Major phosphorylation of SF1 on adjacent Ser-Pro motifs enhances interaction with U2AF65 ´ ´ Valerie Manceau1, Matthew Swenson2, Jean-Pierre Le Caer3, Andre Sobel1, Clara L Kielkopf2 and Alexandre Maucuer1 ` INSERM, U706, UPMC, Institut du Fer a Moulin, Paris, France Department of Biochemistry and Molecular Biology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, MD, USA ´ ´ Ecole Polytechnique, Laboratoire de Chimie des Mecanismes Reactionnels, Palaiseau, France Keywords protein phosphorylation; RNA splicing; SF1; kinase KIS; U2AF homology motif Correspondence A Maucuer, INSERM U706, 17, rue du Fer ` a Moulin, F-75005 Paris, France Fax: +33 14587 6132 Tel: +33 14587 6139 E-mail: maucuer@fer-a-moulin.inserm.fr (Received 14 October 2005, revised December 2005, accepted December 2005) doi:10.1111/j.1742-4658.2005.05091.x 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 (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 The expression of the genome requires the precise and controlled removal of intervening sequences within premessenger RNAs (pre-mRNA splicing) Assembly of the active spliceosome entails successive rearrangements, including the entry and exit of molecular partners (reviewed in [1]) Phosphorylation events are probably molecular switches to control these conformational changes Indeed, experiments with phosphatase inhibitors, purified phosphatases and nonhydrolysable ATP analogues have shown that multiple phosphorylation and dephosphorylation events are required for spliceosome assembly and splicing [2–4] Among the best-characterized of the phosphorylated splicing factors are the serine-arginine rich (SR) proteins (reviewed in [5]), whose intranuclear distribution and activity are influenced by phosphorylation by specific kinases including SRPK1, SRPK2 [6,7], and Clk ⁄ Sty [8] SF3b155 ⁄ SAP155, an integral spliceosome component and substrate of cyclin E ⁄ CDK2 [9], is a non-SR protein whose phosphorylation state is also regulated during the splicing process [10] In addition, other factors that regulate splicing in a phosphorylation-dependent manner have been identified (reviewed in [11,12]) Splicing factor (SF1) was identified as necessary for spliceosome assembly by in vitro reconstitution assays with protein fractions from HeLa cell nuclear extracts [13], and in a synthetic lethality screen with Mud2p, the yeast homologue of the splicing factor U2 auxiliary factor large subunit (U2AF65) [14] Moreover SF1 was found to physically interact with U2AF65 [14–16] SF1 binds to the branch point pre-mRNA consensus sequence (BPS) near the 3¢ splice site [17], and facilitates binding of U2AF65 to the adjacent polypyrimidine tract [15] Next, SF1 is displaced from the Abbreviations BPS, branch point sequence; CIP, calf intestinal phosphatase; DTT, dithiothreitol; GST, glutathione-S-transferase; SF1, splicing factor 1; siRNA, small interfering RNA; snRNP, small nuclear ribonucleoprotein particle; RRM, RNA recognition motif; U2AF, U2 auxiliary factor; UHM, U2AF homology motif FEBS Journal 273 (2006) 577–587 ª 2006 The Authors Journal compilation ª 2006 FEBS 577 SF1 SPSP motif phosphorylation V Manceau et al spliceosome by the ATP-dependent entry of the U2 small nuclear ribonucleoprotein particle (snRNP), whose SF3b155 ⁄ SAP155 protein subunit interacts with U2AF65 [18] and whose RNA component (U2 snRNA) anneals with the BPS [19,20] This first ATPdependent step of 3¢ splice site recognition represents a critical juncture for regulation of pre-mRNA splicing Protein kinase PKG is a potential regulator of this step, by inhibiting the SF1 ⁄ U2AF65 interaction upon phosphorylation of a conserved SF1 serine (Ser20) within its U2AF65 interaction domain [21] Comparison of the structure of an RNA recognition motif (RRM)-containing domain of the U2AF small subunit (U2AF35) complexed to a U2AF65 peptide [22], with the structure of the C-terminal RRM (RRM3) of U2AF65 complexed to an N-terminal peptide of SF1 [23] reveals that the unique RRM of U2AF35 and RRM3 of U2AF65 belong to a subclass of RRMs with specialized features for protein–protein interactions [23,24] Members of this RRM subclass are called U2AF homology motifs (UHMs) Diverse UHM-containing proteins have been identified that contain critical sequence features for interaction with peptide ligands, including the protein kinase KIS [23,24] The KIS polypeptide is organized into a kinase core followed by a C-terminal UHM (Fig 1B) [25,26], and preferentially phosphorylates Ser-Pro sites in vitro [27] A likely role of KIS during the control of cellcycle division is supported by the phosphorylation of the cyclin dependent kinase inhibitor p27kip1 [28] and the observation that KIS mRNA levels are misregulated in neurological tumours [29] We report here that SF1 is phosphorylated on two major adjacent Ser-Pro motifs (hereafter called the SPSP motif) We show that the protein kinase KIS can interact with SF1 through its UHM domain and efficiently phosphorylate SF1 on