Signaling-dependent and coordinated regulation of transcription, splicing, and translation resides in a single coregulator, PCBP1

Abstract

Transcription, splicing, and translation are potentially coordinately regulatable in a temporospatial-dependent manner, although supporting experimental evidence for this notion is scarce. Yeast two-hybrid screening of a mammary gland cDNA library with human p21-activated kinase 1 (Pak1) as bait identified polyC-RNA-binding protein 1 (PCBP1), which controls translation from mRNAs containing the DICE (differentiation control element). Mitogenic stimulation of human cells phosphorylated PCBP1 on threonines 60 and 127 in a Pak1-sensitive manner. Pak1-dependent phosphorylation of PCBP1 released its binding and translational inhibition from a DICE-minigene. Overexpression of PCBP1 also inhibited the translation of the endogenous L1 cell adhesion molecule mRNA, which contains two DICE motifs in the 3' untranslated region. We also found that Pak1 activation led to an increased nuclear retention of PCBP1, recruitment to the eukaryotic translation initiation factor 4E (eIF4E) promoter, and stimulation of eIF4E expression in a Pak1-sensitive manner. Moreover, mitogenic stimulation promoted Pak1- and PCBP1-dependent alternative splicing and exon inclusion from a CD44 minigene. The alternative splicing functions of PCBP1 were in turn mediated by its intrinsic interaction with Caper alpha, a U2 snRNP auxiliary factor-related protein previously implicated in RNA splicing. These findings establish the principle that a single coregulator can function as a signal-dependent and coordinated regulator of transcription, splicing, and translation.

Fig. 1.

Mitogen-mediated phosphorylation of PCBP1 by Pak1. (A) Yeast two-hybrid identification of PCBP1 as Pak1-interacting partner. Yeast cells were cotransfected with a control GAD vector or GAD-PCBP1 along with a GBD vector, GBD-Pak1 constructs. Growth was recorded after 72 h on selection plates lacking leucine and tryptophan (−LT) or adenosine, histidine, leucine, and tryptophan (−AHLT). (B) In vitro GST-pull down for binding of Pak1 and GST-PCBP1. (C) In vivo interaction of T7-PCBP1 with Pak1 in HeLa cells. EGF, 100 ng/ml for 15 min. (D) Endogenous interaction between PCBP1 and Pak1 in HeLa cells. (E) Mitogenes phosphorylate PCBP1 in HeLa cells. Cell lysates were immunoprecipitated (IP) with goat PCBP1 Ab after [³²P]orthophosphoric acid labeling and 10-min treatment with mitogens and blotted or autoradiographed as indicated. (F) HeLa cells were transfected with Pak1 or control siRNA, labeled with [³²P]orthophosphoric acid, and treated with or without EGF. (G) In vitro phosphorylation of GST-PCBP1 by Pak1. (H) Pak1 phosphorylation of GST-PCBP1 mutant proteins. The two bottom images show the status of GST proteins by Ponceau staining. (I) Generation of MCF-7 pooled clones expressing T7-PCBP1-WT or T7-PCBP1-Mut. (Upper) Clones were metabolically labeled with [³²P]orthophosphoric acid, and T7-PCBP1 was IP, and autoradiographed. (Lower) Western blotting of the above blot with an anti-T7 mAb to show equal IP of T7-tagged proteins. IB, immunoblotting.

Fig. 2.

Phosphorylation of PCBP1 by Pak1 relieves translational repression on DICE-containing RNA. (A) EMSA using labeled RNA 4R-DICE with PCBP1-WT and PCBP1-T60A and PCBP1-T60AT127A that were phosphorylated by Pak1 enzyme. ∗, Shifted band of 4R-DICE by PCBP1; SS, super shift with anti-PCBP1 Ab. (B) MDA-435 cells were transfected with Luc-2R luc reporter and with indicated plasmids. (C) Expression of L1CAM mRNA and protein in MCF-7 clones expressing empty vector or T7-PCBP1. (D) MCF-7 clones expressing pcDNA or T7-PCBP1 were serum stimulated for 12 h. (E) Expression of L1CAM protein in MCF-7/DA-Pak1 cells treated with or without doxycycline for 24 h. (F) Membrane with GST, GST-PCBP1, and Pak1 phosphorylated GST, GST-PCBP1, were probed with ³²P-DICE of human L1CAM in vitro transcripts (SI Methods).

