Phosphorylation-dependent binding of human transcription factor MOK2 to lamin A⁄C ` Maryannick Harper, Jeanne Tillit, Michel Kress and Michele Ernoult-Lange ´ CNRS-FRE2937, Institut Andre Lwoff, Villejuif, France Keywords Aurora A; JLP; JNK3; JSAP1; MOK2 Correspondence M Ernoult-Lange, CNRS-FRE2937, Institut ˆ ´ Andre Lwoff, rue Guy Moquet, 94801 Villejuif, France Fax: +33 49 58 33 43 Tel: +33 49 58 33 46 E-mail: ernoult@vjf.cnrs.fr (Received 15 December 2008, revised March 2009, accepted 31 March 2009) doi:10.1111/j.1742-4658.2009.07032.x Human MOK2 is a DNA-binding transcriptional repressor Previously, we identified nuclear lamin A ⁄ C proteins as protein partners of hsMOK2 Furthermore, we found that a fraction of hsMOK2 protein was associated with the nuclear matrix We therefore suggested that hsMOK2 interactions with lamin A ⁄ C and the nuclear matrix may be important for its ability to repress transcription In this study, we identify JNK-associated leucine zipper and JSAP1 scaffold proteins, two members of c-Jun N-terminal kinase (JNK)-interacting proteins family as partners of hsMOK2 Because these results suggested that hsMOK2 could be phosphorylated, we investigated the phosphorylation status of hsMOK2 We identified Ser38 and Ser129 of hsMOK2 as phosphorylation sites of JNK3 kinase, and Ser46 as a phosphorylation site of Aurora A and protein kinase A These three serine residues are located in the lamin A ⁄ C-binding domain Interestingly, we were able to demonstrate that the phosphorylation of hsMOK2 interfered with its ability to bind lamin A ⁄ C The zinc-finger transcription factor MOK2 recognizes both DNA and RNA through its zinc-finger motifs [1] This dual affinity suggests that MOK2 may play a role in transcription, as well as in the post-transcriptional regulation of specific genes We have shown that MOK2 represses expression of the interphotoreceptor retinoid-binding protein (IRBP) gene [2] IRBP contains two MOK2-binding elements, a complete 18-bp MOK2-binding site, located in intron 2, and the essential 8-bp core MOK2-binding site (corresponding to the conserved 3¢-half site) which is in the IRBP promoter MOK2 can bind to the 8-bp sequence in the IRBP promoter and repress transcription from this promoter In the IRBP promoter, the TAAAGGCT MOK2-binding site overlaps with the photoreceptorspecific Crx-binding element, suggesting that MOK2 represses transcription by competing with the cone–rod homeobox protein for DNA binding and decreasing transcriptional activation by the cone–rod homeobox protein The particular arrangement of the two MOK2-binding sites, observed in the human IRBP gene and also in a second human potential MOK2 target gene, Pax3, suggests that MOK2 may repress transcription via a dual mechanism Previously, we identified lamin A ⁄ C proteins as binding partners for hsMOK2 in a yeast two-hybrid screen [3] A-type lamins have been shown to bind hsMOK2 in vitro and in vivo through the coil domain common to lamin A and lamin C, whereas the lamin A ⁄ C-binding site in hsMOK2 has been mapped to its N-terminal acidic domain Divergent evolution has been observed between human and mouse MOK2 genes which results in the loss of this NH2-domain in the mouse gene [4] An in silico search of MOK2 genes in different species has shown that the lamin-binding site is present only in primate MOK2 proteins Furthermore, we have found that a fraction of human hsMOK2 protein is associated with the nuclear matrix We therefore suggested that hsMOK2 interactions with lamin A ⁄ C and the nuclear matrix might be important for its Abbreviations GST, glutathione S-transferase; IRBP, interphotoreceptor retinoid-binding protein; JIP, JNK-interacting proteins; JLP, JNK-associated leucine zipper; JNK, c-Jun N-terminal kinase; PKA, protein kinase A FEBS Journal 276 (2009) 3137–3147 ª 2009 The Authors Journal compilation ª 2009 FEBS 3137 Phosphorylation-dependent binding of MOK2 M Harper et al ability to repress transcription Lamins A and C are the major products of the LMNA gene which is expressed in most differentiated cells [5,6] Mutations in the LMNA gene have been shown to cause a variety of inherited human diseases (i.e laminopathies) We investigated whether missense mutations located in the coil domain of lamin A ⁄ C could affect the interaction with hsMOK2 [7] Our results showed that none of the tested mutations was able to disrupt binding to hsMOK2 in vitro or in vivo However, we observed an aberrant cellular localization of hsMOK2 into nuclear aggregates induced by pathogenic lamin A and C mutant proteins These results indicated that pathogenic mutations in lamin A ⁄ C lead to sequestration of hsMOK2 into nuclear aggregates, which may deregulate MOK2 target genes In this study, we identify two new partners of hsMOK2, which belong to the c-Jun N-terminal kinase (JNK)-interacting proteins (JIP) family The JIP family regulates both the JNK and P38 kinase cascade [8–10] We therefore investigated the phosphorylation status of hsMOK2 and identified two JNK3 phosphorylation sites Furthermore, we also identified an Aurora A ⁄ protein kinase A (PKA) phosphorylation site on hsMOK2 Interestingly, using phosphomimetic substitution, we determined that phosphorylation at this site interferes with the ability of hsMOK2 