both serines of this SPSP motif, and therefore is likely to participate in controlling the phosphorylation state of SF1 Finally, SF1 phosphorylation on its SPSP motif enhanced the interaction of SF1 with U2AF65, and also promoted formation of a ternary complex with a model 3¢ intronic sequence, suggesting the importance of this major SF1 phosphorylation for recognition of the 3¢ splice site Results Phosphorylation of SF1 on serines 80 and 82 in vitro and in vivo A 80 82 65 U2AF KH-QUA2 Zn Proline rich SF1 11-25 SF1f 136-228 279-292 324-590 639 255 B Kinase UHM 23-304 KIS 316-415 419 K R KIS[54R] KIS[1-313] 419 54 313 KIS[∆369-409] 1 KIS[EDKK] 341-342 419 SF1 35 U2AF RS RRM1 RRM2 RRM3/UHM C U2AF 65 419 ED KK 475 25-63 134-229 242-333 85-112 U2AF 65∆RS 374-465 475 Fig Schematic representations of SF1 (A), KIS (B) and U2AF65 (C) (A) Representation of SF1HL1, a major SF1 splice variant in HeLa cells [32] (The various SF1 splice variants differ by the length and sequence of their proline rich C-terminal region [32,53]) The SPSP motif is in a highly phylogenetically conserved region [16], between the N-terminal U2AF65 binding region and the KH-QUA2 domain that mediates recognition of the branchpoint sequence [23,54] The aligned sequences of SF1 are from the following organisms: Hs, Homo sapiens; Ce, Caenorhabditis elegans; Dm, Drosophila melanogaster; Sc, Saccharomyces cerevisiae; Sp, Schizosaccharomyces pombe Zn, zinc knuckle motif The SF1f truncated form of SF1 used for in vitro experiments in this study retains its U2AF65 and RNA binding properties [16] (B) KIS is formed by the juxtaposition of a serine ⁄ threonine kinase domain and a C-terminal UHM domain Mutation of lysine 54 to arginine (KIS[54R]) impairs the kinase activity of KIS [26] KIS [1–313] entirely lacks the UHM domain, whereas KIS[D369–409] is deleted within the UHM Negatively charged residues Glu341 and Asp342 are replaced with positively charged lysines in the KIS[EDKK] variant (C) U2AF65 possesses an N-terminal arginine and serine rich (RS) domain and three C-terminal RRM domains [39] The C-terminal domain interacts with SF1 and is part of a subclass of RRM-like domains called UHMs [23,24] U2AF is a heterodimer of U2AF65 and U2AF35 [38] The domain of U2AF65 interacting with the UHM domain of U2AF35 is indicated [22] U2AF65DRS lacks residues 25–63 [39] The structural features and the mutated forms of SF1, KIS and U2AF65 used in this study are represented in Fig The presence of an SPSP motif in a highly 578 FEBS Journal 273 (2006) 577–587 ª 2006 The Authors Journal compilation ª 2006 FEBS V Manceau et al SF1 SPSP motif phosphorylation conserved region of SF1 suggested that these serine residues could be targets of the proline directed kinase KIS Further, we speculated that KIS could interact with SF1 based upon sequence similarity of the KIS and U2AF65 UHM domains (42% pairwise sequence identity between UHMs of human KIS, accession code NP_787062 and human U2AF65, accession code NP_009210) Pull-down experiments were used to probe the physical interaction of KIS with SF1 in vitro (Fig 2) We observed that KIS efficiently binds to the SF1f fragment of SF1 containing the SPSP motif and the essential domains for BPS and U2AF65 recognition [16,17] (human SF1 residues 1–255) (Fig 2, lane 7) Interestingly, KIS bound SF1f as efficiently as U2AF65DRS, a U2AF65 construct lacking its N-terminal arginine and serine rich (RS) domain (Fig 2, lane 12) In our conditions, binding of U2AF65DRS to SF1f was about twofold less than that of intact U2AF65 (Fig 2, lane 11), suggesting that the U2AF65 RS domain contributes to SF1f binding A possible role for the U2AF65 RS domain for binding to SF1 is consistent with the established interaction between the U2AF65 RS domain and the BPS [30,31], which is also targeted by SF1 during pre-mRNA splicing [17] A complete or a partial deletion of the UHM domain of KIS severely reduced binding to SF1f (Fig 2, lanes and 9) Mutations of acidic residues Glu396 and Glu397 to lysine within the UHM of U2AF65 have been shown to impair its interaction with SF1 [23] We observed that mutations of the corresponding KIS acidic residues (Glu341 and Asp342) to lysine also impaired KIS interaction with SF1 (Fig 2, lane 10) Finally the interactions were specific, as no significant binding of the KIS or U2AF65 variants to glutathioneS-transferase (GST) was detected (Fig 2, lanes 1–6) Altogether, when compared with the established SF1 interaction partner U2AF65, KIS bound efficiently to SF1f and this interaction required structural features of its UHM that are shared with U2AF65 Kinase assays showed that recombinant KIS efficiently phosphorylated SF1f, as evidenced by 32P phosphate incorporation and a marked shift of the SF1f band on SDS ⁄ PAGE (Fig 3A) The interaction of the UHM domain of KIS with SF1 appears to be important for efficient phosphorylation, as deletions within this domain prevented SF1f phosphorylation (Fig 3B, lanes and 4) Under all conditions