The enzymatic activity of SR protein kinases and 1a is negatively affected by interaction with scaffold attachment factors B1 and Dora Tsianou1, Eleni Nikolakaki2, Alexandra Tzitzira1, Sofia Bonanou1, Thomas Giannakouros2 and Eleni Georgatsou1 Department of Medicine, University of Thessaly, Mezourlo, 41110 Larissa, Greece Department of Chemistry, The Aristote University of Thessaloniki, Greece Keywords kinase activity inhibition; nuclear complex formation; SAFB; SRPK1; SRPK1a Correspondence E Georgatsou, Laboratory of Biochemistry, Department of Medicine, School of Health Sciences, University of Thessaly, Mezourlo, 41110 Larissa, Greece Fax: +30 241 068 5545 Tel: +30 241 068 5581 E-mail: egeorgat@med.uth.gr Website: http://www.med.uth.gr (Received 24 January 2009, accepted 16 July 2009) doi:10.1111/j.1742-4658.2009.07217.x SR protein kinases (SRPKs) phosphorylate Ser ⁄ Arg dipeptide-containing proteins that play crucial roles in a broad spectrum of basic cellular processes Phosphorylation by SRPKs constitutes a major way of regulating such cellular mechanisms In the past, we have shown that SRPK1a interacts with the nuclear matrix protein scaffold attachment factor B1 (SAFB1) via its unique N-terminal domain, which differentiates it from SRPK1 In this study, we show that SAFB1 inhibits the activity of both SRPK1a and SRPK1 in vitro and that its RE-rich region is redundant for the observed inhibition We demonstrate that kinase activity inhibition is caused by direct binding of SAFB1 to SRPK1a and SRPK1, and we also present evidence for the in vitro binding of SAFB2 to the two kinases, albeit with different affinity Moreover, we show that both SR protein kinases can form complexes with both scaffold attachment factors B in living cells and that this interaction is capable of inhibiting their activity, depending on the tenacity of the complex formed Finally, we present data demonstrating that SRPK ⁄ SAFB complexes are present in the nucleus of HeLa cells and that the enzymatic activity of the nuclear matrixlocalized SRPK1 is repressed These results suggest a new role for SAFB proteins as regulators of SRPK activity and underline the importance of the assembly of transient intranuclear complexes in cellular regulation Structured digital abstract l MINT-7228149: SRPK1 (uniprotkb:Q96SB4-2) phosphorylates (MI:0217) Nt-LBR (uniprotkb:Q14739) by protein kinase assay (MI:0424) l MINT-7228207: SRPK1 (uniprotkb:Q96SB4-2) physically interacts (MI:0915) with SAFB1C (uniprotkb:Q15424) by pull down (MI:0096) l MINT-7228438: SRPK1a (uniprotkb:Q96SB4-3) physically interacts (MI:0915) with SAFB1C (uniprotkb:Q15424) by pull down (MI:0096) l MINT-7228306: SRPK1 (uniprotkb:Q14151) physically interacts (MI:0915) with SAFB2C (uniprotkb:Q14151) by pull down (MI:0096) l MINT-7228452: SRPK1a (uniprotkb:Q96SB4-3) physically interacts (MI:0915) with SAFB2C (uniprotkb:Q14151) by pull down (MI:0096) l MINT-7228466: SRPK1 (uniprotkb:Q96SB4-2) physically interacts (MI:0915) with SAFB1 (uniprotkb:Q15424) by anti tag coimmunoprecipitation (MI:0007) l MINT-7228500: SRPK1a (uniprotkb:Q96SB4-3) physically interacts (MI:0915) with SAFB1 (uniprotkb:Q15424) by anti tag coimmunoprecipitation (MI:0007) l MINT-7228483: SRPK1 (uniprotkb:Q14151) physically interacts (MI:0915) with SAFB2 (uniprotkb:Q14151) by anti tag coimmunoprecipitation (MI:0007) Abbreviations GFP, green fluorescent protein; GST, glutathione S-transferase; LBR, lamin B receptor; SAFB, scaffold attachment factor B; SRPK, SR protein kinase 5212 FEBS Journal 276 (2009) 5212–5227 ª 2009 The Authors Journal compilation ª 2009 FEBS D Tsianou et al SRPK1/1a inhibition by interaction with SAFB1/2 Introduction Although the SR protein kinase (SRPK) family was discovered < 15 years ago, it has been implicated in cellular processes of the utmost importance SRPKs specifically phosphorylate serine residues in regions rich in Ser ⁄ Arg repeats, also called RS domains RS domain-containing proteins are spread throughout the cell They are functionally associated with a multiplicity of cellular processes, such as splicing, pre-mRNA processing, chromatin structure and remodeling, transcription by RNA polymerase II, mRNA translation, cell-cycle progression, cell structure and other speciesspecific functions [1,2] The SRPK family of protein kinases is highly conserved among eukaryotes, both structurally and functionally [3–9] The correct constitutive and alternative splicing, the shuttling of several RS splicing factors between the nucleus and the cytoplasm, their subnuclear localization in nuclear speckles, their recruitment to sites of transcription and their contribution to correct exon selection via RNA binding, are some of the steps regulated by SRPK phosphorylation [10–15] SRPKs have also been implicated in mRNA export from and protein import into the nucleus [16,17] In addition, phosphorylation of the nucleoplasmic tail of the lamin B receptor (LBR) by SRPK1 [18,19] regulates its binding to chromatin [20] Protamine P1, a histone-replacing protein is also phosphorylated by SRPK1 [21] Phosphorylation of the two proteins has been shown to play a crucial role in mammalian spermiogenesis [22] It is also interesting to note that several viruses alter expression levels of SRPKs, and viral proteins interact and become phosphorylated by SR kinases during the infection cycle (human T-lymphotropic virus-1, herpes simplex virus-1, hepatitis B virus), highlighting the importance of the involvement of SRPKs in a large number of cellular mechanisms [23–26] In humans, the SRPK1 gene product is alternatively spliced producing a minor transcript, the product of which, SRPK1a, contains an additional 171 amino acids at its N-terminus because of the retention of an intron [27] In a previous study, which revealed the expression of SRPK1a as an active kinase displaying only minor differences from SRPK1, we showed that its additional N-terminal region interacts with scaffold attachment factor B1 (SAFB1) [27] SAFB1 is a protein of the nuclear matrix first discovered approximately a decade ago It was reported with different names and was associated with a diversity of functions [28–31] It is clear, however, that SAFB1 resides in the nucleus and is a scaffold ⁄ matrix attachment region element binding protein It is 915 amino acids long and contains a SAF box (amino acids 35–67), a RNA recognition motif domain (amino acids 409–482), a putative nuclear localization signal (amino acids 519–614), a Glu ⁄ Arg-rich region (amino acids 619–699) and a Gly-rich region (amino acids 785–899) A multiplicity of publications provide ample evidence that SAFB1 interacts with several different proteins such as polymerase II, splicing factors and hnRNP proteins and also with the tight junction protein ZO-2 and the tripartite motif family protein TRIM 27 [30,32–36] In addition, it is found in a number of different