to bind lamin A ⁄ C Results and Discussion hsMOK2 interacts with JNK-associated leucine zipper and JSAP1 To identify partners of hsMOK2 that might be involved in regulating hsMOK2 functions, we performed a two-hybrid yeast screen, as described previously [3] One of the clones corresponded to the N-terminal region of JNK-associated leucine zipper (JLP) protein (amino acids 1–141), which is the most recently identified member of the JIP group of scaffold proteins [11] To determine which region of hsMOK2 interacts with JLP, we co-transformed the yeast strain L40 with the library pGAD–JLP 1–141 vector and pLex containing either the nonfinger acidic domain (pLex–NH2) or the finger domain (pLex–finger) of hsMOK2, and performed b-galactosidase assays The JLP 1–141 domain interacted only with the finger domain of hsMOK2 (Fig 1A) No interaction was found with the NH2-acidic domain of hsMOK2 To corroborate the two-hybrid results and test for a direct interaction between JLP and hsMOK2, the JLP 1–141 domain was expressed as a glutathione S-transferase (GST)–fusion protein in bacteria The 3138 A Bait Prey β–Gal pLexA LexA pGAD–JLP (1–141) – pGAD–GH – pLex–hsMOK2 LexA NH2 Finger pLex–hsMOK2 LexA NH2 Finger pGAD–JLP (1–141) +++ pLex–NH2 LexA NH2 pGAD–GH +/– pGAD–JLP (1–141) +/– pGAD–GH – pLex–NH2 LexA NH2 pLex–Finger LexA Finger pLex–Finger LexA Finger pGAD–JLP (1–141) +++ B Fig Identification of JLP as a partner of hsMOK2 using the yeast two-hybrid screen and identification of the hsMOK2 interaction domain (A) Constructs expressing full-length or the indicated domains of hsMOK2 and the human polypeptide JLP 1–141 were co-transformed into yeast The specificity of the interaction between bait and prey was determined by estimating the degree of color development after 90 of incubation in the filter lift b-galactosidase assay, as described in Materials and methods (+++) High color blue development, (+ ⁄ )) very low color blue development, ()) no color development (B) Amino acid sequence alignment of N-terminal of JLP 1–141 and JSAP1 1–146 GST–JLP 1–141 protein was purified, immobilized on glutathione–agarose beads and incubated with nuclear extracts from HeLa cells transfected with full-length hsMOK2 Consistent with the results obtained in the yeast two-hybrid analysis, it was found that this N-terminal region of JLP protein (amino acids 1–141) bound to hsMOK2 (Fig 2A) To further define the region required for interaction with hsMOK2, we constructed a deletion series by removing N- and C-terminal amino acids residues Similar amounts of different GST proteins were used in the binding assay As shown in Fig 2A and summarized in Fig 2B, hsMOK2 was bound by GST–JLP 1–101 and GST– JLP 21–101 deletion mutants at levels similar to those FEBS Journal 276 (2009) 3137–3147 ª 2009 The Authors Journal compilation ª 2009 FEBS M Harper et al Phosphorylation-dependent binding of MOK2 A D B E C Fig The N-terminal domains of JLP and JSAP1 bind to hsMOK2 in vitro (A) Mapping the interaction region of JLP using GST pull-down analysis Various JLP N-terminal regions and the homologous JSAP1 region were tested for their interaction with hsMOK2 Nuclear extracts (20 lg) from HeLa cells transfected with the expression vector hsMOK2 were incubated with an equal amount (10 lg) of recombinant GST fusion proteins bound to glutathione beads After washing the beads thoroughly, the bound proteins were eluted in SDS sample buffer, resolved by SDS ⁄ PAGE and immunoblotted with an affinity purified anti-hsMOK2 serum The proteins were visualized by exposing the blots to CL-Xposure film (Pierce) (B) Structure of the JLP deletion mutants The amino acid number of the encoded proteins is indicated for each construct Interactions observed in (A) are summarized on the right (C) Comparison of the predicted coiled-coil structure of JSAP1 6-146 domain (black) with wild-type (red) and mutant D68N (green) and F65L ⁄ D68N (blue) JLP 1–141 domains The graphs of coiled-coil scores were determined using the PAIRCOIL program [14] (x-axis) Residue number (y-axis) Probability of a coiled-coil formation (D) GST pull-down by JSAP1 21–101 domain and wild-type or mutant of JLP 26–106 domains was performed as described in (A) (E) Bound proteins were visualized with Fluor-S Max MultiImager and quantified with QUANTITY ONE software (Bio-Rad) Results were expressed as a percentage of binding to JSAP1 (21–101) domain (mean ± SD of three different experiments) with the GST–JLP 1–141 protein Furthermore, deletion of amino acids 71–101 strongly reduced interaction with hsMOK2, and GST–JLP 66–141 and GST–JLP 66–101 proteins did not bind to hsMOK2 These results demonstrated that the minimal domain of JLP to mediate hsMOK2 binding was located from amino acids 21 to 101 and that the region surrounding JLP residues 71–101 was required but was not sufficient for this interaction JLP exhibits sequence homology to JSAP1 (also called JIP3) [12,13] In particular, they share 77.3% homology in their N-terminal region (Fig 1B), suggesting that hsMOK2 may also interact with JSAP1 We therefore tested its interaction with the N-terminal region of JSAP1 protein (amino acids 26–106), which corresponds to amino acids 21–101 of JLP hsMOK2 bound even more efficiently to GST– JSAP1 26–106 protein than to