tested, phosphate incorporation never exceeded an evaluated stoichiometry of two phosphates per one SF1f molecule, indicating that phosphorylation occurs on two residues Phosphoamino acid analysis showed that phosphorylation occured exclusively on serine residues (data not shown) Following phosphorylation of SF1 with KIS to high stoichiometry (over 1.5 phosphates per SF1 GST GST-SF1f 10 11 12 GST-SF1f U2AF65 U2AF65∆RS KIS / mutant GST-SF1f KIS[∆369-409] KIS[1-313] GST 35S Coomassie FEBS Journal 273 (2006) 577–587 ª 2006 The Authors Journal compilation ª 2006 FEBS % of Input 25 20 15 10 K 69 IS -4 KI S[ 09 KI -31 S( ED ] KK U 2A ) U 2A F 65 F6 5∆ R S ∆3 binding to SF1f Fig KIS interaction with SF1 in vitro Various forms of KIS and U2AF65 were translated in vitro in the presence of 35S-labelled methionine and tested for their binding to GST–SF1[1–255] (GST–SF1f) in a GST pulldown assay (top right, lanes 7–12) Lane 7: wild-type KIS; lane 8: KIS[D369–409] with a deletion within the UHM domain of KIS; lane 9: KIS[1–313] lacking the UHM domain; lane 10: KIS[EDKK] with mutations of Glu341 and Asp342 to lysine, lane 11: full-length U2AF65 and lane 12: U2AF65DRS lacking the RS domain Lanes 1–6: background binding on GST beads The binding of the different proteins to GST–SF1f was quantified (bottom) as the fraction of the input protein (% of input) that was bound to the beads (the total input protein was determined by running 0.5% of starting material in parallel (not shown)) Data are mean ± SD of three experiments The equivalent loading of the beads with GST and GST–SF1f was checked by Coomassie staining of the gel (top left) GST 10 11 12 579 SF1f 32P incorporation (au) Coomassie 32P 67 C 0 2 CIP(u) 2 2 t(hrs) 67 SF1myc SF1f + SF1 8082A SF1myc SF1f SF1 8082A A vect 54R KIS vect 54R KIS B (min) vect 54R KIS vect 54R KIS A V Manceau et al KIS KIS[54R] KIS[1-313] KIS[∆369-409] 10 20 30 40 60 90 120 180 240 360 SF1 SPSP motif phosphorylation 100 200 300 anti-myc 32P time (min) SF1 ~80kDa Ig HC SF 13 1f 80 255 A 82 80A 82 A SF 13 1f 80 255 A 82 80A 82 A ~60kDa C 82 A 82 A 80 32P SF 13 1f -2 55 80 A 32P incorporation (au) 1.2 1.0 0.8 0.6 GST0.4 SF1/ mutants 0.2 Coomassie Fig KIS phosphorylates SF1 in vitro on serines 80 and 82 (A) SF1 [1–255] (SF1f) was phosphorylated by KIS with 10 lM [c-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) In vitro phosphorylation 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 Ser80 and Ser82 completely abolished phosphate incorporation Data are mean values of three experiments molecule) and digestion with trypsin, we identified, by MS, masses corresponding to peptide 67–92 and peptide 80–92 of human SF1, each with two phosphates Both of these SF1 peptides contained Ser80 and Ser82 of the putative KIS target SPSP motif Phosphorylation efficiency decreased by approximately twofold when using SF1f with either Ser80 or Ser82 mutated to alanine, whereas the double alanine mutation (hereafter called 8082A) completely inhibited phosphorylation (Fig 3C) Thus, KIS can phosphorylate both sites with similar efficiency and no phosphorylation occurs outside the SPSP motif Altogether, KIS phosphorylated SF1f on Ser80 and Ser82 with high efficiency that depended on anchoring its UHM domain to SF1f To determine whether the SPSP motif is phosphorylated in vivo, we metabolically labelled HEK293 cells overexpressing the major spliced form SF1HL1 [32] with a myc tag (hereafter SF1myc), and immunoprecipitated SF1myc using an antimyc antibody As shown in Fig 4A, SF1myc phosphate incorporation 580 32P Coomassie GSTKIS B SF1 SF1f - KIS b c e a b d c SF1 - KIS SF1 - KIS[54R] b d a anti-SF1 d e e d a e c c Fig SF1 SPSP motif phosphorylation in cells (A) HEK293 cells were transfected with SF1myc or SF1myc(8082 A) together with either pCDNA3 (vector), pCDNA3-KIS(54R) (kinase defective) or pCDNA3-KIS After metabolic labelling of the cells with 32P-labelled inorganic phosphate for h, overexpressed SF1myc was immunoprecipitated with antimyc mAb 9E10 and analysed by SDS ⁄ PAGE, Coomassie staining (left) and phosphorimaging (right) (Ig HC: Immunoglobulin heavy chains) (B) Phosphorylated SF1 was analysed by tryptic phosphopeptide mapping Map1: SF1f phosphorylated by KIS in vitro (with a moderate stoichiometry of about 0.1 phosphate per SF1 molecule) Maps 2, and 4: phosphorylated immunoprecipitated SF1myc from lanes 1, and of the gel in A SF1f (Map1) and SF1myc (Map2) present almost identical tryptic maps showing that major phosphorylation occurs on the SPSP motif in vivo Coexpression of KIS leads to a major relative decrease of the more basic a and b peptides (two experiments), indicating an increase in the phosphorylation state of the SPSP motif (C) Five micrograms of protein extract of HEK293 cells overexpressing SF1myc (top) or of untransfected HEK293 cells (bottom) were analysed by phosphatase (CIP) treatment with the indicated amounts (units from New Englands Biolabs) and Western blotting with antimyc (top) or