subnuclear complexes formed by a variety of different combinations of nuclear proteins involved in either transcription [37] or splicing [38] The most prominent function of SAFB1, however, is transcriptional repression It was initially shown that SAFB1 binds to, and acts as, a corepressor of estrogen receptor a [39] It was further shown that it is capable of suppressing the transcription of a reporter gene; suppression being exerted via interaction with TATAbinding protein associated factor II 68 [39] In addition, SAFB1 represses the transcriptional activity of multiple nuclear receptors [40] This repression might be effectuated, in some cases at least, by its interaction with the nuclear receptor corepressor which facilitates binding of histone deacetylases to the sites of transcription of nuclear receptors [41] SAFB2, a protein with 70% structural identity to SAFB1, is much less studied, but it also seems to be a transcriptional repressor [39,42] However, it cannot substitute for SAFB1, because SAFB1 knockout mice display serious defects [43] Moreover, SAFB1 and SAFB2 have different subnuclear localizations [38] A third protein belonging to the SAFB family, SAF-like transcription modulator (34% identity to SAFB1 and 32% to SAFB2), has been shown to downregulate general mRNA synthesis [44] Finally, SAFB1, and the SAFlike transcription modulator, have been reported to exhibit pro-apoptotic activity [44,45] Following our initial finding that the unique N-terminal part of SRPK1a interacts with SAFB1, we decided to investigate whether this interaction has an effect on the enzyme activity of the kinase In this study, we demonstrate that both kinases (SRPK1a and SRPK1) interact with both scaffold attachment factors (SAFB1 and SAFB2), albeit with different affinities Our in vitro experiments clearly show that this interaction inhibits the activity of the kinases, whereas co-immunoprecipitation and subcellular fractionation analyses suggest that this inhibition also takes place in vivo FEBS Journal 276 (2009) 5212–5227 ª 2009 The Authors Journal compilation ª 2009 FEBS 5213 SRPK1/1a inhibition by interaction with SAFB1/2 D Tsianou et al S-transferase (GST)-fused C-terminal amino acids 600–915 (GST–SAFB1C) [39] (Fig 1A) As shown in Fig 1B, SRPK1a phosphorylates bacterially produced GST–NtLBR (lane 1) and this phosphorylation is inhibited in a dose-responsive manner by the addition of GST–SAFB1C (lanes 2–6) Moreover, the inhibition is specific for SAFB1C because, when GST is added to the assay in quantities equal to those of GST–SAFB1C that completely inhibit the reaction, it does not affect the phosphorylation of GST–NtLBR (lane 7) To verify that this inhibition is valid for more than one substrate, we used P2P-R, a nuclear matrix protein which contains RS motifs This protein has previously been shown to be phophorylated by SRPK1a [37] (and our unpublished observations) As shown in Fig 1C, SRPK1a phosphorylates bacterially Results FLAG–SRPK1a activity is inhibited in vitro by SAFB1 SRPK1a interacts with SAFB1 via its N-terminus In order to find out whether the interaction affects the enzymatic activity of the kinase, we performed phosphorylation assays using as the substrate the N-terminal 205 amino acids of LBR (NtLBR) in the presence of increasing quantities of bacterially expressed SAFB1 protein Because it was practically impossible to obtain soluble recombinant SRPK1a from bacteria, we used as a kinase source immunoprecipitates of FLAG–SRPK1a from transfected HeLa cell extracts [27] The SAFB1 protein used in the assays was the bacterially produced glutathione A B FLAG–SRPK1a + + + + + + + GST–NtLBR FLAG–SRPK1a 66- + + + + + + + GST–SAFB1C (µg) GST–NtLBR 45- 7.5 15 22.5 30 37.5 – – – – – – – 37.5 GST (µg) GST GST–SAFB1C 35- 25- C FLAG–SRPK1a + + + + + + + + + GST–SAFB1C (µg) 7.5 22.5 37.5 – GST (µg) – – – – 37.5 GST–P2P-R + GST–P2P-R FLAG–SRPK1a GST GST–SAFB1C D FLAG–SRPK1a + + + + + FLAG–SRPK1a R0 + + + + + GST–SAFB1C (µg) 7.5 15 37.5 – GST (µg) – – – – 37.5 R0 GST GST–SAFB1C Fig Effect of GST–SAFB1C on FLAG–SRPK1a activity (A) Bacterial preparation of GST–SAFB1C (lane 2) Full-length GST–SAFB1C is indicated by a dot Numbers indicate molecular mass in kDa (lane 1) (B) FLAG–SRPK1a kinase immunoprecipitated from HeLa whole-cell extract was incubated with GST–NtLBR and [32P]ATP[cP] in the presence of GST–SAFB1C or GST (quantities were as indicated on the right), as described in Materials and methods Samples were analysed on 10% SDS ⁄ polyacrylamide gel Proteins were Coomassie stained (left) and labelled proteins were detected by autoradiography (right) (C) As in (B) except that GST–P2P-R(442–585) was used as a substrate (D) As in (B) except that peptide R0 was used as a substrate and the SDS ⁄ polyacrylamide gel was 12% 5214 FEBS Journal 276 (2009) 5212–5227 ª 2009 The Authors Journal compilation ª 2009 FEBS D Tsianou et al expressed GST–P2P-R(442–585), a fragment of the P2P-R protein that contains a RS domain (lane 1) and this phosphorylation is gradually abolished by increasing amounts of GST–SAFB1C (Fig 1C, lanes 2–4) Finally, when the artificial peptide R0 corresponding to the RS-rich LBR amino acid sequence 70–91 was used as substrate [46], the results were similar to the previous two experiments (Fig 1D compare lane with lanes 2–4), indicating that SAFB1 inhibits phosphorylation by affecting the kinase itself and not specific sequences on each of the substrates The RE domain of SAFB1 is not required for the inhibition of FLAG–SRPK1a activity Amino acids 619–699 of SAFB1 comprise its so-called Glu ⁄ Arg region, which is rich in RE dipeptides It has been hypothesized that this region mimics phosphorylated RS dipeptides [47], the structure that the SRPK substrates display after the phosphorylation reaction In this context, we explored the possibility that this region may play a role in the inhibition that SAFB1 exerts on SRPK1a activity To this end, we constructed a plasmid producing a fusion protein lacking the RE-rich region, GST–SAFB1CDRE (amino acids 709–915) (Fig 2A) As shown in Fig 2B, the new fusion protein still inhibits the phosphorylation of GST–NtLBR by SRPK1a (Fig 2B, lanes 2–6) GST– SAFB1CDRE also inhibits SRPK1a activity when P2P-R or the R0 peptide is used as a substrate (data not shown) These results show that the deleted Glu ⁄ Arg region of SAFB1 is not required for inhibition of SRPK1a activity Along this line of thought, we tested SAFB2, the close evolutionary relative of SAFB1 which also contains the corresponding RE domain We constructed the bacterially expressed fusion protein GST–SAFB2C (Fig 2C) harboring amino acids 641–953 of the C-terminal region of SAFB2, which corresponds to the respective sequences of GST–SAFB1C, including the Glu ⁄ Arg region As shown in Fig 2D, GST–SAFB2C is practically unable to inhibit the phosphorylation of GST–NtLBR by SRPK1a (compare lane with lanes 2–5) and the barely detectable inhibition is in quantities of GST– SAFB2C significantly exceeding those of GST– SAFB1C (or GST–SAFB1CDRE) that totally inhibit SRPK1a activity (lanes 5–6) These results were confirmed using P2P-R and the R0 peptide as substrates, as shown in Fig 2E,F These data confirm the inability of the RE-rich region to inhibit the phosphorylating activity of SRPK1a SRPK1/1a inhibition by interaction with SAFB1/2 FLAG–SRPK1 activity is inhibited in vitro by SAFB1 and SAFB2 We next asked whether the inhibition exerted by SAFB1 on SRPK1a activity is because of its interaction with the N-terminal part of the kinase We approached this question indirectly by examining the effect of GST–SAFB1C, GST–SAFB1CDRE and GST–SAFB2C on SRPK1, which in its full-length is 100% identical to the SRPK1a molecule, except for the absence of amino acids 5–174 SRPK1 was expressed, like SRPK1a, as a FLAG-tagged protein in HeLa cells, immunoprecipitated by the M2 monoclonal anti-FLAG IgG and used as such, in in vitro phosphorylation assays, with GST–NtLBR as the substrate As shown in Fig 3A (lane 1, compare with lanes 2–6), SRPK1 activity is inhibited by GST–SAFB1C to a similar extent to the inhibition exerted on SRPK1a Accordingly, GST–SAFB1CDRE also inactivates FLAG–SRPK1 to the same extent as FLAG–SRPK1a (Fig 3B) However, as shown in Fig 3C (compare lane with lanes 2–6), FLAG–SRPK1 activity is also clearly inhibited by GST–SAFB2C, unlike that of FLAG–SRPK1a (Fig 2D) Bacterially expressed GST–SRPK1 activity is inhibited in vitro by SAFB1 and SAFB2 The fact that SAFB1C inhibits both SRPK1a and SRPK1 suggests that it does not bind to SRPK1a only via its unique N-terminal part, but also via other regions common to the two kinases However, because in our assays we always used kinases immunoprecipitated from whole-cell extracts we decided to rule out the possibility that a third protein intervenes in the SRPK1 ⁄ 1a–SAFB1 ⁄ interaction To this end, we prepared bacterially expressed GST–SRPK1 which is relatively easily purified [19], (Fig 4A) and used it in in vitro phosphorylation assays (0.7 lg of GST fusion protein per assay) with GST–Nt-LBR as the substrate and increasing quantities of each of the three fusion proteins GST–SAFB1C, GST–SAFB1CDRE and GST–SAFB2C As shown in Fig 4B, the recombinant kinase was active in phosphorylating GST–NtLBR (lane 1) The results are practically identical to those obtained with the whole-cell extract immunoprecipitated kinase, because when GST–SAFB1C is included in the phosphorylation assay at increasing quantities, phosphorylation is gradually abolished (lanes 2–6) As expected, GST–SRPK1 is inhibited by GST–SAFB1 DRE (Fig 4C) and also by GST–SAFB2C (Fig 4D), as observed in the case of HeLa cell isolated kinase Up FEBS Journal 276 (2009) 5212–5227 ª 2009 The Authors Journal compilation ª 2009 FEBS 5215 SRPK1/1a inhibition by interaction with SAFB1/2 A D Tsianou et al B FLAG–SRPK1a 66- + + + + + GST–NtLBR FLAG–SRPK1a 116- + + + + + + 7.5 15 22.5 30 37.5 GST–SAFB1CΔRE (µg) GST–NtLBR + 45GST–SAFB1CΔRE 35- 25- C D FLAG–SRPK1a FLAG–SRPK1a 66- + + + + + + + + + + 7.5 15 22.5 30 37.5 GST–SAFB2C (µg) GST–NtLBR 45- + GST–NtLBR 116- + GST–SAFB2C 35- 25- E FLAG–SRPK1a + + + + + + GST–SAFB2C (µg) GST–P2P-R + GST–P2P-R FLAG–SRPK1a + 7.5 22.5 37.5 + + GST–SAFB2C F FLAG–SRPK1a + R0 + + + + GST–SAFB2C (µg) FLAG–SRPK1a + 7.5 22.5 37.5 R0 GST–SAFB2C Fig Effect of GST–SAFB1CDRE and GST–SAFB2C on FLAG–SRPK1a activity (A) Bacterial preparation of GST–SAFB1CDRE (lane 2) Fulllength GST–SAFB1CDRE is indicated by a dot Numbers indicate molecular mass markers in kDa (lane 1) (B) FLAG–SRPK1a kinase immunoprecipitated from HeLa whole-cell extracts was incubated with GST–NtLBR and [32P]ATP[cP] in the presence of GST–SAFB1CDRE (quantities were as indicated in the right panel) as described in Materials and methods Samples were analysed on 10% SDS ⁄ polyacrylamide gels Proteins were Coomassie stained (left) and labelled proteins were detected by autoradiography (right) (C) Bacterial preparation of GST–SAFB2C (lane 2) Full-length GST–SAFB2C is indicated by a dot Numbers indicate molecular mass markers in kDa (lane 1) (D) As in (B) except that the indicated quantities of GST–SAFB2C were used (E) As in (D) except that GST–P2P-R(442–585) was used as a substrate (F) As in (D) except that peptide R0 was used as a substrate and the SDS ⁄ polyacrylamide gel was 12% to 500 ng of full-length GST–SRPK1 tested in our experiments was inhibited by the maximal quantity of GST–SAFB1C used in all the assays (data not shown) These results indicate that the inhibition exerted by SAFB1 and SAFB2 on SRPK1 is caused by a direct interaction between each of the two proteins and SRPK1 In an attempt to map the region of this interaction on the kinase, we also produced in bacteria a truncated form of GST–SRPK1, from which amino acids 256 to 475, containing the so-called spacer region 5216 of the kinase, are deleted (GST–SRPK1Dspacer) It has previously been shown that removal of the spacer domain has no effect on catalytic activity but drastically affects the subcellular localization of the kinase [48] Indeed, as shown in Fig 4E, GST–SRPK1Dspacer is able to phosphorylate its substrate, GST–NtLBR, as efficiently as GST–SRPK1 (lanes and 5) In addition, its activity is inhibited by both SAFB1 and SAFB2 (lanes and 7), implying binding of the SAFB proteins to (a) region(s) other than the spacer region FEBS Journal 276 (2009) 5212–5227 ª 2009 The Authors Journal compilation ª 2009 FEBS D Tsianou et al SRPK1/1a inhibition by interaction with SAFB1/2 A FLAG–SRPK1 + + + + + + + + + + + GST–SAFB1C (µg) GST–NtLBR + GST–NtLBR FLAG–SRPK1 7.5 15 22.5 30 37.5 + GST–SAFB1C B FLAG–SRPK1 + + + + + + + + + + GST–SAFB1CΔRE (µg) GST–NtLBR + GST–NtLBR FLAG–SRPK1 7.5 15 22.5 30 37.5 + + + + GST–SAFB1CΔRE C FLAG–SRPK1 FLAG–SRPK1 + GST–NtLBR + + + + + + GST–NtLBR GST–SAFB2C (µg) 7.5 15 22.5 30 37.5 + GST–SAFB2C Fig Effect of GST–SAFB1C, GST–SAFB1CDRE and GST–SAFB2C on FLAG–SRPK1 activity (A) FLAG–SRPK1 kinase immunoprecipitated from HeLa whole-cell extracts was incubated with GST–NtLBR and [32P]ATP[cP] in the presence of GST–SAFB1C (quantities were as indicated in the right panel) as described in Materials and methods Samples were analysed on 10% SDS ⁄ polyacrylamide gels Proteins were Coomassie stained (left) and labelled proteins were detected by autoradiography (right) (B) As in (A) except that the indicated quantities of GST–SAFB1CDRE were used (C) As in (A) except that the indicated quantities of GST–SAFB2C were used Both SRPK1, as well as SRPK1a, bind to both SAFB1 and SAFB2 In order to confirm SAFB1 and SAFB2 binding to the kinases, we performed an affinity chromatography experiment in which we immobilized M2 antibodybound FLAG–SRPK1 or SRPK1a on beads and incubated them with GST–SAFB1C, GST–SAFB1CDRE, GSTSAFB2C