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. 2009 Mar 18;583(6):971-7.
doi: 10.1016/j.febslet.2009.02.028. Epub 2009 Feb 24.

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

Jiaqi Shi  1 , John W B Hershey, Mark A Nelson
Affiliations

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

Jiaqi Shi et al. FEBS Lett. .

Abstract

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.

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Figures

Fig. 1
Fig. 1
CDK11p46 phosphorylates eIF3f at more than one site during apoptosis. (A) Kinase assay: CDK11p46 was immunoprecipitated from apoptotic A375 cells and incubated with GST-eIF3f or eIF3fS46A. (B) Kinase assay was performed with GST or four truncated eIF3f proteins as substrates. (C) Diagram of Mov34 domain and four deletion constructs of eIF3f. (D) MS identification of Thr119 phosphorylation. (E) Kinase assays were performed with eIF3f-1 (T119) and eIF3f-1 T119A (T119A) as substrates. (F) Staurosporine treated A375 cells were immunoprecipitated with eIF3 antibody followed by immunoblot with phosphothreonine or eIF3f antibody.
Fig. 2
Fig. 2
Endogenous eIF3f is phosphorylated by CDK11p46 during apoptosis. A375 cells were treated with 0.5 μg/ml anti-Fas with or without pretreatment with caspase 3 inhibitor (DEVD-FMK) for 36 hr. (A) Cells were lysed and immunobloted with CDK11 and α-tubulin antibodies. (B) 2D- SDS-PAGE showed increased phosphorylation of eIF3f during apoptosis. (C) Increased phosphorylation of endogenous eIF3f in CDK11p46 transfected cells.
Fig. 3
Fig. 3
Phosphorylation of eIF3f by CDK11p46 is important for the inhibition of translation and induction of apoptosis. (A) A375 cells were co-transfected with pcDNA3, or pcDNA3-eIF3f, or pcDNA3-eIF3fSATA and pGL3-SV40 carrying the luciferase reporter gene. At 24 h post-transfection, cells were lysed and luciferase activity was measured by a luminometer. (B) Real-time RT-PCR was performed as described. Untransfected A375 cells and reactions without reverse transcription served as controls. (C) Apoptosis was measured by Acridine Orange/Ethidium Bromide staining. (D) (E) Luciferase mRNA was translated in an in vitro translation system in the presence of buffer, GST, or indicated recombinant proteins. Synthesized luciferase activity was measured with a luminometer. (F) A375 cells were transfected with indicated plasmids. Caspase 3/7 activity was measured 48h after transfection.
Fig. 4
Fig. 4
Phosphorylation of eIF3f by CDK11p46 increases the association of eIF3f with other eIF3 subunits during apoptosis. A375 cells were treated with staurosporine or anti-Fas for indicated time. (A) Cell lysates were immunoprecipitated with eIF3 or eIF3f antibody followed by immunoblot with eIF3f or eIF3b antibody. The same experiment was carried out with CDK11p46 transfected cells. (B) Cell lysates were immunoprecipitated with eIF3b antibody followed by immunoblot with eIF3f or eIF3b antibody. (C) Cells were fractionated and immunoprecipitated with eIF3b antibody followed by immunoblot with eIF3f, eIF3b, eIF3c and eIF3a antibodies. Cell fractions were also immunoblotted with eIF3f, CDK11 and α-tubulin antibodies without immunoprecipitation. S: soluble fraction; P: pellet fraction. (D) Cells were analyzed for apoptosis using Annexin V/PI staining and flow cytometry analysis.
Fig. 5
Fig. 5
The effect of the eIF3f phosphorylation sites mutation on its association with eIF3b and apoptosis. (A) A375 cells were transfected with pcDNA3, eIF3f, eIF3fSATA (phosphorylation sites inactivated) or eIF3fSETE (phosphorylation mimic) plasmids. Cell lysates were immunoprecipitated with eIF3b antibody followed by immunoblot with eIF3f antibody. Cell lysates were also used for Western blot using eIF3f and α-tubulin antibodies. (B) (C) A375 cells were transfected with indicated plasmids. Caspase 3/7 activity was measured 48h after transfection.
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