Major phosphorylation of SF1 on adjacent Ser-Pro motifs enhances interaction with U2AF65

Abstract

Protein phosphorylation ensures the accurate and controlled expression of the genome, for instance by regulating the activities of pre-mRNA splicing factors. Here we report that splicing factor 1 (SF1), which is involved in an early step of intronic sequence recognition, is highly phosphorylated in mammalian cells on two serines within an SPSP motif at the junction between its U2AF65 and RNA binding domains. We show that SF1 interacts in vitro with the protein kinase KIS, which possesses a 'U2AF homology motif' (UHM) domain. The UHM domain of KIS is required for KIS and SF1 to interact, and for KIS to efficiently phosphorylate SF1 on the SPSP motif. Importantly, SPSP phosphorylation by KIS increases binding of SF1 to U2AF65, and enhances formation of the ternary SF1-U2AF65-RNA complex. These results further suggest that this phosphorylation event has an important role for the function of SF1, and possibly for the structural rearrangements associated with spliceosome assembly and function.

Figure 1. KIS interaction with SF1 in vitro

Various methionine labelled forms of KIS and U2AF⁶⁵ were tested for their binding to GST-SF1[1-255] (GST-SF1f) in a pull-down assay (top right, lanes 7-12). Lane 7: wildtype KIS; lane 8: KIS with a deletion within its UHM domain, KIS[Δ369-409]; lane 9: KIS lacking its UHM domain, KIS[1-313]; lane 10: KIS with mutations of E341 and D342 to lysine, KIS(EDKK), lane 11: full-length U2AF⁶⁵ and lane 12: U2AF⁶⁵ lacking its RS domain, U2AF⁶⁵ΔRS. Background binding on GST beads was analysed (lanes 1-6) and 0.5% of starting material was run in parallel to allow quantification (not shown). Mean values of three experiments are presented with standard deviations for binding to SF1f (bottom). The equivalent loading of the beads with GST and GST-SF1f was checked by Coomassie staining of the gel (top left).

Figure 2. KIS phosphorylates SF1 in vitro on serines 80 and 82.

A) SF1[1-255] (SF1f) was phosphorylated by KIS with 10 μM [γ-³²P]ATP for the indicated times. Reaction products were analysed by SDS PAGE, Coomassie blue staining and phosphorimaging. Phosphorylation of SF1f induced a shift of the SF1f band on SDS-PAGE. B) The ability of similar amounts of the indicated forms of KIS to phosphorylate SF1f in vitro was compared, showing that deletion of or within the UHM domain of KIS impaired SF1f phosphorylation. C) Phosphorylation in vitro of different forms of GST-SF1f as indicated. Reactions were stopped while phosphate incorporation was still linear with time (See “Experimental Procedures”). Mutation of both serines 80 and 82 completely abolished phosphate incorporation. Quantification: mean values of three experiments. D) Primary structure of the SF1 isoform SF1HL1 (Alternatively spliced forms of SF1 differs by the length and sequence of their proline rich C-terminal region [28,51]). The SPSP motif is located in between the N-terminal U2AF⁶⁵ binding region and the KH-QUA2 domain that mediates recognition of the BPS [20,52]. The SPSP motif is in a highly phylogenetically conserved region as presented in the alignment [15]. The sequences of SF1 are from the following organisms Hs, Homo sapiens; Ce, Caenorhabditis elegans; Dm, Drosophila melanogaster; Sc, Saccharomyces cerevisiae; Sp, Schizosaccharomyces pombe.

Figure 3. SF1 SPSP motif phosphorylation in cells.

A) HEK293 cells were transfected with SF1myc or SF1myc(8082A) together with either pCDNA3 (vect), pCDNA3-KIS(54R) (kinase defective) or pCDNA3-KIS. Overexpressed SF1myc was immunoprecipitated with antimyc mAb 9E10 and analysed by SDS PAGE, coomassie staining (left) and phosphorimaging (right). B) Phosphorylated SF1 was analysed by tryptic phosphopeptide mapping. Map 1: SF1f phosphorylated by KIS in vitro (with a moderate stoichiometry of about 0.1 phosphate per SF1 molecule). Maps 2, 3 and 4 : phosphorylated SF1myc from lanes 1, 3 and 2 of gel in A. SF1f and SF1myc present almost identical maps showing that major phosphorylation occurs on the SPSP motif in vivo. Coexpression of KIS leads to disappearance of the more basic a and b peptides (two experiments) indicating an increase in the phosphorylation state of the SPSP motif. C) 5 μg of protein extract of HEK293 cells overexpressing SF1myc (top) or of untransfected HEK293 cells (bottom) were analysed by phosphatase (CIP) treatment with the different amounts indicated (units from New Englands Biolabs) and western blotting with antimyc or antiSF1 antibody (Cemines). In additional characterization experiments we showed that the 80kDa band migrated just below SF1myc, was nuclear and heat soluble which are characteristics of SF1 [53]. The 60kDa band most probably corresponds to a differentially spliced form [28].

Figure 4. SF1 SPSP motif phosphorylation enhances binding to U2AF⁶⁵.

Pull-down experiments were performed using purified GST-SF1f or GSTSF1f-8082A that were phosphorylated by KIS (+) or mock phosphorylated (−). We used as input a mixture of in vitro translated methionine labelled U2AF⁶⁵ and U2AF⁶⁵ΔRS. We checked that interaction was the same when these proteins were alone or in the mixture (not shown). Interactions with GST-SF1f phosphorylated by KIS versus mockphosphorylated were in duplicates. Quantification was performed by phosphorimaging. The mean values with standard deviation are represented. Representative results of two experiments.

Figure 5. SF1 SPSP motif phosphorylation enhances formation of a SF1-U2AF⁶⁵- RNA ternary complex.

A. We used a ³²P labelled RNA oligonucleotide corresponding to a model 3′ intronic sequence previously characterized in gel-shift experiments with SF1 and U2AF⁶⁵ (see Experimental Procedures) [14]. RNA was incubated with different protein mixtures. Single shift with U2AF⁶⁵ and supershift with U2AF⁶⁵ plus SF1f were clearly identified (lanes 2 and 3). Formation of SF1f-U2AF⁶⁵-RNA complex was enhanced by previous phosphorylation of SF1f by KIS (compare lane 3 with lanes 4 and 5). B. Increasing concentrations of SF1f or SF1f phosphorylated by KIS (pSF1f) showed no major difference in RNA shift. C. To quantify the enhancement of formation of the SF1f-U2AF⁶⁵-RNA complex upon phosphorylation of the SPSP motif, we used increasing concentrations of SF1f or SF1f phosphorylated by KIS (pSF1f) in the presence of 1.2 μM U2AF⁶⁵. The different bands were quantify by phosphorimaging and we plotted the formation of the ternary complex as a fraction of total RNA. We determined the decrease of the apparent Kd of SF1 for RNA upon phosphorylation by KIS, by determining the y intercept of the Hill plot for each experiment (not shown). We used three different preparations of phosphorylated and mockphosphorylated SF1f in four experiments and found that phosphorylation induced an average 3 fold decrease of the apparent Kd of SF1 for RNA in the presence of 1.2μM U2AF⁶⁵.