p38gamma regulates interaction of nuclear PSF and RNA with the tumour-suppressor hDlg in response to osmotic shock

Abstract

Activation of p38γ modulates the integrity of the complex formed by the human discs large protein (hDlg) with cytoskeletal proteins, which is important for cell adaptation to changes in environmental osmolarity. Here we report that, in response to hyperosmotic stress, p38γ also regulates formation of complexes between hDlg and the nuclear protein polypyrimidine tract-binding protein-associated-splicing factor (PSF). Following osmotic shock, p38γ in the cell nucleus increases its association with nuclear hDlg, thereby causing dissociation of hDlg-PSF complexes. Moreover, hDlg and PSF bind different RNAs; in response to osmotic shock, p38γ causes hDlg-PSF and hDlg-RNA dissociation independently of its kinase activity. These findings identify a novel nuclear complex and suggest a previously unreported function of p38γ, which is independent of its catalytic activity and could affect mRNA processing and/or gene transcription to aid cell adaptation to osmolarity changes in the environment.

Fig. 1.

Cell stress regulates association of hDlg with the splicing factors PSF and p54^nrb. (A) Endogenous hDlg was immunoprecipitated, using 2 μg anti-hDlg antibody per sample, from 5 mg (lanes 1) or 10 mg (2) protein lysates from HEK293 cells, unstimulated or exposed to 0.5 M sorbitol (15 minutes). Protein bands a and b were excised from the gel, digested in-gel with trypsin, and their identity determined by mass fingerprinting. The number of peptides, percentage of sequence coverage and the accession number for each protein are given in the table. (B) Endogenous hDlg was immunoprecipitated, using 2 μg anti-hDlg antibody per sample, from 0.2 mg lysates from HEK293 or HeLa cells, unstimulated or stimulated as in A. Pellets were immunoblotted with anti-PSF or anti-p54^nrb, with an antibody against hDlg phosphorylated at Ser158 (P-hDlg^S158) and an antibody that recognises both unphosphorylated and phosphorylated hDlg.

Fig. 2.

p38γ phosphorylation of hDlg does not regulate its interaction with PSF. (A) WT or p38γ/δ^−/− MEFs were incubated (1 hour) alone or with 10 μM SB203580, then exposed to 0.5 M sorbitol (15 minutes). Endogenous hDlg was immunoprecipitated with anti-hDlg antibody and pellets immunoblotted with anti-PSF or anti-hDlg antibody. (B) HEK293 cells were transfected with empty pSuper vector (pS) or with a mixture of siRNA constructs to knock down p38γ (pS3mix). Cells were treated as in A, endogenous hDlg was immunoprecipitated from 0.5 mg cell lysates and pellets were immunoblotted with anti-PSF, anti-P-hDlg^S158 or anti-hDlg antibody. Total lysates were immunoblotted with an antibody against total p38γ. (C) HEK293 cells were incubated alone or with 10 μM SB203580 or 1 μM BIRB 0796 (1 hour), then exposed to 0.5 M sorbitol (15 minutes). Endogenous hDlg was immunoprecipitated with anti-hDlg antibody and pellets were immunoblotted as in B. Total lysates were immunoblotted with an antibody that recognises phosphorylated p38γ and p38α.

Fig. 3.

Generation and characterization of the inactive p38γ knock-in mouse. (A) Diagram showing the endogenous p38γ gene, the targeting construct vector generated, the targeted allele with the neomycin selection cassette still present (Neo), and the targeted allele with the neomycin cassette removed by Cre recombinase. The grey boxes represent exons and the black triangles, the LoxP sites. The knock-in allele with the D171A mutation in exon 7 is marked (dark grey). Genomic DNA purified from tail biopsy sample was used as a template for PCR, resolved on a 1% agarose gel and examined by ethidium bromide staining. (B) Lysates from WT, p38γ^−/− or p38γ^171A/171A MEFs (30-50 μg of protein) were immunoblotted with specific antibodies for each protein indicated. (C) WT or p38γ^171A/171A MEFs were exposed to 0.5 M sorbitol (15 minutes) and activation of the indicated MAPK was examined using phospho-specific antibodies. To analyse activation of p38γ, this isoform was immunoprecipitated with anti-p38γ antibody from 2 mg cell lysates and immunoblotted with the p38 phospho-specific antibody. (D) WT, p38γ^171A/171A or p38γ/δ^−/− MEFs were treated as in C and endogenous hDlg immunoprecipitated with anti-hDlg antibody from 0.5 mg protein lysate. Pellets were immunoblotted with anti-P-hDlg^S158 or anti-hDlg.

