Phosphorylation of the eukaryotic initiation factor 3f by cyclin-dependent kinase 11 during apoptosis

eIF3f is a subunit of eukaryotic initiation factor 3 (eIF3). We previously showed that eIF3f is phosphorylated by cyclin dependent kinase 11 (CDK11^p46) which is an important effector in apoptosis. Here, we identified a second eIF3f phosphorylation site (Thr119) by CDK11^p46 during apoptosis. We demonstrated that eIF3f is directly phosphorylated by CDK11^p46 in vivo. Phosphorylation of eIF3f plays an important role in regulating its function in translation and apoptosis. Phosphorylation of eIF3f enhances the association of eIF3f with the core eIF3 subunits during apoptosis. Our data suggested that eIF3f may inhibit translation by increasing the binding to the eIF3 complex during apoptosis.

Structured summary

MINT-6948874: EIF3b (uniprotkb:P55884) physically interacts (MI:0218) with EIF3f (uniprotkb:O00303) by anti bait coimmunoprecipitation (MI:0006)MINT-6948891: EIF3b (uniprotkb:P55884) physically interacts (MI:0218) with EIF3c (uniprotkb:Q99613), EIF3a (uniprotkb:Q14152) and EIF3f (uniprotkb:O00303) by anti bait coimmunoprecipitation (MI:0006)MINT-6948836, MINT-6948849, MINT-6948862: CDK11p46 (uniprotkb:P21127) phosphorylates (MI:0217) EIF3f (uniprotkb:O00303) by protein kinase assay (MI:0424)     

CDK11p46 and RPS8 associate with each other and suppress translation in a synergistic manner

CDK11p46, a 46 kDa isoform of the PITSLRE kinase family, is a key mediator of cell apoptosis, while the precise mechanism remains to be elucidated. By using His pull-down and mass spectrometry analysis, we identified the ribosomal protein S8 (RPS8), a member of the small subunit ribosome, as an interacting partner of CDK11p46. Further analysis confirmed the association of CDK11p46 and RPS8 in vitro and in vivo, and revealed that RPS8 was not a substrate of CDK11p46. Moreover, RPS8 and CDK11p46 synergize to inhibit the translation process both in cap- and internal ribosomal entry site (IRES)-dependent way, and sensitize cells to Fas ligand-induced apoptosis. Taken together, our results provide evidence for the novel role of CDK11p46 in the regulation of translation and cell apoptosis.     

Translation inhibition in apoptosis: caspase-dependent PKR activation and eIF2-alpha phosphorylation

The protein kinase PKR is a major player in the cellular antiviral response, acting mainly by phosphorylation of the alpha-subunit of the eukaryotic translation initiation factor 2 (eIF2-alpha) to block de novo protein synthesis. PKR activation requires binding of double-stranded RNA or PACT/RAX proteins to its regulatory domain. Since several reports have demonstrated that translation is inhibited in apoptosis, we investigated whether PKR and eIF2-alpha phosphorylation contribute to this process. We show that PKR is proteolysed and that eIF2-alpha is phosphorylated at the early stages of apoptosis induced by various stimuli. Both events coincide with the onset of caspase activity and are prevented by caspase inhibitors. Using site-directed mutagenesis we show that PKR is specifically proteolysed at Asp(251) during cellular apoptosis. This site is cleaved in vitro by recombinant caspase-3, caspase-7, and caspase-8 and not by the proinflammatory caspase-1 and caspase-11. The released kinase domain efficiently phosphorylates eIF2-alpha at the cognate Ser(51) residue, and its overexpression in mammalian cells impairs the translation of its own mRNA and of reporter mRNAs. Our results demonstrate a new and caspase-dependent activation mode for PKR, leading to eIF2-alpha phosphorylation and translation inhibition in apoptosis.     