anti-SF1 antibody (bottom) In additional characterization experiments we showed that the 80-kDa band migrated just below SF1myc, and was nuclear and heat soluble, which are characteristics of SF1 [13] The 60-kDa band most probably corresponds to an alternatively spliced form [32] was dramatically reduced (by approximately fourfold) when both Ser80 and Ser82 of SF1 were mutated to alanine Thus, the SPSP motif contains major phosphorylation sites of SF1 in vivo The remaining incorporated phosphate most likely corresponds to phosphorylation of other SF1 sites such as Ser20, a known in vivo phosphorylation target [21] To confirm the major phosphorylation of the SPSP motif, we compared the tryptic phosphopeptide map of SF1f phosphorylated in vitro by KIS to that of SF1myc FEBS Journal 273 (2006) 577–587 ª 2006 The Authors Journal compilation ª 2006 FEBS Modulation of SF1 binding properties upon SPSP motif phosphorylation The SPSP motif lies at the junction between the U2AF65 binding N-terminal region of SF1 and its RNA binding domain, suggesting that phosphorylation of these sites might regulate the formation of the KIS + + + U2AF65 ∆RS 35S binding (% of input) phosphorylated in cells (Fig 4B, maps and 2) These peptide maps were highly similar They displayed multiple spots corresponding to incomplete digestion by trypsin, as previously observed by MS, and to the presence of single and double phosphorylation of the peptides as suggested by our analysis of SF1f phosphorylated by KIS to different extents in vitro (data not shown) Co-expression of KIS did not clearly increase the incorporation of radioactive phosphate (Fig 4A, lane 3), but phosphopeptide mapping revealed its effect to increase the relative amounts of more acidic peptides (c, d, e) (Fig 4B, compare maps and 3), which suggests that KIS increases the phosphorylation level of SF1 on the SPSP motif Coexpression with KIS(54R), bearing a mutation within the kinase active site that extensively suppresses activity [26], had an intermediate effect (Fig 4B, map 4) This agreed with our unpublished observations that KIS(54R) retains low levels of kinase activity towards SF1, due to the unusually high activity of KIS for this substrate The slight effect of KIS overexpression on phosphate incorporation in SF1myc suggested that SF1 is already mostly in a phosphorylated state in proliferating HEK293 cells This was also supported by the observation that wild-type SF1myc expressed in HEK293 cells migrates slower than the 8082A mutant (compare lanes 1–3 with 4–6 in Fig 4A) We tested this possibility by treating nuclear extracts of SF1myc overexpressing cells with alkaline phosphatase As shown in Fig 4C, phosphatase treatment induced a faster migration of SF1myc on SDS ⁄ PAGE showing that SF1myc is, indeed, already highly phosphorylated in proliferating cells This was also the case for endogenous forms of SF1 in HEK293 (Fig 4C, lower panel) and HeLa cells (data not shown) To summarize, these analyses show that the SPSP motif contains major in vivo phosphorylation sites of SF1 In agreement with this result, two recent independent large-scale analyses of the phosphoproteome, one in nuclear extracts of HeLa cells, the other in WEHI-231 B lymphoma cells, lead to the identification of the same 67–92 peptide of SF1 phosphorylated on both Ser80 and Ser82 [33,34] Altogether, this high level of phosphorylation of its SPSP motif indicates a particular involvement in SF1 function SF1 SPSP motif phosphorylation GS T GS T-S F1 80 f 82 A (1%) V Manceau et al 90 80 70 60 50 40 30 20 10 KIS Prey U2AF65 U2AF65∆RS + + GST SF1f + 8082A Fig SF1 SPSP motif phosphorylation enhances binding to U2AF65 Pull-down experiments were performed using purified GST, GST–SF1f or GST–SF1f(8082A) that were phosphorylated by KIS (+) or mock-phosphorylated (–).We used as input a mixture of in vitro translated [35S]methionine-labelled U2AF65 and U2AF65DRS 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 vs mock-phosphorylated were in duplicate One per cent of the input was loaded to allow quantification by phosphorimaging of the fraction of the 35S-labelled proteins that was bound to the beads The mean values of duplicates with standard deviation are represented Representative results of two experiments SF1-U2AF-RNA complex We first tested the effect of phosphorylation on the interaction of SF1 with U2AF65 by GST pull-down experiments Using GST– SF1f that was phosphorylated by KIS to high stoichiometry (over 1.5 phosphate per SF1f molecule) we observed a twofold increase in U2AF65 and a threefold increase of U2AF65DRS binding (Fig 5) In contrast no increase in binding could be observed with the SF1f(8082A) mutant upon treatment with KIS, showing that phosphorylation on the SPSP motif of SF1 is responsible for U2AF65 binding enhancement As SF1 and U2AF65 have been shown to bind in a cooperative manner to a model 3¢ splice site RNA, we hypothesized that the modification of SF1 binding to U2AF65 upon phosphorylation