and GST bacterial preparations The beads were washed and the eluted proteins were probed with the anti-GST IgG for SAFB protein detection and the anti-FLAG IgG for the immunoprecipitated SRPK1 protein calibration As shown in Fig 5, both SAFB proteins bind clearly and specifically to both kinases (no proteins bind to beads alone: lanes 9, 10, 11 and 12), albeit with different affinities SAFB1 binds tightly to SRPK1a and almost as tightly to SRPK1 (compare lanes and 1) and the same holds true for the GST–SAFB1CDRE protein (lanes and 4), whereas SAFB2 barely binds to SRPK1a (lane 6) but almost as tightly as SAFB1 to SRPK1 (compare lanes and 1) These results confirm the direct interaction of SAFBs with the SRPK1 ⁄ 1a proteins and provide a direct link between the affinity of the SAFB–SRPK1 ⁄ 1a interaction and the extent of the inhibition exerted on kinase activity in each case (see Discussion) Also, both GST–SAFB1 and GST– SAFB2 were able to bind to a FLAG–SRPK1DSpacer fusion protein in a similar experiment, suggesting that the catalytic region of the kinase, comprising amino acids 1–256 and 476–655, interacts with the SAFB proteins (data not shown) In SRPK ⁄ SAFB complexes, able to form in living cells, SRPK activity is inhibited In a following step, we asked whether the corresponding SRPK ⁄ SAFB complexes were able to form in living cells with full-length SAFB proteins, and whether the kinases in these complexes were also repressed HeLa cells were co-transfected with plasmids expressing FLAG, FLAG– SRPK1a or FLAG–SRPK1 together with green fluorescent protein (GFP), GFP–SAFB1 or GFP–SAFB2, lysed and FLAG proteins were immunoprecipitated with the M2 anti-FLAG IgG Kinase assays were performed on the immunoprecipitated kinases using FEBS Journal 276 (2009) 5212–5227 ª 2009 The Authors Journal compilation ª 2009 FEBS 5217 SRPK1/1a inhibition by interaction with SAFB1/2 A D Tsianou et al B 11666- GST–NtLBR GST–NtLBR + + GST–SAFB1C (µg) + + + + + + + + + + GST 7.5 15 22.5 30 37.5 – – – – – – – 37.5 GST 45- + + GST–SRPK1 GST–SRPK1 GST–SAFB1C 35- 25- C GST–SRPK1 GST–NtLBR + + + + + + + + + + 7.5 15 22.5 30 37.5 GST–NtLBR + + GST–SAFB1CΔRE (µg) GST–SRPK1 GST–SAFB1CΔRE D GST–SRPK1 + + + + + + + + + + GST–SAFB2C (µg) GST–NtLBR + GST–NtLBR GST–SRPK1 7.5 15 22.5 30 37.5 + GST–SAFB2C E GST–SRPK1 + + + + – – – – GST–SRPK1Δspacer – – – – + + + + GST–NtLBR + + + + + + + + GST–SAFB1C (µg) – 37.5 – – – 37.5 – – GST–SAFB2C (µg) – – 37.5 – – – 37.5 – GST – – – 37.5 – – – 37.5 Fig Effect of GST–SAFB1C, GST–SAFB1CDRE and GST–SAFB2C on GST–SRPK1 and GST–SRPK1Dspacer activity (A) Bacterial preparation of GST–SRPK1 (lane 2) Full-length GST–SRPK1 is indicated with a dot Numbers indicate molecular mass markers in kDa (lane 1) (B) GST–SRPK1 purified from Escherichia coli whole-cell extract was incubated with GST–NtLBR and [32P]ATP[cP] in the presence of GST– SAFB1C or GST (quantities were as indicated in the right panel) as described in Materials and methods Samples were analysed on 10% SDS ⁄ polyacrylamide gels Proteins were Coomassie stained (left) and labelled proteins were detected by autoradiography (right) (C) As in (B) except that the indicated quantities of GST–SAFB1CDRE were used (D) As in (B) except that the indicated quantities of GST–SAFB2C were used (E) GST–SRPK1 (lanes 1–4) and GST–SRPK1Dspacer (lanes 5-8) purified from E coli whole-cell extract were incubated with GST–NtLBR and [32P]ATP[cP] in the presence of the indicated quantities of GST–SAFB1C, GST–SAFB1C or GST as described in (B) Labelled proteins were detected by autoradiography GST–NtLBR as a substrate In order to monitor the SRPK ⁄ SAFB complex assembly, half of the extract was used to detect the proteins with the anti-FLAG and the anti-GFP IgG As shown in Fig 6A both kinases are active when extracted from cells co-expressing GFP (lanes and 4) However, when SRPK1 is co-expressed with either SAFB1 or SAFB2, its activity is clearly 5218 inhibited (lanes 2, 3) SRPK1a activity is also inhibited by SAFB1 (lane 5), yet inhibition by SAFB2 is much weaker (lane 6) As shown in Fig 6B, both GFP–SAFB1 and GFP–SAFB2 proteins bind to both kinases (left and right panels, lanes 2, and 5, 6), unlike GFP (lanes and 4) which does not bind by itself However, FEBS Journal 276 (2009) 5212–5227 ª 2009 The Authors Journal compilation ª 2009 FEBS D Tsianou et al SRPK1/1a inhibition by interaction with SAFB1/2 Input GST– GST– GST– SAFB1C SAFB1CΔRE SAFB2C GST Anti-GST FLAG–SRPK1a FLAG–SRPK1 FLAG–SRPK1a FLAG–SRPK1 FLAG–SRPK1a FLAG–SRPK1 FLAG–SRPK1a Eluate FLAG–SRPK1 Fig Binding of the GST–SAFB1/2 proteins on immobilized FLAG–SRPK1/1a proteins FLAG–SRPK1 and FLAG–SRPK1a were immunoprecipitated on beads by from HeLa cell extracts and the beads were incubated with GST–SAFB1C, GST– SAFB1CDRE, GST–SAFB2C or GST The eluates were subjected to electrophoresis and proteins were detected using the antiFLAG and the anti-GST IgG as indicated (lanes 1-8) The GST fusion proteins were also incubated with beads alone treated with immunoprecipitates from whole-cell extracts of non-transfected cells (lanes 9–12) (lower) One-thirtieth of the input proteins were subjected to electrophoresis and detected using the anti-GST IgG (upper) Beads Anti-FLAG GST– SAFB1CΔRE GST– SAFB1C A GST GST– SAFB2C Anti-GST GST– GST– GST– SAFB1C SAFB1CΔRE SAFB2C GST + + + + + + + + – – – – – + + + GFP + – – + – – GFP–SAFB1 – + – – + – GFP–SAFB2 – – + – – + 12 – FLAG–SRPK1a 11 + FLAG–SRPK1 Fig The binding effect of full-length SAFB1 and SAFB2 on SRPK1 and SRPK1a kinase activities HeLa cells were co-transfected with plasmids expressing FLAG, FLAG–SRPK1 or FLAG–SRPK1a together with GFP, GFP–SAFB1 or GFP–SAFB2 Whole-cell extracts were immunoprecipitated with an anti-FLAG IgG A) On half of the immunoprecipitated material a kinase assay was performed, as described in Materials and methods Samples from the kinase assay were analysed on SDS ⁄ polyacrylamide gel and labelled proteins were detected by autoradiography (B) The remaining half of the immunoprecipitates were analysed on SDS ⁄ polyacrylamide gel and immunoblotted with an anti-FLAG and an anti-GFP IgG On the same gel, ⁄ 10 of the quantity of the whole-cell extract that was used in each immunoprecipitation assay was subjected to electrophoresis and immunoblotted with an anti-GFP IgG GST–NtLBR 10 B Whole cell extract Whole cell extract GFP GFP GFP– GFP– SAFB1 SAFB2 GFP– GFP– SAFB1 SAFB2 Anti-GFP Eluate FLAG–SRPK1 Eluate FLAG–SRPK1a Anti-FLAG GFP GFP– GFP– SAFB1 SAFB2 GFP GFP– GFP– SAFB1 SAFB2 Anti-GFP although SAFB1 seems to bind to SRPK1 almost as well as to SRPK1a, SAFB2, which is clearly present in the eluate of SRPK1, is barely detectable in the eluate of SRPK1a These results show that SRPK ⁄ SAFB complexes are able to form in living cells and that cell-extracted SAFB-bound kinases are inactive in vitro SRPK ⁄ SAFB complexes are present in the