GST–JLP 21–101 (Fig 1A,D) We analyzed and compared the secondary structure of the JLP 1-141 and JSAP1 6–106 domains using the paircoil program [14] This program uses pairwise residue probabilities to detect coiled-coil motifs in protein sequence data, and the database of pairwise residue correlations suggests structural FEBS Journal 276 (2009) 3137–3147 ª 2009 The Authors Journal compilation ª 2009 FEBS 3139 Phosphorylation-dependent binding of MOK2 M Harper et al features that stabilize or destabilize coiled-coils Analysis showed that the predicted coiled-coil region in the JSAP1 6–146 domain is more extended than in the JLP 1–141 domain (Fig 2C) The coiled-coil region begins at residue 69 in the JLP 1–141 domain, whereas it begins 15 amino acids upstream in the JSAP1 6–146 domain Interestingly, a difference of three amino acids was found between the two sequences of this region (Fig 2C) The substitution in JLP sequence of F65 and D68 amino acids by the corresponding JSAP1 residues (Leu70 and Asn73) increases the probability of residues 54–68 forming a coil-coiled region We determined experimentally the effects of a single point mutation D68N and double point mutation F65L ⁄ D68N in JLP (amino acids 21–101) on binding to hsMOK2 Only the double substitution within the JLP domain significantly enhanced the association of JLP with hsMOK2 (Fig 2D,E) suggesting that the single mutation D68N does not provide enough stabilization of the coil-coiled region The association with the double point mutation F65L ⁄ D68N in JLP became comparable with that observed with the JSAP1 domain These results confirmed that the region between residues 59 and 73 in JSAP1 strongly promotes binding to hsMOK2 To determine whether hsMOK2 interacts with JSAP1 in mammalian cells, GST pull-down and co-immunoprecipitation analyses were performed The endogenous MOK2 protein is difficult to assess because of its very low expression level, and so we examined the in vivo interaction in transfected cells Indeed, the endogenous MOK2 protein has been detected only by electron microscopy [1] HEK293 cells were transfected with constructs that expressed hsMOK2 tagged with GST in the N-terminus (GST– hsMOK2) and JSAP1 fused to a Flag epitope in the N-terminus (Flag–JSAP1), either together or separately As shown in Fig 3A, Flag–JSAP1 protein was strongly detected in glutathione-bound proteins from cells co-transfected with Flag–JSAP1 and GST– hsMOK2 (lane 3) compared with those from cells transfected with Flag–JSAP1 alone (lane 1) In reverse experiments, GST–hsMOK2 protein was strongly detected in anti-Flag immunoprecipitates from cells co-transfected with Flag–JSAP1 and GST–hsMOK2 (Fig 3B, lane 3) compared with those from cells transfected with GST–hsMOK2 alone (lane 2) To verify equivalent recovery of GST–hsMOK2 (Fig 3A, lower) or Flag–JSAP1 (Fig 3B, lower), the blots were stripped and reprobed with anti-hsMOK2 or anti-Flag serum, respectively These results demonstrated that interaction between the full-length hsMOK2 and JSAP1 proteins occurs in mammalian cells 3140 A B Fig Interaction of hsMOK2 and JSAP1 in human cells Cultured HEK293 cells were transfected with expression vector for Flag– JSAP1 (lane 1), GST–hsMOK2 (lane 2) or co-transfected with both vectors (lane 3) (A) Whole-cell extracts (100 lg) were incubated with 30 lL of 50% slurry glutathione beads After washing the beads thoroughly, the bound proteins were eluted in SDS sample buffer, resolved by SDS ⁄ PAGE and immunoblotted with mouse anti-(Flag M2) mAb The blot was stripped and reprobed with antihsMOK2 serum to verify equivalent recovery of the GST fusion protein (lower) (B) Whole-cell extracts from transfected HEK293 cells (100 lg) were immunoprecipitated with 20 lL of anti-(Flag M2) agarose affinity gel After washing the beads thoroughly, the bound proteins were eluted in SDS sample buffer, resolved by SDS ⁄ PAGE and immunoblotted with an affinity purified anti-hsMOK2 serum The blot was stripped and reprobed with Flag M2 mAb to verify equivalent recovery of the Flag fusion protein (lower) hsMOK2 is phosphorylated in cells The interaction of hsMOK2 with JLP and JSAP1 scaffold proteins suggests that hsMOK2 activity could be modulated by phosphorylation by the JNK family of MAP kinases We examined the in vivo phosphorylation status of transfected hsMOK2 HeLa cells transfected with GST–hsMOK2 were lyzed and incubated with glutathione–agarose to purify the GST–hsMOK2 fusion protein As negative and positive controls, we used GST alone and GST-tagged kinesin-12 (also called Kif 15), respectively [15] We used two commercially available antibodies to detect phosphorylation at serine or threonine residues in the hsMOK2 protein Purified GST fusion proteins were resolved in duplicate SDS ⁄ PAGE gels and immunoblotted with either FEBS Journal 276 (2009) 3137–3147 ª 2009 The Authors Journal compilation ª 2009 FEBS M Harper et al Phosphorylation-dependent binding of MOK2 Fig hsMOK2 is a phosphoserine protein Whole-protein extracts (500 lg) from HeLa cells transfected with GST (lane 1), GST–Kin-12–stalk2 (lane 2) or GST–hsMOK2 (lane 3) were incubated with 50 lL of 50% slurry glutathione beads After thoroughly washing the beads, the bound proteins were eluted in SDS sample buffer, resolved in duplicate