p97/DAP5 is a ribosome-associated factor that facilitates protein synthesis and cell proliferation by modulating the synthesis of cell cycle proteins

p97 (also referred to as DAP5, NAT1 or eIF4G2) has been proposed to act as a repressor of protein synthesis. However, we found that p97 is abundantly expressed in proliferating cells and p97 is recruited to ribosomes following growth factor stimulation. We also report that p97 binds eIF2beta through its C-terminal domain and localizes to ribosome through its N-terminal MIF4G domain. When overexpressed, p97 increases reporter luciferase activity. In contrast, overexpression of the C-terminal two-thirds of eukaryotic initiation factor 4GI (eIF4GI), a region that shares significant homology with p97, or the N-terminal MIF4G domain of p97 markedly inhibits reporter activity, the rate of global translation and cell proliferation. Conversely, downregulation of p97 levels by RNA interference also decreases the rate of global translation and inhibits cell proliferation. This coincides with an increase in p27/Kip1 protein levels and a marked decrease in CDK2 kinase activity. Taken together, our results demonstrate that p97 is functionally different from the closely related C-terminal two-thirds of eIF4GI and it can positively promote protein synthesis and cell proliferation.     

The cyclin-dependent kinase 11 interacts with 14-3-3 proteins

Cyclin-dependent kinase 11 isoforms (CDK11) are members of the p34(cdc2) superfamily. They have been shown to play a role in RNA processing and apoptosis. In the present study, we investigate whether CDK11 interacts with 14-3-3 proteins. Our study shows that the putative 14-3-3 binding site (113-RHRSHS-118) within the N-terminal domain of CDK11(p110) is functional. Endogenous CDK11(p110) binds directly to 14-3-3 proteins and phosphorylation of the serine 118 within the RHRSHS motif seems to be required for the binding. Besides, CDK11(p110) is capable of interacting with several different isoforms of 14-3-3 proteins both in vitro and in vivo. The interaction of 14-3-3 gamma with CDK11(p110) occurs throughout the entire cell cycle and reaches maximum at the G2/M phase. Interestingly, 14-3-3 gamma shows strong interaction with N-terminal portion of caspase-cleaved CDK11(p110) (CDK11(p60)) product at 48 h after Fas treatment, which correlates with the maximal cleavage level of CDK11(p110) and the maximum activation level of CDK11 kinase activity during apoptosis. Collectively, these results suggest that CDK11 kinases could be regulated by interaction with 14-3-3 proteins during cell cycle and apoptosis.     

p38 mitogen-activated protein kinase/Hog1p regulates translation of the AU-rich-element-bearing MFA2 transcript

AU-rich-element (ARE)-mediated mRNA regulation occurs in Saccharomyces cerevisiae in response to external and internal stimuli through the p38 mitogen-activated protein kinase (MAPK)/Hog1p pathway. We demonstrate that the ARE-bearing MFA2 3' untranslated region (UTR) controls translation efficiency in a p38 MAPK/Hog1p-dependent manner in response to carbon source growth conditions. The carbon source-regulated effect on MFA2 3'-UTR-controlled translation involves the role of conserved ARE binding proteins, the ELAV/TIA-1-like Pub1p, which can interact with the cap/eIF4G complex, and the translation/mRNA stability factor poly(A) binding protein (Pab1p). Pub1p binds the MFA2 3'-UTR in a p38 MAPK/Hog1p-regulated manner in response to carbon source growth conditions. Significantly, the p38 MAPK/Hog1p is also required to modulate Pab1p in response to carbon source. We find that Pab1p can bind the MFA2 3'-UTR in a regulated manner to control MFA2 3'-UTR reporter translation. Binding of full-length Pab1p to the MFA2 3'-UTR correlates with translation repression. Importantly, Pab1p binds the MFA2 3'-UTR only in a PUB1 strain, and correlating with this requirement, Pub1p controls translation repression of MFA2 in a carbon source/Hog1p-regulated manner. These results suggest that the p38 MAPK/Hog1p pathway regulates 3'-UTR-mediated translation by modulating recruitment of Pab1p and Pub1p, which can interact with the translation machinery.     