nucleus of HeLa cells Finally, we sought to detect the existence of endogenous SRPK ⁄ SAFB complexes SAFB proteins were immunoprecipitated from whole-cell lysates of exponentially growing HeLa cells and the eluate was probed with the FEBS Journal 276 (2009) 5212–5227 ª 2009 The Authors Journal compilation ª 2009 FEBS 5219 D Tsianou et al In pu t( 20 % ) SRPK1/1a inhibition by interaction with SAFB1/2 A 127 IP: a-SAFB 77 WB: a-SRPK1 Fig A fraction of endogenous SRPK1 ⁄ 1a co-immunoprecipitates with SAFB1 ⁄ Complexes between SAFB and SRPK1 ⁄ 1a proteins were immunoprecipitated from HeLa cell extracts with a monoclonal anti-HET ⁄ SAFB IgG and analysed on 10% SDS ⁄ polyacrylamide gels The proteins were then transferred to nitrocellulose and SRPK1 ⁄ 1a was detected with the monoclonal anti-SRPK1 IgG, recognizing both isoforms (lane 4) No direct immunoprecipitation of SRPKs was observed when an irrelevant monoclonal anti-GFP IgG was used as control (lane 3) A standard amount of cell extract, one-fifth of which is shown (lane 2), was used in each immunoprecipitation assay Molecular mass markers are shown in kDa on the left anti-SRPK1 IgG (the mAbs available not distinguish between SAFB1 ⁄ and SRPK1 ⁄ 1a) An immunoreactive band was detected in the eluate of SAFB co-immunoprecipitated proteins (Fig 7, lane 4), indicating that part of endogenous SRPK1 ⁄ 1a is complexed with SAFB in HeLa cells Approximately 2% of the total SRPK1 ⁄ 1a levels was calculated to co-immunopercipitate with the SAFB proteins (based on the intensinty of the bands on the western blot) In order to determine in which subcellular compartment such complexes may form, we proceeded in subcellular fractionation of HeLa cells and subsequent immunoblotting of the fractions with the anti-SAFB and anti-SRPK1 IgG (Fig 8) As expected, SAFB is detected in the nucleus where it is found mostly in the nuclear matrix ( 80%), whereas SRPK1 ⁄ 1a is detected mainly in the cytoplasm ( 60%) and to a lesser extent in the nucleus ( 40%) Notably, a small but clearly detectable fraction of the kinase ( 10%) is found in the nuclear matrix where the SAFB concentration is high (Fig 8A) Consequently, SRPK ⁄ SAFB-containing complexes may exist in the nuclear matrix as well as in the nucleoplasm When the different fractions were assayed for SR kinase activity using GST–NtLBR as a substrate, phosphorylation was easily detected in the cytoplasmic and nucleoplasmic fractions, but none was detected in the nuclear matrix (Fig 8B), despite the presence of SRPK1 ⁄ 1a molecules in this fraction (Fig 8A) Discussion In this study, we have followed up our initial observation that SRPK1a, the alternatively spliced form of 5220 B Fig Distribution of endogenous SAFB1 ⁄ and SRPK1 ⁄ 1a proteins in HeLa cells, following biochemical fractionation (A) The distribution of SAFB1 ⁄ and SRPK1 ⁄ 1a proteins between the various fractions was analysed by immunoblotting using a mouse monoclonal anti-HET/SAFB and a monoclonal anti-SRPK1 IgG respectively (the available antibodies not distinguish between SAFB1 ⁄ and SRPK1 ⁄ 1a, respectively; see Materials and methods for the analytical fractionation protocol) (B) The different fractions were assayed for RS kinase activity, using bacterially produced GST–NtLBR as substrate The samples were analysed by SDS ⁄ PAGE and autoradiographed The radioactive bands corresponding to labelled GST–NtLBR from were excised, and the radioactivity was determined by Cerenkov counting RS kinase activity of the different fractions is expressed as total units (%) SRPK1, interacts with the nuclear matrix protein SAFB1 via its unique additional N-teminal domain In our pursuit of a biological consequence of this interaction, we examined the activity of SRPK1a in the presence of SAFB1 and showed that the kinase is inhibited by this factor in vitro In our assays, we used the C-terminal region of SAFB1 because it includes the area found to interact with SRPK1a (amino acids 585–720) [27] Inhibition was evident when SRPK1a activity was tested on three different substrates (LBR, P2P-R and a RS domain-containing synthetic peptide) eliminating the possibility that SAFB1 interferes with different domains in each substrate However, relatively large quantities ( 20 lg) of our total bacterial GST–SAFB1C preparation were needed to eliminate phosphorylation of the substrates We cannot be FEBS Journal 276 (2009) 5212–5227 ª 2009 The Authors Journal compilation ª 2009 FEBS D Tsianou et al certain whether this is because only the full-length SAFB1C, which is a relatively small fraction in the total population of the GST-purified peptide, inhibits the kinase or because SAFB1 homopolymerizes via its RE-rich region [38,42] (and our unpublished observations), large quantities are needed to have sufficient SAFB1 monomers available to form complexes with the kinase under the conditions of the in vitro phosphorylation assays The Glu ⁄ Arg-rich region is a candidate for repression of the kinase activity because of its particular structure, which resembles a phosphorylated RS domain RS domains are known to be protein–protein and protein–RNA interaction surfaces and the phosphorylation of serines affects those interactions as well as interactions between RS domain-containing proteins [49,50] Very recently the core complex of SRPK1 with one of its substrates, the spliceosome factor ASF ⁄ SF2, has been crystallized [51], revealing important information and confirming previous observations [52–54] about the significance of enzyme–substrate contacts for catalysis This study highlights the importance of the interaction of a phosphoserine of the RS domain of the substrate with the catalytic region of the kinase, on the one hand, and of the positive charge of the RS domain with the negatively charged ‘docking groove’ of SRPK1, on the other hand The RE-rich region of SAFB1 may interfere with any of these processes by mimicking a phosphorylated RS domain, thus disturbing the catalytic activity of the kinase However, when we deleted the sequence rich in RE dipeptides, the remaining SAFB1 sequences (709–915) still inhibited SRPK1a activity, implying that some particular structural element or configuration in this region should be responsible for the observed effect At this point, it should be noted that among the SAFB1 sequences contained in the GST–SAFB1CDRE fusion protein, several RE dipeptides are scattered so that we cannot exclude the possibility that inhibition is exerted by these sequences, particularly because the pI of the remaining SAFB1 sequence in the GST–SAFB1CDRE peptide is still basic Otherwise, it could be a combination of two effects, where both specific structural elements and the scattered REs would contribute to the observed inhibition We were intrigued to find that, although SAFB1 interacts with the unique N-terminal part of SRPK1a, it also inhibits the activity of SRPK1 