gels by SDS ⁄ PAGE and immunoblotted with either anti-(phosphoSerine Q5) serum (middle) or anti-(phosphoThreonine Q7) serum (right) The same blots were stripped and re-probed with anti-GST serum to confirm equivalent loading of GST fusion protein (left) The proteins were visualized by exposing the blots to CL-Xposure film (Pierce) anti-(phosphoserine Q5) or anti-(phosphothreonine Q7) serum None of the antibodies reacted with GST alone (Fig 4, lane 1) Phosphorylation of GST– hsMOK2 was detected only by the anti-phosphoserine serum (Fig 4, lane 3), whereas phosphorylation of GST–Kin-12–stalk2 was detected only by the antiphosphothreonine serum (Fig 4, lane 2) An anti-GST serum confirmed that equivalent amounts of purified GST fusion proteins were loaded and that the bands revealed by the anti-phosphoserine or the anti-phosphothreonine serum corresponded to the migration of GST–hsMOK2 and GST–Kin-12–stalk2 (Fig 4, left) These results established that hsMOK2 is phosphorylated on serine residues in vivo hsMOK2 is phosphorylated by JNK3, Aurora A and PKA kinases in vitro It is known that JNK kinases phosphorylate Ser ⁄ ThrPro motifs in target proteins [16] hsMOK2 contains four of these motifs: S28P, S38P, S129P and S191P (Fig 5A) The sequence of human MOK2 is highly conserved between primates, but only S129P motif is conserved (Fig 5A) Because JNK3 is the JNK kinase expressed primarily in the brain like MOK2 [16,17], we examined hsMOK2 phosphorylation by JNK3 kinase in an in vitro kinase assay hsMOK2 was expressed in Escherichia coli as GST fusion protein, purified on glutathione–agarose and incubated with activated recombinant JNK3 in the presence of [32P]ATP[cP] The result showed that recombinant hsMOK2 was a substrate for JNK3 in vitro (Fig 5B, lane 1) To determine the possible contribution of the four serine residues, we replaced individual serine resi- A B Fig Phosphorylation of hsMOK2 in vitro by JNK3 kinase (A) Alignment of primate MOK2 proteins highlighting the potential SP motifs for JNK kinases in bold The percentage identity with human is indicated in parentheses (B) GST-tagged wild-type or mutant hsMOK2 bound to glutathione beads were incubated with recombinant JNK3 kinase in the presence of [32P]ATP[cP] The proteins were then separated on SDS ⁄ PAGE The gel was subjected to Coomassie Brilliant Blue staining (lower) followed by autoradiography (upper) dues with alanine and expressed the mutant proteins as GST fusion in E coli Similar quantities of the protein (as shown in the Coomassie Brilliant Blue-stained gel in Fig 5B, lower), were subjected to in vitro phosphorylation with JNK3 kinase The results showed that replacement of Ser28 or Ser191 with Ala did not decrease the phosphorylation of hsMOK2 by JNK3, whereas the phosphorylation was markedly decreased when Ser38 or Ser129 were replaced with Ala The simultaneous replacement of Ser38 and Ser129 caused FEBS Journal 276 (2009) 3137–3147 ª 2009 The Authors Journal compilation ª 2009 FEBS 3141 Phosphorylation-dependent binding of MOK2 M Harper et al a larger decrease in phosphorylation The data demonstrate that JNK3 phosphorylates hsMOK2 on Ser38 and Ser129 in vitro Because JSAP1 is expressed along with JNK3 and MOK2 in the brain, JSAP1 could bring together MAPKs and hsMOK2 in this tissue Such a function of JIP proteins has been previously proposed for two other transcription factors, c-Myc and Max [11] Interestingly, the region of JLP that binds Myc is similar to the region of JSAP1 binding hsMOK2 It is tempting to speculate that JSAP1 may enhance hsMOK2 phosphorylation by JNK3 Unfortunately, our attempts to demonstrate such an effect were unsuccessful The JSAP1 protein had no effect when added to our in vitro phosphorylation assay (data not shown), which may be because the recombinant JNK3 is an activated form of the kinase To address this issue in vivo, HEK293 cells, which not express JSAP1, were transiently transfected with tagged hsMOK2, with and without JSAP1 JSAP1 did not stimulate hsMOK2 phosphorylation, even after activation of endogeneous JNK kinases following incubation of the cells with sorbitol This does not preclude that JSAP1 may play a role in hsMOK2 phosphorylation in brain tissue A computer search for other potential phosphorylation sites indicated the existence of several putative PKA, protein kinase C and caseine kinase II phosphorylation sites spread along the hsMOK2 sequence, as well as two Aurora phosphorylation sites in the lamin A ⁄ C-binding N-terminal acidic domain of hsMOK2 Because these two Aurora sites, centered on amino acids Ser46 and Ser146, are strictly conserved between primates [18] (Fig 6A), we tested whether they could be phosphorylated by Aurora A kinase In vitro phosphorylation experiments showed that hsMOK2 was a substrate for recombinant Aurora A kinase (Fig 6B) To determine which serine is phosphorylated, we replaced Ser46 or Ser146, or both Ser46 and Ser146, with alanine in GST–hsMOK2 constructs Incubation of these fusion proteins with recombinant Aurora A revealed a minor reduction in phosphorylation of the mutant containing only the S146A mutation (Fig 6B, lane 3), compared with the wild-type, and a remarkably reduced phosphorylation of the two mutants containing the S46A mutation (Fig 6B, upper, lanes 