on the SPSP motif could in turn modulate the formation of the SF1– U2AF65–RNA ternary complex We analysed the formation of this complex and the effect of SF1 phosphorylation by RNA gel-shift with a model 3¢ intronic sequence previously used by Berglund and colleagues to characterize the cooperative binding of SF1 and U2AF65 to RNA [15] As shown in Fig 6, the RNA shifts induced by U2AF65, and SF1f plus U2AF65 binding were clearly identified as previously described [15] Interestingly we observed an increase in ternary complex formation when using SF1 previously phosphorylated by KIS (Fig 6A, compare lane with lanes FEBS Journal 273 (2006) 577–587 ª 2006 The Authors Journal compilation ª 2006 FEBS 581 SF1 SPSP motif phosphorylation V Manceau et al B A U2AF65 1.2 µM SF1f 0.3µM pSF1f 0.46 1.4 4.6 0.46 1.4 4.6 KIS KIS 54R GST treatment SF1f µM SF1f/U2AF65 U2AF65 SF1f free RNA free RNA U2AF65 (1.2 µM) pSF1f 0.27 0.53 0.80 0.27 0.53 0.80 SF1f SF1f/U2AF65 U2AF65 Fraction of RNA bound to SF1f (%) C 20 SF1f pSF1f 16 12 Discussion 0 0,2 0,4 0,6 0,8 [SF1] µM free RNA Kd(SF1) = 3.0 +/- 0.6 (n=4) Kd(pSF1) Fig SF1 SPSP motif phosphorylation enhances formation of an 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) [15] The RNA oligonucleotide was incubated with different protein mixtures A single shift with U2AF65 and supershift with U2AF65 plus SF1f were clearly identified (lanes and 3) Formation of SF1f-U2AF65-RNA complex (upper band) was enhanced by previous phosphorylation of SF1f by KIS (compare lane with lanes and 5) (B) Increasing concentrations of mock-phosphorylated SF1f or SF1f phosphorylated by KIS (pSF1f) showed no major difference in RNA shift (C) To quantify the enhancement of SF1f–U2AF65–RNA complex formation upon phosphorylation of the SPSP motif, we used increasing concentrations of mock-phosphorylated SF1f or SF1f phosphorylated by KIS (pSF1f) in the presence of 1.2 lM U2AF65 The different bands were quantified by phosphorimaging and the formation of the ternary complex was plotted as a fraction of total RNA The decrease of the apparent Kd of SF1 for RNA upon phosphorylation by KIS was calculated by determining the y intercept of the Hill plot for each experiment (data not shown) We used three different preparations of phosphorylated and mock-phosphorylated SF1f in four experiments and found that phosphorylation induced an average threefold decrease of the apparent Kd of SF1 for RNA in the presence of 1.2 lM U2AF65 582 and 5) In contrast, in the absence of U2AF65, we observed no major difference in RNA binding of mock-phosphorylated SF1f and phosphorylated SF1 (Fig 6B) Further analysis using increasing concentrations of mock-phosphorylated SF1f or phosphorylated SF1f with a constant concentration of U2AF65 (1.2 lm) confirmed the enhancement of ternary complex formation upon phosphorylation of SF1 (Fig 6C) This was observed using three different productions of phosphorylated SF1 vs parallel productions of mock-phosphorylated SF1 The ratios of the apparent equilibrium dissociation constants (Kd) were calculated by linear regression of the Hill plot for four experiments, and showed an approximately threefold decrease of apparent Kd for SF1 binding to RNA upon phosphorylation by KIS To summarize, these results show that phosphorylation of SF1 on the SPSP motif enhances binding to U2AF65 and formation of the ternary SF1–U2AF65–RNA complex Given that the kinase KIS contains a UHM domain with significant similarity to the SF1-binding UHM domain of the pre-mRNA splicing factor U2AF65, we hypothesized that KIS likewise might be targeted to pre-mRNA splicing factors by its UHM [26] Accordingly, the splicing factor SF1 was a very efficient substrate for KIS in vitro when compared with other candidate substrates that we have tested ([27] and unpublished data) Using MS and mutagenesis, we demonstrated that KIS phosphorylates SF1f (human SF1 residues 1–255) exclusively on Ser80 and Ser82 within an SPSP motif Interestingly, we observed that KIS can interact with SF1f as efficiently as with U2AF65DRS, and that its UHM domain is required for binding to, and for the phosphorylation of SF1f This result further illustrates the molecular similarity between the UHM domains of KIS and U2AF65 Altogether, KIS is a likely candidate for controlling the phosphorylation state of the SF1 SPSP motif in vertebrates, where KIS is highly conserved (for example, 63% sequence identity between human KIS, accession code NP_787062 and zebrafish KIS, accession code XP_698499) Other proline directed kinases may phosphorylate the highly conserved SPSP motif of SF1 in nonvertebrates (Fig 1A) The relative importance of KIS, compared with other kinases and phosphatases, for regulating the phosphorylation state of the SF1 SPSP motif in vivo is an intriguing subject for further investigation By comparing the phosphopeptide map of SF1f following in vitro phosphorylation by KIS with that of FEBS Journal 273 (2006) 577–587 ª 2006 The Authors Journal compilation