which lacks this unique part We excluded the possible involvement of a cellular protein in our in vitro assays by using bacterially purified GST-fused SRPK1 and confirmed that both SAFB1 and SAFB2 repress its activity Affinity chromatography experiments confirmed that SAFB1 SRPK1/1a inhibition by interaction with SAFB1/2 and SAFB2 bind to SRPK1, which suggests that there exists a domain on the kinase recognized by each of the two factors We excluded the possibility that such a domain is located in the spacer region of the kinase because both SAFB proteins still bind on a kinase molecule from which the spacer domain is deleted and they still inhibit its enzymatic activity Thus, the inhibition mechanism involves binding of the SAFB molecules to the catalytic domain of SRPK1 Furthermore, SAFB1 also binds to SRPK1a (as previously shown), but SAFB2 barely does We obtained the same qualitative results concerning the relative affinities of the four proteins when we co-immunoprecipitated the kinases with SAFB proteins from HeLa cells This result is not very easy to explain for the pair SRPK1a– SAFB2 Because SAFB2 interacts with SRPK1, it must recognize and bind to a specific region on it, which is evidently also contained in SRPK1a However, SAFB2 binds only weakly to SRPK1a One should then accept that either SAFB1 and SAFB2 have two different, though almost equal in strength, types of interaction with SRPK1 that are differentiated in SRPK1a because of the N-terminal domain, or that even if their interaction with SRPK1 is similar, the N-terminal domain has a stabilizing effect on SAFB1, but a destabilizing effect on the interaction with SAFB2 In any case, additional dissection of the SAFB and SRPK1 molecules is required to determine the regions responsible for their interactions It should be pointed out, however, that this study is the first to reveal functional differences between SAFB1 and SAFB2 Although at this stage in our study our results can only be qualitative, we have noticed that the inhibiting activity exerted by SAFB proteins on the kinases, is closely related to the affinity with which they interact in affinity chromatography and co-immunoprecipitation experiments Although this is not unexpected, it is indicative of the importance of SRPK ⁄ SAFB intracellular complexing We were able to demonstrate the existence of SRPK ⁄ SAFB complexes in HeLa cells, estimating the percentage of total cellular SRPK1 ⁄ 1a molecules occupied in these complexes to be  2% Because the antibodies used cannot distinguish between SRPK1 and SRPK1a, or between SAFB1 and SAFB2, we not know the exact composition of the detected SRPK ⁄ SAFB complexes However, using overexpressed proteins in HeLa cells we demonstrated that all four complexes, SRPK1a–SAFB1, SRPK1–SAFB1, SRPK1–SAFB2 and SRPK1a– SAFB2 (listed by relative order of affinity) can be formed in living cells by the full-length proteins Moreover, SRPK1 ⁄ 1a molecules extracted from the FEBS Journal 276 (2009) 5212–5227 ª 2009 The Authors Journal compilation ª 2009 FEBS 5221 SRPK1/1a inhibition by interaction with SAFB1/2 D Tsianou et al lysates of such cells were shown to be inactivated to an extent that depended on their binding affinities to their SAFB partner The results of our subcellular fractionation in HeLa cells show that SAFB molecules are localized exclusively in the nucleus, as previously described [30,38,55],  80% of them residing in the nuclear matrix Several previous studies examined the subnuclear partitioning of SAFB It is known and generally accepted to be a scaffold ⁄ matrix attachment region binding protein, it was found in nuclear speckles [30], it has been shown to change subnuclear localization in response to heat shock from perichromatin fibrils to nuclear structures that not correspond to nuclear speckles [31,55] and also to migrate to the nucleolus in response to early apoptotic signals [45] Sergeant et al [38] demonstrated that in HEK293 and HeLa cells, SAFB2 is found in large stable multiprotein complexes that also contain a fraction of SAFB1, whereas SAFB1 is also found in its monomeric form Proteins related to RNA processing were mostly found in these stable complexes Yet, many proteins known to interact with SAFB1 ⁄ 2, such as Trab2 [56], estrogen receptor a and the C-terminal domain of RNA polymerase II, were not detected, and neither was DNA This observation leads to the assumption that such interactions may be transient and some may take place under particular conditions or in certain cell types or cell-cycle phases Likewise, the intracellular localization of SR kinases has always been a point of discussion because ordinarily only a fraction of SRPK1 is found in the nucleus and additional molecules move into the nucleus in response to cell-cycle signals [48,57] Within the nucleus, SRPK1 is localized in nuclear speckles, which are nuclear substructures believed to play a role in coupling transcription and pre-mRNA splicing The composition of such structures is thought to be relatively ‘fluid’, changing in response to metabolic and environmental signals, whereas individual components shuttle between them and active gene loci [58] Proteomic analysis, using MS, has identified 146 proteins including SAFB1, many of its already known interacting partners and as expected, many SRPK1 substrates [59] Our subfractionation experiment in HeLa cells has shown that SRPK1 ⁄ 1a is distributed at a ratio of : between the cytoplasm and the nucleus and a further subnuclear partitioning of : between the nucleoplasm and the nuclear matrix It is evident that the SRPK ⁄ SAFB complexes could form in the nucleoplasm or the nuclear matrix, or both However, it was very interesting to find that, whereas the SRPK1 ⁄ 1a substrate NtLBR was easily phosphorylated by the cytoplasmic and nucleoplasmic fractions, it was not 5222 phosphorylated by the nuclear matrix fraction, despite 10% of the total SRPK1 ⁄ 1a molecules being detected in this fraction, implying that the inhibitory factor associated with the nuclear matrix may be SAFB1 ⁄ The higher activity detected in the nucleoplasmic fraction compared with the cytoplasmic fraction (which otherwise contains more SRPK1 ⁄ 1a) may be caused by other kinases phosphorylating NtLBR, but most importantly to inhibitors already described as being in the cytoplasm that inhibit SRPK1 [7] We propose that the SAFB ⁄ SRPK complexes we detected are transiently formed in cells, most likely under specific conditions It is tempting to suppose that the transient interaction of a SR kinase with a SAFB protein under certain cellular conditions leads to temporary inhibition of phosphorylation, influencing the processes of mRNA splicing or gene transcription (or even another RS domain-dependent function) via cross-talk between the SRPK and SAFB molecules For example, it is well established that for correct splicing to