2, and 4) We concluded that only Ser46 is a major Aurora A phosphorylation site on hsMOK2 Recently, it has been reported that human Aurora A and Aurora B kinases prefer substrate sequences with an arginine residue at the position )2 [19,20] Accordingly, only the sequence surrounding Ser46 in hsMOK2 conforms to this preference (RDSV) Lastly, because the consensus for 3142 A B C Fig Phosphorylation of hsMOK2 in vitro by Aurora A and PKA (A) Alignment of primate MOK2 proteins highlighting the two conserved Aurora phosphorylation motifs in bold (B) GST-tagged wild-type or mutant hsMOK2 bound to glutathione beads were incubated with recombinant Aurora A in the presence of [32P]ATP[cP] The proteins were then separated on SDS ⁄ PAGE The gel was subjected to Coomassie Brilliant Blue staining (lower) followed by autoradiography (upper) (C) GST-tagged wild-type or mutant hsMOK2 bound to glutathione beads were incubated with recombinant PKA, in the presence of [32P]ATP[cP] and visualized as in (B) Aurora A is reminiscent of that of PKA, we examined the ability of PKA to phosphorylate hsMOK2 protein in vitro We obtained the same phosphorylation pattern of wild-type and mutant hsMOK2 as observed with Aurora A kinase (Fig 6C) We conclude that hsMOK2 is efficiently phosphorylated in vitro by Aurora A kinase and PKA at residue Ser46 Analysis of phosphomimetic mutations on hsMOK2 capacity to bind DNA Binding of hsMOK2 to DNA may be affected, either positively or negatively, by phosphorylation Therefore, to determine whether serine phosphorylation would affect the ability of hsMOK2 to bind DNA, we introduced phosphomimetic mutations in hsMOK2 by replacing individual serine residues by aspartic acid FEBS Journal 276 (2009) 3137–3147 ª 2009 The Authors Journal compilation ª 2009 FEBS M Harper et al Fig Effects of hsMOK2 phosphomimetic mutations on DNA binding activity EMSA was performed on whole-cell extracts derived from HeLa cells transfected with various hsMOK2 expression plasmids as indicated The amount of extracts was adjusted to obtain equal level of the various hsMOK2 proteins The 32P-labeled double-stranded oligonucleotide corresponds to the 18-bp MOK2 binding site of human IRBP gene The binding reaction was performed in buffer containing various concentrations of NaCl Gel-shift experiments were performed with nuclear extracts of HeLa cells expressing wild-type or mutant hsMOK2 proteins and a [32P]-labeled double-stranded oligonucleotide, corresponding to the 18-bp MOK2binding site present in hsIRBP intron Immunoblot analysis attested that similar amounts of the wild-type and mutant proteins were used in the gel-shift assay (data not shown) As shown in Fig 7, phosphomimetic substitutions at positions Ser46, Ser38 and Ser129 had no effect on protein–DNA complex abundance at any o the NaCl concentrations tested Hence, the two phosphomimetic mutants retained the ability to bind the specific DNA sequence with high affinity, indicating that this binding is not regulated by phosphorylation at Aurora A ⁄ PKA or JNK sites Effect of hsMOK2 phosphorylation on its capacity to bind lamin A ⁄ C The two JNK phosphorylation sites of hsMOK2 are located in the lamin A ⁄ C-binding N-terminal acidic domain We therefore examined whether phosphorylation of hsMOK2 at JNK3 or Aurora A ⁄ PKA sites had an impact on the interaction with lamin A ⁄ C Nuclear extracts from HeLa cells expressing wild-type or mutant hsMOK2 proteins were prepared and incubated with an equal amount of GST–DlaminC bound Phosphorylation-dependent binding of MOK2 to glutathione beads Phosphomimetic substitutions at Ser38, Ser129 or the double Ser38Ser129 mutation did not markedly decrease binding to DlaminC (Fig 8A, upper) By contrast, phosphomimetic substitution at Ser46 markedly decreased the binding, although alanine substitution at Ser46 had no effect (Fig 8A, lower) The data indicated that the phosphorylation of hsMOK2 at the Aurora A ⁄ PKA site interfered with its ability to bind lamin A ⁄ C in vitro We then sought evidence that a similar effect occurs in vivo We used the characteristic that mutations in lamin A ⁄ C lead to sequestration of hsMOK2 in nuclear aggregates (Fig 8B) [7] HeLa cells were co-transfected with the expression vector for the Q294P lamin C mutant and wild-type hsMOK2 or hsMOK2 mutated at position S46 The nonphosphorylatable hsMOK2–S46A protein was found in the nuclear aggregates induced by the Q294P lamin C mutant like hsMOK2–WT protein (Fig 8B), whereas the phosphomimetic hsMOK2– S46D protein exhibited a homogeneous nuclear pattern (Fig 8B) The hsMOK2–S46D protein was therefore not displaced in nuclear aggregates, demonstrating that phosphomimetic substitution at Ser46 also prevents the interaction with lamin A ⁄ C in vivo To confirm that phosphorylation in vivo can disrupt the interaction between hsMOK2 and lamin A ⁄ C, we examined the localization of hsMOK2–WT in cells treated with the phosphatase inhibitor orthovanadate to enhance cellular phosphorylation HeLa cells co-transfected with expression vector for lamin C–Q294P and hsMOK2–WT were incubated with mm sodium orthovanadate for h In this condition, no sequestration of hsMOK2 by mutant lamin A ⁄ C was observed, confirming the importance of phosphorylation for hsMOK2 and lamin A ⁄ C interaction (Fig 8C, upper) However, the same observation was made using hsMOK2–S46A (Fig 8C, lower), indicating that the effect of orthovanadate treatment can be mediated by phosphorylation at another position Human MOK2 is a DNA-binding transcriptional repressor and its interaction with lamin A ⁄ C and the nuclear matrix may be important for its ability to repress transcription Such involvement of lamins A ⁄ C has been proposed previously for the transcriptional activator pRb [21–23] pRb controls cell-cycle progression by negatively regulating the E2F transcription factor in a phosphorylation-dependent manner [24] The active (hypophosphorylated) form of pRb co-localizes with lamins A ⁄ C at the nuclear periphery in vivo and binds to lamins in vitro [21] Thus, transcriptional repression by pRb correlates with its lamin-binding activity Similarly, transcriptional repression by hsMOK2 might be correlated with its lamin-binding FEBS Journal 276 (2009) 3137–3147 ª 2009 The Authors Journal compilation ª 2009 FEBS 3143 Phosphorylation-dependent binding of MOK2 M Harper et al B A C Fig Effect of hsMOK2 phosphorylation on its capacity to bind lamin A ⁄ C (A) For in vitro interaction, nuclear extracts from HeLa cells overexpressing wild-type or mutant hsMOK2 were incubated with 10 lg of GST–DlaminC or GST alone bound to glutathione beads The amount of nuclear extracts was adjusted to obtain equal levels of the various hsMOK2 proteins Input lanes correspond to 5% (upper) and 10% (lower) of the extracts used for binding reaction After thoroughly washing the beads, the bound proteins were eluted in SDS sample buffer, resolved by SDS ⁄ PAGE and immunoblotted with affinity purified anti-hsMOK2 serum The proteins were visualized by exposing the blots to CL-Xposure film (Pierce) (B) For in vivo interaction, HeLa cells were co-transfected with expression vector for lamin C–WT or lamin C–Q294P mutant and hsMOK2–WT, hsMOK2–S46A or hsMOK2–S46D After 36 h, cells were fixed and double stained sequentially with lamin A ⁄ C mAb and anti-hsMOK2 serum Cells were observed with a Leica DMR microscope and an Apochromat 63 · 1.32 oil immersion objective (C) HeLa cells were co-transfected with expression vector for lamin C–Q294P and hsMOK2–WT or hsMOK2–S46A Sixteen hours after transfection, the cells were treated with mM sodium orthovanadate for h, fixed and double-stained sequentially with lamin A ⁄ C mAb and anti-hsMOK2 serum activity The simplest scenario is one in which the lamin A ⁄ C–hsMOK2 complex stabilizes a repressive complex on DNA, preventing gene activation To allow gene activation, hsMOK2 would be phosphorylated and then released from lamin A ⁄ C This regulation may take place following activation of kinases such as PKA in response to various signaling pathways In addition, Aurora A kinase is specifically activated before mitosis [25,26] Mitotic nuclear envelope breakdown requires disassembly of the nuclear lamina Lamins A and C are rapidly released throughout the nucleoplasm in early prophase [27,28] hsMOK2 dissociation from lamin A ⁄ C at hsMOK2regulated loci in early mitosis may contribute to the dispersion of lamins A ⁄ C into the cytoplasm 3144 Materials and methods Plasmid constructs The recombinant pLex–hsMOK2, pLex–NH2, pLex–finger, pCMV–hsMOK2, pCMV–laminC(Q294P), pGEX–hsMOK2, pGEX–DlaminC and pGEX–Kin-12-stalk2 vectors have been described previously [2,3,7,15] Point mutations were introduced into hsMOK2 constructs using the QuickChange XL site-directed mutagenesis kit (Stratagene, Amsterdam, The Netherlands) The prokaryotic expression vector pET29, containing Aurora A cDNA, was a kind gift ´ ´ from C Prigent (Faculte de Medecine, Rennes, France) The pGEX–JLP(1–141) and truncation mutants were generated from pGAD–JLP(1–141) by PCR using 5¢ FEBS Journal 276 (2009) 3137–3147 ª 2009 The Authors Journal compilation ª 2009 FEBS M Harper et al primers and 3¢ primers containing an EcoRI site and a SalI site, respectively After digestion with EcoRI and SalI, the products were cloned into the corresponding sites of the pGEX–6P1 vector (Amersham Pharmacia Biotech, Orsay, France) The mammalian expression pcDNA3–Flag–JSAP1 plasmid containing the entire coding sequence of mouse JSAP1 was kindly provided by K Yoshioka [29] Cell culture, transfections and protein extracts Human HeLa or HEK293 cells were routinely maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum For transient transfections, 105 cells were plated on a 35-mm Petri dish containing a glass coverslip, 24 h prior to transfection with lg of plasmid, using calcium phosphate precipitation, as described previously [30] For whole-protein extracts, transfected HeLa cells from three 100-mm Petri dishes were scraped into NaC ⁄ Pi, resuspended in 600 lL Hepes buffer (25 mm Hepes, pH 7.5, 150 mm NaCl, 10% glycerol, 10 lm ZnSO4 and 0.1% Nonidet P-40) supplemented with a protease inhibitor cocktail without EDTA (Roche Diagnostics, Meylan, France) and disrupted by sonication After centrifugation at 15 000 g for 10 min, the total extracts were frozen at )80 °C Whole-protein extracts from one 100-mm Petri dish plated with transfected HEK293 cells were prepared in 500 lL of 20 mm Tris ⁄ HCl (pH 7.5), 150 mm NaCl, 1% Triton X-100, 0.5% sodium deoxycholate and mm