Fig. 3.

PCBP1 phosphorylation controls its nuclear localization and eIF4E transactivation. (A) Nuclear and cytoplasmic extracts from MCF-7 clones expressing pcDNA or T7-PCBP1 were blotted with indicated antibodies. (B) Induction of DA-Pak1 in MCF-7 cells up-regulates eIF4E mRNA (Left), and Pak1siRNA down-regulates eIF4E mRNA in MCF-7 cells (Right). (C) eIF4E mRNA in MCF-7 cells stably expressing PCBP1 or PCBP1-Mut. (D) Levels of eIF4E mRNA in HeLa cells transfected with PCBP1 siRNA for 3 d. (E) HeLa cells were treated with serum or EGF for 45 min, and ChIP analysis was performed on an eIF4E promoter fragment (678–873 bp, GenBank accession no. NM_001968). (F) MCF-7 cells expressing T7-PCBP1 or T7-PCBP1-Mut were treated with EGF and were subjected to ChIP on eIF4E promoter. (G) HeLa cells treated with con- or PCBP1-siRNA, transfected with eIF4E core promoter p543-Luc, and stimulated with EGF for 12 h before the luc assay. (H) MCF-7/PCBP1 clones were transfected with p543-Luc and stimulated with EGF.

Fig. 4.

Pak1 and PCBP1 regulate pre-mRNA splicing. (A) MCF-7 cells transfected with pETCatEBLucv5 were treated with serum or EGF for 16 h, and minigene transcripts and splicing activity was assayed. RT-PCR was performed to analyze splicing products. Lower band is the splicing product with CD44 v5 skipped, and the upper band is the product with v5 included. (B) MCF-7 cells were cotransfected with pETCatEBLucv5 and Pak1 plasmid, and luc activity was measured. (C) MCF-7 cells were transfected with indicated plasmids along with pETCatEBLucv5, and luc activity was measured. (D) HeLa cells were cotransfected with HSV-CD44 splicing minigene and Pak1 and were subjected to RT-PCR analysis of minigene transcripts. Splicing activity on this minigene was represented by the ratio of transcripts of included exons (bands a and b) to transcripts of both exons excluded (band c). (E) MCF-7 cells were cotransfected with pETCatEBLucv5 and different amounts of PCBP1-WT or PCBP1-Mut and were assayed for the luc activity. (F) MCF-7 cells were cotransfected with HSV-CD44 and PCBP1-WT or PCBP1-Mut plasmids and subjected to RNA isolation and RT-PCR analysis of minigene transcripts. (G) MCF-7 clones were transfected with pETCatEBLucv5 splicing minigene reporter, and luc activity was assayed. (H) HeLa cells cotransfected with PCBP1-siRNA with Pak1 or control plasmid and pETCatEBLucv5 minigene and assayed for the luc-activity. (I) HeLa extracts were immunoprecipitated with a Caper α antibody (Left) or PCBP1 antibody (Right) and were followed by IB with indicated antibodies. (J) HeLa cells were treated with con or Caper α-specific siRNA, cotransfected with pcDNA-PCPB1 and pETCatEBLucv5 splicing minigene, and luc activity was measured.

Fig. 5.

Working model for signaling-dependent alterations of the nuclear and cytoplasmic activities of PCBP1. Upstream activators of the Pak1 pathway induce Pak1 kinase activity wherein Pak1 exists in an autophosphorylated state. The active Pak1 exerts both cytoplasmic and nuclear functions. PCBP1 gets phosphorylated by Pak1 in the cytoplasmic compartment, and phosphorylation reduces its RNA-binding capabilities, thus releasing the translational repression on specific target mRNAs. In the nucleus, the phosphorylated PCBP1 is involved in splicing and transcriptional activities. Whether PCBP1 is phosphorylated by Pak1 in the nucleus after translocation or the phosphor-PCBP1 moves to the nucleus from the cytoplasm is still not known. Phosphorylation induced by Pak1 results in enhanced binding to recently transcribed mRNA in the nucleus to influence the splicing machinery via CAPER α. Furthermore, phosphorylated PCBP1 in the nucleus also gets recruited to promoters of target genes and regulates transcriptional activity of the gene. Broken lines represent the events that are not fully understood.