Identification of human and mouse p19, a novel CDK4 and CDK6 inhibitor with homology to p16ink4.

The cell cycle in mammalian cells is regulated by a series of cyclins and cyclin-dependent kinases (CDKs). The G1/S checkpoint is mainly dictated by the kinase activities of the cyclin D-CDK4 and/or cyclin D-CDK6 complex and the cyclin E-CDK2 complex. These G1 kinases can in turn be regulated by cell cycle inhibitors, which may cause the cells to arrest at the G1 phase. In T-cell hybridomas, addition of anti-T-cell receptor antibody results not only in G1 arrest but also in apoptosis. In searching for a protein(s) which might interact with Nur77, an orphan steroid receptor required for activation-induced apoptosis of T-cell hybridomas, we have cloned a novel human and mouse CDK inhibitor, p19. The deduced p19 amino acid sequence consists of four ankyrin repeats with 48% identity to p16. The human p19 gene is located on chromosome 19p13, distinct from the positions of p18, p16, and p15. Its mRNA is expressed in all cell types examined. The p19 fusion protein can associate in vitro with CDK4 but not with CDK2, CDC2, or cyclin A, B, E, or D1 to D3. Addition of p19 protein can lead to inhibition of the in vitro kinase activity of cyclin D-CDK4 but not that of cyclin E-CDK2. In T-cell hybridoma DO11.10, p19 was found in association with CDK4 and CDK6 in vivo, although its association with Nur77 is not clear at this point. Thus, p19 is a novel CDK inhibitor which may play a role in the cell cycle regulation of T cells.     

Thr-370 is responsible for CDK11(p58) autophosphorylation, dimerization, and kinase activity

CDK11(p58), a member of the p34(cdc2)-related kinase family, is associated with cell cycle progression, tumorigenesis, and proapoptotic signaling. It is also required for the maintenance of chromosome cohesion, the maturation of centrosome, the formation of bipolar spindle, and the completion of mitosis. Here we identified that CDK11(p58) interacted with itself to form homodimers in cells, whereas D224N, the kinase-dead mutant, failed to form homodimers. CDK11(p58) was autophosphorylated, and the main functions of CDK11(p58), such as kinase activity, transactivation of nuclear receptors, and proapoptotic signal transduction, were dependent on its autophosphorylation. Furthermore, the in vitro kinase assay indicated that CDK11(p58) was autophosphorylated at Thr-370. By mutagenesis, we created CDK11(p58) T370A and CDK11(p58) T370D, which mimic the dephosphorylated and phosphorylated forms of CDK11(p58), respectively. The T370A mutant could not form dimers and be phosphorylated by the wild type CDK11(p58) and finally lost the kinase activity. Further functional research revealed that T370A failed to repress the transactivation of androgen receptor and enhance the cell apoptosis. Overall, our data indicated that Thr-370 is responsible for the autophosphorylation, dimerization, and kinase activity of CDK11(p58). Moreover, Thr-370 mutants might affect CDK11(p58)-mediated signaling pathways.     

Increased expression of CDK11p58 and cyclin D3 following spinal cord injury in rats