EDTA, supplemented with protease inhibitor cocktail, as described for transfected HeLa cells Nuclear extracts were prepared according to the Dignam method [31] and dialyzed against Hepes buffer at °C for h After dialysis and centrifugation, the nuclear extract was frozen at )80 °C The protein concentration was determined by the Coomassie Brilliant Blue protein assay (Pierce, Brebieres, France) Antibodies Affinity-purified rabbit polyclonal anti-hsMOK2 serum was obtained as described previously [2] Mouse anti(lamin A ⁄ C(636)) mAb was purchased from Santa Cruz Biotechnology, Heidelberg, Germany) Mouse monoclonal anti-(Flag M2) and anti-(Flag M2) agarose affinity gel were purchased from Sigma (St-Quentin Fallavier, France) Mouse anti-(phosphoThreonine Q7) and anti(phosphoSerine Q5) sera were purchased from Qiagen (Courtaboeuf, France) Rhodamine (TRITC)-conjugated goat anti-(mouse IgG), CY2TM-conjugated goat anti-(rabbit IgG) and peroxidase-conjugated rabbit anti-(mouse IgG) sera were purchased from Jackson Immunoresearch Laboratories (Bar Harbor, ME, USA) Phosphorylation-dependent binding of MOK2 Yeast two-hybrid screen Yeast two-hybrid screening using human hsMOK2 as bait was described previously [3] A human HeLa S3 Matchmaker cDNA library (BD Clontech, St-Germain-en-Laye, France), constructed in the pGAD–GH vector expressing the GAL4 activation domain fusion protein, was transformed into L40 containing the pLex–hsMOK2 construct The cDNA inserts of positive clones were isolated by direct PCR of yeast colonies The cDNA inserts were further characterized by sequencing and searching for gene sequence similarity in the GenBankÔ database with the program blast Immunofluorescence microscopy Cells were fixed in )20 °C methanol for and incubated with primary anti-hsMOK2 or anti-(lamin A ⁄ C) sera, followed by incubation with the secondary antibody The incubations were for h each and were carried out sequentially (with washes in NaCl ⁄ Pi after each step) The cellular DNA was labeled with 0.12 lgặmL)1 4Â-6-diamidino-2phenylindole for The slides were mounted in antifadent AF1 ⁄ glycerol ⁄ NaCl ⁄ Pi mounting medium (Citifluor Ltd, London, UK) Immunofluorescence microscopy was performed using a Leica DMR microscope (Leica, Heidelberg, Germany) and an Apochromat 63 · 1.32 oil immersion objective Photographs were taken using a Micromax (Princeton Instruments, Evry, France) CCD camera and metaview (Universal Imaging Corp.) software Purification of GST fusion proteins and kinase assay For bacteria expressing GST fusion proteins, crude protein extracts were prepared and purified as described previously [3] The purity and amount of the recombinant proteins were determined by examining SDS ⁄ PAGE gel staining with Coomassie Brilliant Blue The GST fusion proteins bound to glutathione beads were used as 50% slurry in appropriate buffer The N-terminal His6-tagged full-length human Aurora A protein was expressed in E coli strain BL21(DE3) and purified using Ni2+ ⁄ NAT agarose as described by Cremet et al [32] The Aurora A protein solution was concentrated using a centricon 10 (Millipore, St-Quentin-en Yvelines, France) at mgỈmL)1 and stored at )80 °C The N-terminal His6-tagged full-length human JNK3 ⁄ SPAK1b active protein was purchased from Upstate (St-Quentin-en Yvelines, France) and the N-terminal His tagged human catalytic subunit of PKA was purchased from Calbiochem (Nottingham, UK) The kinase assays were performed in 25 lL of 50 mm Tris ⁄ HCl pH 7.5, 0.1% 2-mercaptoethanol, mm EGTA, FEBS Journal 276 (2009) 3137–3147 ª 2009 The Authors Journal compilation ª 2009 FEBS 3145 Phosphorylation-dependent binding of MOK2 M Harper et al 0.2 mm dithiothreitol, 0.2 mm sodium orthovanadate, 15 mm MgCl2, 100 lm ATP containing 10 lCi [32P]ATP [cP] 3000 CiỈmmol)1 (Amersham Pharmacia Biotech), 200 ng Aurora A or 50 ng JNK3 ⁄ SPAK1b or 10 ng PKA and 10–15 lL of a 50% slurry of GST fusion proteins bound to the beads The reactions were incubated at 30 °C for 10 Ten microliters of 5· Laemmli sample buffer was added and the reaction was heated at 100 °C for The proteins were then separated on a Nupage 4–12% Bis-Tris gels (Invitrogen, Illkirch, France) The gel was subjected to Coomassie Brilliant Blue staining, dried and analysed using a Phosphoimager apparatus (Molecular Dynamics, Orsay, France) Acknowledgements GST pull-down assay and co-immunoprecipitation Arranz V, Harper F, Florentin Y, Puvion E, Kress M & Ernoult-Lange M (1997) Human & mouse MOK2 proteins are associated with nuclear ribonucleoprotein components & bind specifically to RNA & DNA through their zinc finger domains Mol Cell Biol 17, 2116–2126 Arranz V, Dreuillet C, Crisanti P, Tillit J, Kress M & Ernoult-Lange M (2001) The zinc finger transcription factor, MOK2, negatively modulates expression of the interphotoreceptor retinoid-binding protein (IRBP) gene J Biol Chem 276, 11963–11969 Dreuillet C, Tillit J, Kress M & Ernoult-Lange M (2002) In vivo & in vitro interaction between human transcription factor MOK2 & nuclear lamin A ⁄ C Nucleic Acids Res 30, 4634–4642 Ernoult-Lange M, Arranz V, Leconiat M, Berger R & Kress M (1995) Human & mouse Kruppel-like (MOK2) orthologue genes encode two different zinc finger proteins J Mol Evol 41, 784–794 Fisher DZ, Chaudhary N & Blobel G (1986) cDNA