occur, phosphorylation and sequential dephosphorylation of SR proteins must take place [2,10,60] SRPKs, which are responsible for the phosphorylation of RS splicing factors, might, in this case, be temporarily inhibited during the action of phosphatases on the phosphorylated RS proteins SAFB1 has been proposed to act as a structural platform where transcription and pre-mRNA processing components assemble close to scaffold ⁄ matrix attachment region elements [30] SRPK1 or SRPK1a molecules could be transient components of such structures, depending on cellular activities The notion of a nucleus where proteins and RNA rapidly diffuse in and out of complexes that form transiently, has emerged as a result of multiple experimental data that also underline the physiological importance of such intranuclear trafficking and aggregate formation [61] Kinase inhibition might take place in vivo depending on the specific protein milieu of the complex in which the SRPK ⁄ SAFB pair resides and it could be finetuned by the specific composition of the transiently formed complexes Each partner of the SAFB ⁄ SRPK complex pair could be implicated in specific cellular processes to varying degrees These could be also cell type specific as, for example, in Sertoli cells where SAFB2 (but not SAFB1) is expressed [38] Neither the functional differences between SRPK1 and SRPK1a, nor those between SAFB1 and SAFB2 are clear at present, but the differential pairing between them, resulting in differentially regulated SR kinase function, should reflect important fine tuned cellular mechanisms in vivo Elucidation of the regulation and physiological properties of SRPK ⁄ SAFB complex formation in vivo FEBS Journal 276 (2009) 5212–5227 ª 2009 The Authors Journal compilation ª 2009 FEBS D Tsianou et al will unravel important information concerning nuclear and cellular function Materials and methods Plasmids pGEX-4T1 ⁄ SAFB1CDRE The cDNA fragment corresponding to SAFB1CDRE (amino acids 709–915) was amplified by PCR from plasmid pGEX2TK ⁄ SAFB1C, using as primers: forward 5¢-TTTGGATCC GCGGTGCGGCGGC-3¢, containing the underlined BamHI site and reverse: 5¢-TTGAATTCTCAGTAGCGGCGAGT GAA-3¢, containing the underlined EcoRI site The PCR fragment was digested with BamHI and EcoRI, repurified and subcloned into the BamHI and EcoRI sites of the bacterial expression vector pGEX-4T1 (Amersham Biosciences, Piscataway, NJ, USA) pGEX-4T1 ⁄ SAFB2C The cDNA fragment corresponding to SAFB2C (amino acids 641–953) was amplified by PCR from plasmid pEGFP– SAFB2 using as primers: forward 5¢-TTTGGATCCGAG CGCGAGCAGCGGG-3¢, containing the underlined BamHI site and reverse: 5¢-TTGAATTCTTAGTAGCGGCGGGT GAA-3¢, containing the underlined EcoRI site The PCR fragment was digested with BamHI and EcoRI, repurified and subcloned into the BamHI and EcoRI sites of the bacterial expression vector pGEX-4T1 pGEX-2T ⁄ P2P-R The cDNA fragment of P2P-R coding for amino acids 442– 585 was excised from plasmid pCMV–FLAGTM–24 ⁄ P2P-R, a gift of RE Scott (University of Tennessee Health Science Center, Memphis, TN, USA), with EcoRI and SalI and subcloned into the EcoRI and SalI sites of the bacterial expression vector pGEX-2T pGEX-2T ⁄ SRPK1Dspacer Spacer-deleted SRPK1 (lacking amino acids 256-475) was generated by ligating the cDNAs coding for the N- and C-terminus of human SRPK1 The cDNA coding for the N-terminus was amplified by PCR from plasmid pGEX2T ⁄ SRPK1, using as primers: forward: 5¢-GCGTGGATC CATGGAGCGGAAAGTGCTTGCG-3¢, containing the underlined BamHI site and reverse: 5¢-TCCCCCGGGAG CAGTACTGACTGCAGATCC-3¢, containing the underlined SmaI site The cDNA coding for the C-terminus was also amplified by PCR from plasmid pGEX-2T ⁄ SRPK1, using as primers: forward: 5¢-TCCCCCGGGAATTTTCT TGTTATTCCCCTTGAG-3¢, containing the underlined SRPK1/1a inhibition by interaction with SAFB1/2 SmaI site and reverse: 5¢-CCGAGGAATTCGGAGTTAA GCCAAGGGTGCCG-3¢, containing the underlined EcoRI site The PCR fragments were digested with BamHI ⁄ SmaI and SmaI ⁄ EcoRI, respectively, repurified and subcloned into the BamHI and EcoRI sites of the bacterial expression vector pGEX-2T The following plasmids have been previously described: pGEX-2TK ⁄ SAFB1C [39], pGFP3–SAFB1 [38], pGFP3– SAFB2 [38], pFLAG–CMV-2 ⁄ SRPK1a [27], pFLAG– CMV-2 ⁄ SRPK1 [27], pGEX-2T ⁄ SRPK1 [21], pGEX-2T ⁄ NtLBR [46] Cell cultures and transfections HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco BRL, Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Biochrom KG Seromed, Berlin, Germany) and antibiotic–antimycotic solution (Gibco BRL) Cells ( 60% confluent) were transfected with 10 lg of DNA (equal quantities of each plasmid when more than one plasmid were used) in 10 cm dishes using the TranspassÔ D2 Transfection Reagent (New England Biolabs, Ipswish, MA, USA) and incubated for 24 h in Dulbecco’s modified Eagle’s medium at 37°C in a 5% CO2 ⁄ 95% air incubator SDS ⁄ PAGE and western analysis Protein samples were resolved by 10 or 12% SDS ⁄ PAGE and analysed by Coomassie Brilliant Blue staining or western blotting using a monoclonal anti-(FLAG M2) mouse IgG (1 : 10 000; Sigma-Aldrich, St Louis, MO, USA) or a monoclonal anti-(HET ⁄ SAFB) mouse IgG (1 : 2000; Upstate Biotechnology, Lake Placid, NY, USA) or a monoclonal anti-GST goat IgG (1 : 5000, Amersham Biosciences) or a monoclonal anti-SRPK1 mouse IgG (1 : 1000; BD Transduction Laboratories, Lexington, KY, USA), or a rabbit polyclonal anti-GFP serum (1 : 3000), a gift of H Boleti (Hellenic Pasteur Institute, Athens, Greece) Membranes were then incubated with horseradish peroxidase- or alkaline phosphatase-conjugated goat anti-mouse IgG (1 : 3000; Bio-Rad Laboratories, Hercules, CA, USA) or horseradish peroxidase–mouse anti-(goat IgG) (1 : 3000; Jackson Immunoresearch, Baltimore Pike, PA, USA) or horseradish peroxidase–goat anti-(rabbit IgG) (1 : 3000, Cell Signaling, Beverly, MA, USA) antibodies Detection of the immunoreactive bands was performed using ECL (Amersham Biosciences) or the 5-bromo-4-chloro-3-indolyl phosphate ⁄ nitro blue tetrazolium substrate Protein expression and purification Expression of the fusion proteins GST–SAFB1C, GST– SAFB1CDREand GST–SAFB2C was induced with mm FEBS Journal 276 (2009) 5212–5227 ª 2009 The Authors Journal compilation ª 2009 FEBS 5223 SRPK1/1a inhibition by interaction with SAFB1/2 D Tsianou et al isopropyl thio-b-d-galactoside (Fermentas International Inc., Ontario, Canada) at 24 °C for h, whereas expression of GST–NtLBR, GST–SRPK1a, GST–SRPK1, GST– SRPK1Dspacer and GST–P2P-R(442-585) was induced with 0.2 mm isopropyl thio-b-d-galactoside at 28 °C for h Cells were suspended in ice-cold buffer [1 · NaCl ⁄ Pi, 1% Triton-X, mm phenylmethanesufonyl fluoride, protease inhibitors cocktail (Roche Diagnostics, Manheim, Germany)], the cell suspension was sonicated and insoluble debris was pelleted by centrifugation (12 000 