ª 2006 FEBS V Manceau et al SF1myc immunoprecipitated from HEK293 cells, we demonstrated that SF1myc is phosphorylated on the SPSP motif in these cells Accordingly, mutation of Ser80 and Ser82 to alanine strongly reduced phosphate incorporation in SF1myc The high level of phosphorylation of overexpressed and endogenous SF1 was demonstrated by treating cell extracts with phosphatase, which resulted in a faster migration of SF1 on SDS ⁄ PAGE, and showed that all detectable SF1 is initially in a phosphorylated state in HEK293 cells This was also the case in HeLa cells and mouse brain (data not shown) The functional significance of this major phosphorylation of SF1 was further suggested by the induced modification of its interaction with U2AF65 Remarkably, U2AF65 recovery on GST–SF1 beads in pull-down experiments was markedly enhanced when GST–SF1 was first phosphorylated by KIS (Fig 5) Moreover, in gel-shift experiments with a model 3¢ splice site RNA, phosphorylation of the SF1 SPSP motif by KIS reproducibly enhanced formation of the ternary SF1–U2AF65–RNA complex (Fig 6) Two a-helices are predicted to form on each side of the proline-rich region containing the SPSP motif [16] Thus, local structural modifications of the SPSP region by phosphorylation could be amplified by these helical structures, thereby facilitating the SF1 ⁄ U2AF65 interaction This hypothesis is supported by the observation that phosphorylation within proline rich regions often induces structural rearrangements [35], which could propagate up to the U2AF65 binding site in the case of SF1 However, it is also possible that the phosphorylated SPSP motif directly participates in the interaction with U2AF65 Of note, the experiments with SF1 fragments showing that regions C-terminal to residue 25 were dispensable for interaction were performed with unphosphorylated SF1 and thus not exclude this possibility [23] Our data suggest that the phosphorylated SPSP motif of SF1 at least does not interact with the basic RS domain of U2AF65, because enhancement of binding upon phosphorylation was also observed for the U2AF65DRS fragment Further investigations are needed to examine the structural basis for the effect of SPSP motif phosphorylation on the interaction of SF1 with U2AF65 A comprehensive set of data supports the functional importance of the interaction of SF1 with U2AF65 In mammals, the interaction between U2AF65 and SF1 was demonstrated by two-hybrid, pull-down and farwestern assays [14–16] These interactions involve the highly conserved N terminus of SF1 and the UHM domain of U2AF65 In addition, SF1 and U2AF65 were shown to be both present in the early spliceosomal SF1 SPSP motif phosphorylation complex E [14,36] SF1 was shown to bind preferentially to the pre-mRNA branchpoint sequence [17,37] while U2AF65 interacts with the nearby poly-pyrimidine tract and its RS domain contacts the branchpoint [30,31,38,39] Furthermore the interactions of U2AF65 and SF1 with the branchpoint region were found to be cooperative [15] Finally structural bases for this interaction have been determined [23] The SF1 ⁄ U2AF65 interaction appears to be highly conserved throughout evolution In Schizosaccharomyces pombe, SF1 and the U2AF subunit homologs U2AF59 and U2AF23 form a stable complex [40] In Saccharomyces cerevisiae, the SF1 orthologue Msl5p is highly conserved while the U2AF65 orthologue Mud2p shows a sequence conservation restricted to the UHM domain involved in its interaction with SF1 Like their mammalian counterparts, Msl5p and Mud2p interact and are recruited early in spliceosome assembly [41,42] Msl5p has a marked preference for binding to the consensus S cerevisiae branchpoint sequence [17] and Mud2p requires an intact branchpoint region for binding [41,43] Furthermore MSL5 was identified by virtue of its genetic interaction with MUD2 [14] Importantly, SF1 is thought to perform an essential conserved function for cell viability Actually, in HeLa cells, small interfering RNA (siRNA) depletion of SF1 leads to cell death [44] and in S cerevisiae MSL5 disruption is lethal [14] In this context, our finding that the SF1 ⁄ U2AF65 interaction can be enhanced by phosphorylation is likely to be significant for understanding SF1 function Of note, despite strong evidence that SF1 is necessary for spliceosome assembly using in vitro reconstitution assays with protein fractions from HeLa nuclear extracts [13], several reports suggest that the SF1– U2AF65 interaction is dispensable for splicing of at least a subset of pre-mRNAs Actually, thorough immunodepletion of SF1 from a HeLa splicing extract only modestly reduces the kinetics of spliceosome assembly [45] Similarly, no differences in the splicing of three pre-mRNA substrates could be detected when a U2AF65-depleted nuclear extract was complemented with full-length U2AF65 or U2AF65 lacking its UHM domain [46] Moreover, SF1 knockdown in HeLa cells using siRNA did not apparently affect splicing of several pre-mRNAs, nor