sequencing of nuclear lamins A & C reveals primary & secondary structural homology to intermediate filament proteins Proc Natl Acad Sci USA 83, 6450–6454 McKeon FD, Kirschner MW & Caput D (1986) Homologies in both primary & secondary structure between nuclear envelope & intermediate filament proteins Nature 319, 463–468 Dreuillet C, Harper M, Tillit J, Kress M & ErnoultLange M (2008) Mislocalization of human transcription factor MOK2 in the presence of pathogenic mutations of lamin A ⁄ C Biol Cell 100, 51–61 Dhanasekaran DN, Kashef K, Lee CM, Xu H & Reddy EP (2007) Scaffold proteins of MAP-kinase modules Oncogene 26, 3185–3202 Morrison DK & Davis RJ (2003) Regulation of MAP kinase signaling modules by scaffold proteins in mammals Annu Rev Cell Dev Biol 19, 91–118 10 Yoshioka K (2004) Scaffold proteins in mammalian MAP kinase cascades J Biochem 135, 657–661 11 Lee CM, Onesime D, Reddy CD, Dhanasekaran N & Reddy EP (2002) JLP: a scaffolding protein that tethers For in vitro GST pull-down assay, 10 lg of GST fusion protein, immobilized on glutathione–agarose beads was added to 20 lg of nuclear proteins from transfected HeLa cells, in a total volume of 400 lL Hepes buffer For in vivo GST pull-down assay or co-immunoprecipitation, wholecell extracts (100 lg) from transfected HEK293 were incubated either with 30 lL of 50% slurry glutathione beads or with 20 lL of anti-(Flag M2) agarose affinity gel After h at °C, the beads were extensively washed and eluted by boiling in Laemmli buffer Bound proteins were separated by SDS ⁄ PAGE and analyzed by immunoblotting with the indicated antibodies using the Supersignal West Pico Chemiluminescent Signal kit (Pierce) The proteins were visualized by exposing the blots to CL-XPosure film (Pierce) or using a Fluor-S Max MultiImager with quantity one software (Bio-Rad, Marne-la-Coquette, France) Electrophoretic mobility shift assay A 25-bp oligonucleotide corresponding to the sequence of human IRBP intron containing the 18-bp MOK2-binding site (5¢-CTGCAGGACTTGTCAGGGCCTTTAA-3¢) was used as a probe The double-strand oligonucleotide was labeled with T4 polynucleotide kinase (Biolabs, Ipswich, MA, USA) in the presence of [32P]ATP[cP] and purified on a 15% polyacrylamide gel End-labeled oligonucleotides (0.2 ng) were incubated for 20 at room temperature in 20 lL Hepes buffer containing lg poly(dI–dC), various concentrations of NaCl and whole-protein extracts from HeLa cells transfected with wild-type or mutant hsMOK2 The amount of extract was adjusted to obtain an equivalent level of the transfected hsMOK2 proteins Complexes were analyzed by electrophoresis on a nondenaturing premigrated 6% polyacrylamide gel (acrylamide ⁄ bis ratio 19 : 1) in 0.5· TB buffer (45 mm Tris borate, pH 8.3) at °C at 200 V EDTA was omitted in all binding and electrophoresis buffers to avoid denaturing hsMOK2 3146 We thank Katsuji Yoshioka for providing the pcDNA3–Flag–JSAP1 expression vector and Claude Prigent for pET29–Aurora A vector We also thank Vanessa Philipot for technical assistance and Dominique Weil for critical reading of the manuscript This research was supported by grants from the Centre National de la Recherche Scientifique, the Fondation Raymonde et Guy Strittmatter and the association Retina France References FEBS Journal 276 (2009) 3137–3147 ª 2009 The Authors Journal compilation ª 2009 FEBS M Harper et al 12 13 14 15 16 17 18 19 20 21 JNK ⁄ p38MAPK signaling modules & transcription factors Proc Natl Acad Sci USA 99, 14189–14194 Ito M, Yoshioka K, Akechi M, Yamashita S, Takamatsu N, Sugiyama K, Hibi M, Nakabeppu Y, Shiba T & Yamamoto KI (1999) JSAP1, a novel jun N-terminal protein kinase (JNK)-binding protein that functions as a Scaffold factor in the JNK signaling pathway Mol Cell 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breakdown proceeds by microtubule-induced tearing of the lamina Cell 108, 83–96 29 Bayarsaikhan M, Shiratsuchi A, Gantulga D, Nakanishi Y & Yoshioka K (2006) Selective expression of the scaffold protein JSAP1 in spermatogonia & spermatocytes Reproduction 131, 711–719 30 Arranz V, Kress M & Ernoult-Lange M (1994) The gene encoding the MOK-2 zinc-finger protein: characterization of its promoter & negative regulation by mouse Alu type-2 repetitive elements Gene 149, 293–298 31 Dignam JD, Lebovitz RM & Roeder RG (1983) Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei Nucleic Acids Res 11, 1475–1489 32 Cremet JY, Descamps S, Verite F, Martin A & Prigent C (2003) Preparation & characterization of a human aurora-A kinase monoclonal antibody Mol Cell Biochem 243, 123–131 FEBS Journal 276 (2009) 3137–3147 ª 2009 The Authors Journal compilation ª 2009 FEBS 3147 ... Mislocalization of human transcription factor MOK2 in the presence of pathogenic mutations of lamin A ⁄ C Biol Cell 100, 51–61 Dhanasekaran DN, Kashef K, Lee CM, Xu H & Reddy EP (2007) Scaffold proteins of. .. proposed for two other transcription factors, c-Myc and Max [11] Interestingly, the region of JLP that binds Myc is similar to the region of JSAP1 binding hsMOK2 It is tempting to speculate that JSAP1... The amount of extracts was adjusted to obtain equal level of the various hsMOK2 proteins The 32P-labeled double-stranded oligonucleotide corresponds to the 18-bp MOK2 binding site of human IRBP