Fig. 4.

p38γ regulates hDlg-PSF association independently of its catalytic activity. (A) WT, p38γ^171A/171A or p38γ/δ^−/− MEFs were treated as in Fig. 3C and endogenous hDlg immunoprecipitated using anti-hDlg antibody from 0.2 mg of protein lysate. Pellets were immunoblotted with anti-PSF or anti-hDlg antibody. Similar results were obtained in two independent experiments. (B) HEK293 cells were transfected with empty GFP-vector, GFP-p38γ(FL), GFP-p38γ(ΔC) or GFP-p38γ(KD). Endogenous hDlg was immunoprecipitated as in A, and pellets were immunoblotted with anti-PSF or anti-hDlg. (C) Cells were transfected as in B and exposed to 0.5 M sorbitol (15 minutes). Endogenous hDlg was immunoprecipitated, and pellets were immunoblotted with anti-P-hDlg^S158 or anti-hDlg. Similar results were obtained in at least two independent experiments. Levels of p38γ expression in B and C were checked by immunoblotting 20 μg cell extract with anti-p38γ antibody.

Fig. 5.

p38γ accumulates in the nucleus after osmotic stress and regulates hDlg-PSF interaction. (A) HeLa cells, unstimulated or treated with 0.5 M sorbitol (15 minutes), were stained with anti-p38γ or anti-hDlg antibodies and analysed by fluorescence microscopy. Inserts in the top panels show a higher magnification of p38γ localization in cell nuclei. Cells were fractionated and 30 μg protein from cytoplasm (Cyt) and nuclear (Nuc) fractions were immunoblotted using anti-p38γ, anti-p38α and anti-hDlg antibodies. (B) Different hDlg domains were expressed in HEK293 cells (top). After transfection, cells were lysed and GST-proteins purified by pull-down. Endogenous PSF was immunoprecipitated using anti-PSF antibody. Pellets were immunoblotted using anti-PSF or anti-GST antibody. (C) HEK293 cells were transfected with GST-PDZ1, stained with anti-GST and/or anti-PSF antibody, and analysed by fluorescence microscopy. Nuclei are stained with DAPI. Results were similar in four independent experiments in HEK293 cells and in two independent experiments in HeLa cells. (D) HEK293 cells expressing GST-PDZ1 and GFP-p38γ were treated with sorbitol as in A. GST-PDZ1 was purified by pull-down immunoprecipitation and pellets were immunoblotted using anti-PSF, anti-GST or anti-GFP antibodies. Total lysates were immunoblotted with anti-GFP antibody to examine p38γ expression. (E) Band intensity of blots in D (anti-PSF, anti-GST for PDZ1 and anti-GFP for p38γ) were quantified and the relative amount of p38γ or PSF in pellets was calculated. Histogram values are means ± s.e.m. of four independent experiments.

Fig. 6.

Osmotic stress decreases RNA binding to hDlg and PSF. (A) Endogenous PSF or hDlg were immunoprecipitated, with anti-PSF or anti-hDlg antibody, from HEK293 cells, alone or treated with 0.5 M sorbitol (15 minutes); IgG immunoprecipitation was used as negative control. RNA in the pellets was purified by a differential display-based method using set 1 or set 2 of random primers and resolved on a 6% denaturing polyacrylamide gel. Bands of interest were excised, re-amplified and cloned for identification (bands are indicated as 1 to 13; unidentified bands are labelled n.d.); band identity and GenBank accession number are shown (bottom). (B) Extracts from were prepared as in A, hDlg or PSF immunoprecipitated and the levels of bound RNA identified in A were determined using specific primers for each RNA by real-time PCR. The RNAs identified were also amplified by RT-PCR using specific primers and resolved in an agarose gel. (C) WT, p38γ^171A/171A or p38γ^−/− MEF extracts were prepared as in A, hDlg was immunoprecipitated and the levels of bound ODC1 RNA determined using specific primers by real-time PCR.

Fig. 7.

Model of regulation of the hDlg-PSF-RNA complex by the p38γ kinase. (A) Under normal physiological conditions, in resting cells, p38γ is localized mainly in the cytoplasm of the cell, whereas the hDlg-PSF-RNA complex is associated in the nucleus of the cell. (B) Changes in the osmolarity of the environment causes the accumulation of p38γ in the nucleus, which increases its binding to hDlg, The nuclear interaction of p38γ with hDlg through its PDZ domain 1 leads to hDlg dissociation from PSF and RNAs in the nucleus.