the levels of a variety of proteins translated from spliced mRNA [44] Finally, using S cerevisiae extracts that had been either depleted of SF1 or prepared from temperature-sensitive mutants grown at a nonpermissive temperature, no dramatic defect in splicing of a pre-mRNA with consensus splice sites was detected [42,47] Nevertheless, splicing in vivo of pre-mRNA with weakened splice site sequences was FEBS Journal 273 (2006) 577–587 ª 2006 The Authors Journal compilation ª 2006 FEBS 583 SF1 SPSP motif phosphorylation V Manceau et al reduced in temperature-sensitive mutants grown at a nonpermissive temperature, suggesting that SF1 function is important for splicing efficiency [47] It has been proposed that a low level of SF1 remains in depleted extracts or in siRNA treated cells, and is sufficient for its transient catalytic action in pre-spliceosome assembly on most pre-mRNAs [42,44,47] This leaves open the possibility that SF1 has an essential role for splicing in vivo that is not evident under in vitro conditions and in siRNA treated cells In this context, the phosphorylation of the SPSP motif that enhances its binding to U2AF65 could be important for splicing by facilitating recognition of weak acceptor sequences The highly phosphorylated state we observe for SF1 in vivo suggests that SF1 dephosphorylation is transient, for example to allow the recycling of SF1 after prespliceosome assembly Specifically, dephosphorylation of SF1 may facilitate replacement by the U2 snRNP component SF3b155 ⁄ SAP155 within the assembling spliceosome [18], by decreasing the affinity of SF1 for U2AF65 Accordingly, the fact that 54 amino acid residues separate the SPSP phosphorylation site from the minimal U2AF65 binding domain suggests that the SPSP motif remains accessible to regulatory phosphatases in the early spliceosome complex Several proposals have been made to explain the SF1 requirement for cell viability When SF1 levels are decreased, it is possible that SF1 becomes rate limiting for splicing of a subset of pre-mRNAs, some of which encode essential peptides [47] Alternatively, a cumulative reduction in the kinetics of splicing of a larger subset of pre-mRNAs may be lethal Finally, functions of SF1 in pathways other than pre-mRNA splicing have been suggested to explain its requirement for cell viability [42,44,47] For example, SF1 was found to bind transcription factors and to repress transcription in a reporter assay in mammalian cells [48,49] An interaction of SF1 with the transcription elongation factor CA150 was also reported [50] In addition, the UHMcontaining proteins PUF60, Tat-SF1 and CAPER that may also associate with SF1 have been implicated in transcription (see [23,24] and references herein) In S cerevisiae, MSL5 temperature-sensitive mutants at a nonpermissive temperature presented a pre-mRNA retention defect [47] In this context, U2AF65 also has been shown to be a shuttling protein and its role during RNA export has been documented [51,52] Thus, the observed effect of phosphorylation of the SPSP motif on ternary SF1–U2AF65–RNA complex formation may regulate any of several putative SF1 functions: premRNA splicing, transcription, or export In conclusion, given the strong evidence supporting the conserved SF1 ⁄ U2AF65 interaction, it is likely that the regulations 584 of the SF1 ⁄ U2AF65 interaction by phosphorylation of SF1 on the highly conserved SPSP motif may play a major functional role Our identification of this novel phosphorylated form of SF1 provides a starting point to evaluate the questions of whether and how pre-mRNA splicing and ⁄ or the other potential functions of SF1 are regulated by SPSP motif phosphorylation and by KIS Experimental procedures Plasmids and mutagenesis The plasmid for expression of the human SF1 fragment, SF1f (residues 1–255) was constructed in the pGEX6P-1 vector (Amersham Biosciences, Uppsala, Sweden) SF1f contains the U2AF65 binding domain and KH-QUA2 domain for BPS recognition Prior to PCR, the SF1 template was corrected for a Arg19Gly mutation inadvertently present from previous published work [14,15] Plasmids pSP64U2AF65 and U2AF65DRS (lacking residues 25–63) [39] for in vitro translation were kindly provided by J Valcarcel To allow expression of rat KIS from the same vector, an NcoI– XhoI blunt insert from plasmid BSKIS [26] was transferred to pSP64-U2AF65 NcoI–EcoRI blunt yielding pSP64-KIS For expression in mammalian cells, SF1HL1 cDNA was amplified from cDNA of HeLa cells and subcloned in pCDNA3 with a myc tag at the C terminus (SF1myc in the text) Site-directed mutagenesis was performed using the Quikchange protocol from Stratagene (La Jolla, CA, USA) Protein expression and purification GST–KIS and mutants were produced as previously described [27] GST–SF1f was purified by glutathione affinity chromatography, followed by removal of GST using Precision Protease (Amersham Biosciences) and further purification on SP-sepharose (Amersham Biosciences) Purified fractions were