Protein kinases are critical signalling molecules for normal cell growth and development. CDK11^p58 is a p34^cdc2-related protein kinase, and plays an important role in normal cell cycle progression. However its distribution and function in the central nervous system (CNS) lesion remain unclear. In this study, we mainly investigated the protein expression and cellular localization of CDK11 during spinal cord injury (SCI). Western blot analysis revealed that CDK11^p58 was not detected in normal spinal cord. It gradually increased, reached a peak at 3 day after SCI, and then decreased. The protein expression of CDK11^p58 was further analyzed by immunohistochemistry. The variable immunostaining patterns of CDK11^p58 were visualized at different periods of injury. Double immunofluorescence staining showed that CDK11 was co-expressed with NeuN, CNPase and GFAP. Co-localization of CDK11/active caspase-3 and CDK11/proliferating cell nuclear antigen (PCNA) were detected in some cells. Cyclin D3, which was associated with CDK11^p58 and could enhance kinase activity, was detected in the normal and injured spinal cord. The cyclin D3 protein underwent a similar pattern with CDK11^p58 during SCI. Double immunofluorescence staining indicated that CDK11 co-expressed with cyclin D3 in neurons and glial cells. Coimmunoprecipitation further showed that CDK11^p58 and cyclin D3 interacted with each other in the damaged spinal cord. Thus, it is likely CDK11^p58 and cyclin D3 could interact with each other after acute SCI. Another partner of CDK11^p58 was β-1,4-galactosyltransferase 1 (β-1,4-GT 1). The co-localization of CDK11/β-1,4-GT 1 in the damaged spinal cord was revealed by immunofluorescence analysis. The cyclin D3-CDK4 complexes were also present by coimmunoprecipitation analysis. Taken together, these data suggested that both CDK11 and cyclin D3 may play important roles in spinal cord pathophysiology. The authors Yuhong Ji and Feng Xiao contributed equally to this work.     

Staurosporine inhibits phosphorylation of translational regulators linked to mTOR

Treatment of Swiss 3T3 cells with staurosporine resulted in dephosphorylation of two proteins which play key roles in regulating mRNA translation. This occurred before the execution of apoptosis, assessed by caspase-3 activity. These translation regulators are p70 S6 kinase, which phosphorylates ribosomal protein S6, and eukaryotic initiation factor (eIF) 4E binding protein 1 (4E-BP1), which both lie downstream of the mammalian target of rapamycin (mTOR). This resulted in decreased p70 S6 kinase activity, dephosphorylation of ribosomal protein S6, increased binding of 4E-BP1 to eIF4E and a concomitant decrease in eIF4F complexes. Our data show that staurosporine impairs mTOR signalling in vivo but that this not due to direct inhibition of mTOR or to inhibition of protein kinase C. It is becoming clear that agents which cause apoptosis inactivate mTOR signalling as a common early response prior to the execution of apoptosis, i.e., before caspase activation.     

Binding of eukaryotic translation initiation factor 4E (eIF4E) to eIF4G represses translation of uncapped mRNA.

mRNA translation in crude extracts from the yeast Saccharomyces cerevisiae is stimulated by the cap structure and the poly(A) tail through the binding of the cap-binding protein eukaryotic translation initiation factor 4E (eIF4E) and the poly(A) tail-binding protein Pab1p. These proteins also bind to the translation initiation factor eIF4G and thereby link the mRNA to the general translational apparatus. In contrast, uncapped, poly(A)-deficient mRNA is translated poorly in yeast extracts, in part because of the absence of eIF4E and Pab1p binding sites on the mRNA. Here, we report that uncapped-mRNA translation is also repressed in yeast extracts due to the binding of eIF4E to eIF4G. Specifically, we find that mutations which weaken the eIF4E binding site on the yeast eIF4G proteins Tif4631p and Tif4632p lead to temperature-sensitive growth in vivo and the stimulation of uncapped-mRNA translation in vitro. A mutation in eIF4E which disturbs its ability to interact with eIF4G also leads to a stimulation of uncapped-mRNA translation in vitro. Finally, overexpression of eIF4E in vivo or the addition of excess eIF4E in vitro reverses these effects of the mutations. These data support the hypothesis that the eIF4G protein can efficiently stimulate translation of exogenous uncapped mRNA in extracts but is prevented from doing so as a result of its association with eIF4E. They also suggest that some mRNAs may be translationally regulated in vivo in response to the amount of free eIF4G in the cell.     