g for 15 at °C) The supernatant was mixed with glutathione Sepharose beads (Amersham Biosciences) at °C for 30 and bound proteins were eluted with glutathione elution buffer (50 mm Tris ⁄ HCl pH 8.0, 10 mm glutathione) The purity and concentration of GST–fusion proteins was determined using SDS ⁄ PAGE followed by Coomassie Brilliant Blue staining and the Bradford method glycerol, 0.1% Triton X-100, 0.5 mm phenylmethanesufonyl fluoride) and bound proteins were eluted in SDS sample buffer The same procedure was followed for the co-immunoprecipitation of endogenous SRPK and SAFB from HeLa cell extracts Affinity chromatography FLAG–SRPK1a or FLAG–SRPK1 immobilized on protein G beads was incubated with  100 lg of purified GST–SAFB1C, GST–SAFB1CDRE, GST–SAFB2C or GST in cold TNMT buffer (25 mm Tris ⁄ HCl pH 7.5, 150 mm NaCl, 0.1% Triton X-100, mm phenylmethanesufonyl fluoride) After h of incubation at °C, the beads were washed three times with TNMT buffer The bound proteins were eluted in SDS sample buffer, analysed by SDS ⁄ PAGE and visualized by western blotting using the relevant antibody Immunoprecipitation For the immunoprecipitation of the FLAG–SRPK1a and FLAG–SRPK1 kinases from HeLa cells, extracts of cells transfected with pFLAG–CMV-2 ⁄ SRPK1a or pFLAG– CMV-2 ⁄ SRPK1 were lysed with mL lysis buffer (50 mm Tris ⁄ HCl pH 7.5, 150 mm NaCl, 1% Triton X-100, mm phenylmethanesufonyl fluoride, mm dithiothreitol) for 30 on ice and centrifuged for 30 at 12 000 g One hundred micrograms of FLAG–SRPK1a or FLAG–SRPK1 overexpressing cell extract were incubated with 0.3 lL of the M2 monoclonal anti-FLAG IgG in immunoprecipitation buffer (25 mm Tris ⁄ HCl pH 7.5, 150 mm NaCl, 1% Triton X-100, mm phenylmethanesufonyl fluoride, mm dithiothreitol) for h at °C Twenty microliters of protein G beads were added and incubated at °C overnight Antigen– antibody complexes were collected by centrifugation and washed three times with immunoprecipitation buffer For co-immunoprecipitation of the kinase with the SAFB proteins, extracts of HeLa cells transfected with equal quantities of plasmids expressing FLAG, FLAG–SRPK1a or FLAG–SRPK1 and GFP, GFP–SAFB1 or GFP–SAFB2 were lysed 24 h after transfection with 200 lL RIPA buffer (20 mm Tris ⁄ HCl pH 7.4, 150 mm NaCl, 0.5% Nonident P-40, 0.5% NaDoc, 0.1% SDS, mm EDTA, 0.5 mm phenylmethanesufonyl fluoride) for 30 on ice The cell suspension was then diluted in 800 lL RIPA-rescue buffer (10 mm Na-phosphate pH 7.2, 20 mm NaCl, 0.5 mm phenylmethanesufonyl fluoride), centrifuged at 12 000 g for 30 at °C and quantified by the Bio-Rad Protein assay Dye Reagent (Bio-Rad Laboratories, Hercules, CA, USA) Samples were incubated with 0.6 lg mouse monoclonal anti-(FLAG M2) IgG (Sigma) for h at °C Twenty microliters of protein G beads (Sigma-Aldrich) were added and incubated at °C overnight Beads were collected by centrifugation, washed three times with cold HNTG buffer (50 mm Hepes pH 7.5, 150 mm NaCl, mm EDTA, 10% 5224 Cell fractionation Cell fractionation was based on a combination of the protocols described by Dignam et al [62] and Jiang et al [63] Approximately · 106 HeLa cells were harvested, washed in NaCl ⁄ Pi, resuspended in 500 lL of ice-cold buffer A (10 mm Hepes ⁄ KOH pH 7.5, 10 mm KCl, 1.5 mm MgCl2, 0.5 mm dithiothreitol) and allowed to stand for 10 at °C The cells were then collected by centrifugation, suspended in 200 lL of buffer A and lysed by 10 strokes of a glass Dounce homogenizer The homogenate was centrifuged for 10 at 5000 g and the supernatant was collected as soluble cytoplasm fraction The pellet was resuspended in 100 lL buffer A and laid onto a 1.2 mL cushion consisting of 0.8 m sucrose in buffer A After centrifugation at 5000 g for 10 min, the pellet (purified nuclei) was collected, washed twice with NaCl ⁄ Pi and extracted with extraction buffer consisting of 10 mm Pipes pH 6.8, 250 mm ammonium sulfate, 300 mm sucrose, mm MgCl2 and mm phenylmethanesufonyl fluoride The supernatant was collected as a soluble nucleoplasmic fraction, whereas the pellets were then digested with RNAse-free DNAse I (7.65 unitsỈlL)1) in digestion buffer (10 mm Pipes pH 6.8, 300 mm sucrose, 50 mm NaCl, mm MgCl2, 0.5% TritonX 100 and mm phenylmethanesufonyl fluoride) at 32 °C for 60 and centrifuged at 4300 g for The pellets (nuclear matrix fraction) were washed twice with extraction buffer and resuspended in extraction buffer Gel loading was adjusted to give equivalent cell numbers in each lane In vitro kinase assay Protein G beads, collected by centrifugation, as described above, with FLAG–SRPK1a or FLAG–SRPK1 immunoprecipitated from HeLa cells or 50 ng of full-length FEBS Journal 276 (2009) 5212–5227 ª 2009 The Authors Journal compilation ª 2009 FEBS D Tsianou et al Coomassie-quantified GST–SRPK1 [21] (corresponding to 0.7 lg of Bradford measured GST purified protein) were incubated with lg GST–NtLBR or lg GST–P2P-R or 10 lg synthetic peptide R0 (70SSPSRRSRSRSRSRSPGRPAKG91) [19] and in vitro phosphorylation assays were carried out as described previously [46] Samples were incubated in a total volume of 25 lL containing 25 mm Tris ⁄ HCl pH 7.5, 10 mm MgCl2, 100 mm NaCl, 50 lm [32P]ATP[cP] (Amersham, Bacacos SA, Greece) for 20 at 30 °C and the reaction was stopped by adding SDS buffer and heating at 95 °C for All samples were analysed by 10 or 12% SDS ⁄ polyacrylamide gel followed by Coomassie Brilliant Blue staining and labelled proteins were detected by autoradiography Incorporation of radioactivity was measured by excising the respective radioactive bands from an SDS ⁄ PAGE gel and scintillation counting RS kinase activity is expressed as total units (%) SRPK1/1a inhibition by interaction with SAFB1/2 Acknowledgements We wish to thank Dr S Oesterreich (Baylor College of Medicine and Methodist Hospital, Houston, TX, USA) for kindly providing us with plasmid pGEX2TK ⁄ SAFB1C, Dr D.J Elliot (University of Newcastle, UK) for plasmids pGFP3–SAFB1 and pGFP3–SAFB2, Dr H Boleti (Hellenic Pasteur Institute, Athens, Greece) for the rabbit anti-GFP serum and Dr R.E Scott (University of Tennessee Health Science Center, Memphis, TN, USA) for plasmid pCMV–FLAGÔ-24 ⁄ P2P-R We are also thankful to Dr G Simos and Dr I Mylonis for helpful discussions and comments on the manuscript This work was supported by a grant from the Greek Ministry of National Education and Religious affairs (IRAKLEITOS in the context of the E.U.-funded EPEAEK 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GST– GST– SAFB1C SAFB1CΔRE SAFB2C GST Anti-GST FLAG–SRPK1a FLAG–SRPK1 FLAG–SRPK1a FLAG–SRPK1 FLAG–SRPK1a FLAG–SRPK1 FLAG–SRPK1a Eluate FLAG–SRPK1 Fig Binding of the GST–SAFB1 /2 proteins on immobilized... 27 6 (20 09) 5 21 2 – 522 7 ª 20 09 The Authors Journal compilation ª 20 09 FEBS 5 21 5 SRPK1 /1a inhibition by interaction with SAFB1 /2 A D Tsianou et al B FLAG–SRPK1a 66- + + + + + GST–NtLBR FLAG–SRPK1a