frozen in 200 mm NaCl, 25 mm Hepes pH 6.8, 20% v ⁄ v glycerol Plasmid for U2AF65 was a gift of M.R Green, and the histidine-tagged protein was expressed in bacteria and purified using standard protocols GST pull-down In vitro translations were performed using the TNT system (Amersham Biosciences) and [35S]-methionine (NEN, Boston, MA, USA) For experiments in Fig 2, lL of in vitro translation product was mixed with 250 lL of GST or GST– SF1f extract corresponding to mL of the BL21 cell cultures, in GST sonication buffer (GSB; 25 mm Hepes pH 7.5, 100 mm KCl, mm EDTA, 0.1% NP40, 10% glycerol) with mm dithiothreitol (DTT) and antiprotease mix from Roche (Mannheim, Germany) For experiments in Fig 5, in vitro translation products were incubated with lg purified GST FEBS Journal 273 (2006) 577–587 ª 2006 The Authors Journal compilation ª 2006 FEBS V Manceau et al or GST–SF1 in the presence of lgỈlL)1 BSA as a nonspecific competitor After a 90-min incubation at °C, 10 lL of glutathione beads (Amersham Biosciences) were added for a further 30 min, beads were washed rapidly five times with GSB buffer, and proteins were analysed by SDS ⁄ PAGE, Coomassie blue staining and phosphorimaging (Cyclone, Packard Instrument Company, Meriden, CT, USA) Phosphorylation reactions Phosphorylation reactions were performed as described previously [27] Briefly, 20 lL reactions contained  20 ng recombinant GST–KIS and lg of substrate in 50 mm Mes pH 8.0, 10 mm MgCl2, mm DTT, mm EDTA, 25% glycerol, 10 lm [c-32P]ATP (5 nCiỈpmole)1; NEN) For substrate and kinase mutant comparison we performed 30-min reactions and checked the linearity of phosphate incorporation For stoichiometric phosphorylation we performed h incubations with 0.4 mm ATP Mass spectrometry After phosphorylation by KIS, SF1f was dialysed against 50 mm NH3HCO3 pH 8.0, digested with trypsin, and HPLC reverse phase fractions were analysed with a FT-ICR mass spectrometer APEX III (Bruker Daltonics, Bremen, Germany) equipped with a Tesla supraconducting magnet and an infinity cell Metabolic labelling of cells HEK293 cells were transfected using lipofectamine reagent (Invitrogen, Carlsbad, CA, USA) After 24 h, the medium was replaced with mL fresh medium containing mCi [32P]-inorganic phosphate (NEN) for h Cells were lysed in immunoprecipitation buffer containing 50 mm Tris pH 7.5, 150 mm NaCl, 1% Nonidet P40, mm EDTA, mm DTT and protease inhibitors Immunoprecipitation was performed with the monoclonal antimyc 9E10 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) Phosphopeptide mapping Proteins were digested ‘in-gel’ overnight with trypsin at 30 °C as previously described [27] Eluted peptides were lyophilized and resuspended in electrophoresis buffer (pH 3.5) After electrophoresis and chromatography, plates were revealed by phosphorimaging Phosphatase treatment Cell extracts (5 lg of proteins) were treated with the indicated amount of calf intestinal phosphatase (CIP) (New England Biolabs, Beverly, MA, USA) for h SF1myc was SF1 SPSP motif phosphorylation analysed with the monoclonal antimyc 9E10 antibody (Santa Cruz Biotechnology) Endogenous forms of SF1 were detected with a rabbit polyclonal antibody (Cemines, Golden, CO, USA) RNA gel-shift assays RNA oligonucleotide with sequence derived from the Adenovirus major late pre-mRNA (5¢-UUCGUGCU GACCCUGUCCCUUUUUUUUCCACAGC-3¢) was synthesized by Dharmacon (Lafayette, CO, USA) 5¢ labelling was performed with [c-32P]ATP and T4 polynucleotide kinase Labelled RNA was purified on Nensorb20 column (NEN) About 10000 cpm ⁄ fmole oligonucleotide was used with the indicated concentrations of purified proteins and with tRNA (0.5 lgỈlL)1) as nonspecific competitor Interactions were in 15 lL containing 25 mm Tris pH 7.5, 90 mm NaCl and mm EDTA, for h at room temperature We checked that equilibrium was reached at this time point The mixture was loaded on 6% acrylamide gels in 0.5 · TBE buffer, and run at °C for h Acknowledgements We thank colleagues from INSERM U706, D Weil and J Chamot-Rooke for stimulating discussion and support We are grateful for the generous gifts of ´ U2AF65 constructs from J Valcarcel and M.R Green, and SF1 cDNA from J.A Berglund We thank S Lindley for 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splicing factor Science 294, 1098–1102 FEBS Journal 273 (2006) 577–587 ª 2006 The Authors Journal compilation ª 2006 FEBS 587 ... effect of SPSP motif phosphorylation on the interaction of SF1 with U2AF65 A comprehensive set of data supports the functional importance of the interaction of SF1 with U2AF65 In mammals, the interaction. .. regulator of this step, by inhibiting the SF1 ⁄ U2AF65 interaction upon phosphorylation of a conserved SF1 serine (Ser20) within its U2AF65 interaction domain [21] Comparison of the structure of an... given the strong evidence supporting the conserved SF1 ⁄ U2AF65 interaction, it is likely that the regulations 584 of the SF1 ⁄ U2AF65 interaction by phosphorylation of SF1 on the highly conserved