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First published online 18 March 2003
doi: 10.1242/jcs.00409

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Protein phosphatase 4 interacts with the Survival of Motor Neurons complex and enhances the temporal localisation of snRNPs

¹ Medical Research Council Protein Phosphorylation Unit, Division of Cell Signalling, University of Dundee, Dundee DD1 5EH, Scotland, UK
² Division of Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, UK

View larger version (40K): [in a new window]   Fig. 1. Expression R2 and PPP4c mRNAs in human tissues. Human multiple tissue northern blots (Clontech) were hybridised with a R2 cDNA probe (0.8 kb, coding for amino acids 78-316) and PPP4c cDNA probe (0.8 kb, coding for amino acids 12-279) and a ß-actin control probe. ß-actin mRNA was used as a control to assess the variation in sample loading. Hybridisation using each probe was carried out separately, and the blot was stripped before being re-hybridised with the next probe. Transcripts are indicated by arrows on the right, and the positions of RNA standards of known sizes are indicated in kb on the left.

View larger version (22K): [in a new window]   Fig. 2. Co-elution of PPP4c and Flag-R2 from an anti-Flag agarose column. HEK 293 cell lysates were incubated with anti-Flag agarose as described in Materials and Methods. Fractions (12x1 ml) eluted with Flag peptide from the anti-Flag agarose column were collected and analysed by SDS-PAGE and subsequent immunoblotting with anti-Flag (for detection of Flag-R2) and anti-PPP4c antibodies.

View larger version (34K): [in a new window]   Fig. 3. Analysis of Flag–R2-PPP4c material by gel filtration. Peak fractions from the anti-Flag agarose column containing Flag-R2 and PPP4c were pooled and subjected to Superose 6 gel filtration chromatography. Fractions eluted were analysed by SDS-PAGE and subsequent immunoblotting with anti-Flag (for detection of Flag-R2) and anti-PPP4c antibodies. Superose 6 fraction numbers are indicated above each lane. Molecular mass markers, thyroglobulin (670 kDa) and ferritin (450 kDa) are indicated by arrows.

View larger version (63K): [in a new window]   Fig. 4. Analysis of material co-eluting with Flag–R2-PPP4c. Flag–R2-PPP4c material eluted with Flag peptide from the anti-Flag agarose column was concentrated and analysed by SDS-PAGE. Proteins bands were detected using Coomassie blue and excised from the gel prior to tryptic digestion and identification by mass spectrometry. The bands at 70 kDa and 40 kDa are believed to be artefacts since they stain more strongly than Flag–R2 and PPP4c and they have also been noted in the eluates with other Flag-tagged proteins. Protein methyltransferase JBP1 is contained within the band at 70 kDa. The other proteins in these bands were not identifiable. The weak protein bands at 50 kDa are - and ß-tubulin.

View larger version (22K): [in a new window]   Fig. 5. Co-immunoprecipitation of Gemin3 and Gemin4 with Flag-R2. A vector expressing Flag-R2 was transfected into HEK 293 cells. Supernatant (S) and pellet (P) fractions were obtained by centrifugation following immunoadsorption from cell lysates (L) with anti-Flag agarose. Murine IgG was used for all controls in place of anti-Flag antibodies. Proteins in the lysate (10 µl), supernatant (10 µl) and pellet (recovered from 500 µl) fractions were analysed by SDS-PAGE and subsequent immunoblotting with anti-Gemin3 (A) and anti-Gemin4 (B) antibodies.

View larger version (24K): [in a new window]   Fig. 6. Co-immunoprecipitation of Gemin3, PPP4R2 and PPP4c with Gemin4. A vector expressing Xpress-tagged Gemin4 was transfected into HEK 293 cells. Supernatant (S) and pellet (P) fractions were obtained by centrifugation following immunoadsorption from cell lysates (L) with anti-Xpress Sepharose (protein G). Murine IgG was used for controls in place of anti-Xpress antibodies. The proteins in the lysate (10 µl), supernatant (10 µl) and pellet (recovered from 500 µl) fractions were analysed by SDS-PAGE and subsequent immunoblotting with anti-Gemin4 (left hand side 3 lanes), anti-Xpress (right hand side 3 lanes) (A), anti-Gemin3 (B), anti-R2 (C) and anti-PPP4c (D) antibodies.

View larger version (38K): [in a new window]   Fig. 7. Co-immunoprecipitation of PPP4c with proteins previously identified in the SMN protein complex. HeLa cells expressing CFP-SMN were used for immunoadsorption of SMN and HeLa cells transfected with vector expressing GFP were used in controls. Supernatant (S) and pellet (P) fractions were obtained by centrifugation following immunoadsorption from cell lysates (L) using anti-GFP-Sepharose (protein G), which readily interacts with both CFP and GFP. Proteins in the lysate (10 µl), supernatant (10 µl) and pellet (recovered from 500 µl) fractions were analysed by SDS-PAGE and subsequent immunoblotting with anti-GFP (A), anti-Gemin2 (B), anti-Gemin3 (C), anti-Gemin4 (D) and anti-PPP4c (E) antibodies. The 50 kDa bands in the pellet fractions of A are caused by the presence of large amounts of the antibody heavy chain.

View larger version (105K): [in a new window]   Fig. 8. Effect of expression of R2-PPP4c on the localisation pattern of newly formed snRNPs in HeLa cells. HeLa cells were injected with expression plasmid DNAs pEYFP-SmB, pEGFP-R2 and/or pCMV5-HA-PPP4c (each at 15 µg/ml), which are capable of expressing YFP-SmB, GFP-Flag-R2 (termed hereafter GFP-R2) and/or HA-PPP4c. 2 hours after injection cells were fixed in 3.7% paraformaldehyde. Images were collected on a Zeiss Delta Vision microscope and digitally deconvoluted. Panels A and J were visualised using anti-HA antibodies and secondary antibodies conjugated to Cy3. DNA was detected with DAPI. (A-D) HeLa cells injected with a mixture of plasmid DNAs expressing GFP-R2, HA-PPP4c and YFP-SmB. (E,F) HeLa cells injected with plasmid DNA expressing YFP-SmB alone. (G-I) HeLa cells injected with a mixture of plasmid DNAs expressing GFP-R2 and YFP-SmB. (J-L) HeLa cells injected with a mixture of plasmid DNAs expressing HA-PPP4c and YFP-SmB. Bar, 10 µm.