal region of eIF2β (eIF2β-NTD). Here we have reconstructed the complexes of (eIF5-CTD)-(eIF2β-NTD) and (eIF2Bε-CTD)-(eIF2β-NTD) in vitro, and investigated binding mechanism by circular dichroism spectroscopy and small angle X-ray scattering in solution. The results showed the conformation of eIF2β-NTD was changed when bound to partner proteins, whereas the structures of eIF5-CTD and eIF2Bε-CTD were similar in both isolated and complex states. We propose that eIF2β-NTD works as an intrinsically disordered domain which is disorder in the isolated state, but folds into a definite structure when bound to its partner proteins. Such flexibility of eIF2β-NTD is expected to be responsible for its binding capability.