Downregulation of beta1,4-galactosyltransferase 1 inhibits CDK11(p58)-mediated apoptosis induced by cycloheximide

Cyclin-dependent kinase 11 (CDK11; also named PITSLRE) is part of the large family of p34(cdc2)-related kinases whose functions appear to be linked with cell cycle progression, tumorigenesis, and apoptotic signaling. The mechanism that CDK11(p58) induces apoptosis is not clear. Some evidences suggested beta1,4-galactosyltransferase 1 (beta1,4-GT 1) might participate in apoptosis induced by CDK11(p58). In this study, we demonstrated that ectopically expressed beta1,4-GT 1 increased CDK11(p58)-mediated apoptosis induced by cycloheximide (CHX). In contrast, RNAi-mediated knockdown of beta1,4-GT 1 effectively inhibited apoptosis induced by CHX in CDK11(p58)-overexpressing cells. For example, the cell morphological and nuclear changes were reduced; the loss of cell viability was prevented and the number of cells in sub-G1 phase was decreased. Knock down of beta1,4-GT 1 also inhibited the release of cytochrome c from mitochondria and caspase-3 processing. Therefore, the cleavage of CDK11(p58) by caspase-3 was reduced. We proposed that beta1,4-GT 1 might contribute to the pro-apoptotic effect of CDK11(p58). This may represent a new mechanism of beta1,4-GT 1 in CHX-induced apoptosis of CDK11(p58)-overexpressing cells.     

Control of Cyclin-Dependent Kinase Inhibitor p27 Expression by Cap-Independent Translation

p27 is a key regulator of cell proliferation through inhibition of G(1) cyclin-dependent kinase (CDK) activity. Translation of the p27 mRNA is an important control mechanism for determining cellular levels of the inhibitor. Nearly all eukaryotic mRNAs are translated through a mechanism involving recognition of the 5' cap by eukaryotic initiation factor 4E (eIF4E). In quiescent cells eIF4E activity is repressed, leading to a global decline in translation rates. In contrast, p27 translation is highest during quiescence, suggesting that it escapes the general repression of translational initiation. We show that the 5' untranslated region (5'-UTR) of the p27 mRNA mediates cap-independent translation. This activity is unaffected by conditions in which eIF4E is inhibited. In D6P2T cells, elevated cyclic AMP levels cause a rapid withdrawal from the cell cycle that is correlated with a striking increase in p27. Under these same conditions, cap-independent translation from the p27 5'-UTR is enhanced. These results indicate that regulation of internal initiation of translation is an important determinant of p27 protein levels.     

Protein kinase C-related kinase 2 phosphorylates the protein synthesis initiation factor eIF4E in starfish oocytes

Phosphorylation of eIF4E is required for protein synthesis during starfish oocyte maturation. The activity of protein kinase C-related kinase 2 (PRK2) increases prior to the phosphorylation of eIF4E (G. Stapleton et al., 1998, Dev. Biol. 193, 34-46). We investigate here whether eIF4E is activated by PRK2. A 3.5-kb eIF4E clone isolated from starfish cDNA is 57% identical to human eIF4E and contains the putative phosphorylation site serine-209. The serine-209 environment (SKTGS(209)MAKSRF) is similar to the consensus sequence of the phosphorylation site of protein kinase C and related kinases. A starfish eIF4E fusion protein (GST-4E) was phosphorylated in vitro by PRK2 in the presence of 1,2-diolyl-sn-glycerol 3-phosphate. In contrast, replacing the GST-4E serine-209 with an alanine significantly reduced this phosphorylation. Analysis by two-dimensional phosphopeptide mapping reveals a major phosphopeptide in trypsin-digested GST-4E, but not in its serine-209 mutant. Importantly, this major phosphopeptide in GST-4E corresponds to a major phosphopeptide of eIF4E isolated from (32)P-labeled oocytes. Thus, PRK2 may regulate translation initiation during oocyte maturation by phosphorylating the serine-209 residue of eIF4E in starfish. We also demonstrate that high levels of cAMP inhibit the activation of PRK2, eIF4E, and the eIF4E binding protein during starfish oocyte maturation, while PI3 kinase activates these proteins.