The Multifunctional Nuclear Protein p54nrb is Multiphosphorylated in Mitosis and Interacts with the Mitotic Regulator Pin1

doi:10.1016/j.jmb.2004.12.034 J. Mol. Biol. (2005) 346, 1163–1172

The Multifunctional Nuclear Protein p54nrb isMultiphosphorylated in Mitosis and Interacts with theMitotic Regulator Pin1

Ariane Proteau1, Stephanie Blier1, Alexandra L. Albert1

Sebastien B. Lavoie1, Abdulmaged M. Traish2 and Michel Vincent1*

1CREFSIP and Departement deMedecine, Laval UniversityPavillon C.-E.-MarchandRoom 4263 Laval UniversityQue., Canada, G1K 7P4

2Department of BiochemistryBoston University School ofMedicine, Center for AdvancedBiomedical Research, 700Albany Street, W607, BostonMA02118, USA

0022-2836/$ - see front matter q 2004 E

Abbreviations used: CTD, carboxyRNA polymerase II); mAb, monoclNHERF-1, NaC/HC exchanger regprotein phosphatase type 1; PSF, pobinding protein-associated protein;transferase.E-mail address of the correspond

[email protected]

The human protein p54nrb and its mouse homolog NonO have beenimplicated in a variety of nuclear processes including transcription, pre-mRNA processing, nuclear retention of edited RNA and DNA relaxation.We have identified p54nrb as an antigen of the phosphodependentmonoclonal antibodies CC-3 and MPM-2 and shown that this protein isphosphorylated on multiple sites during mitosis. The use of the cyclin-dependent protein kinase inhibitor roscovitine and immunodepletionstudies with an anti-cyclin B1 antibody established that Cdk1 wasresponsible for the phosphorylation of the carboxy-terminal extremity ofp54nrb whereas a different kinase appeared to be involved in the generationof CC-3 epitope(s) in the amino-terminal moiety of the protein. Like manyCC-3 and MPM-2 antigens, we show that p54nrb is a target of thepeptidylprolyl isomerase Pin1, suggesting that it may be regulated byphosphorylation-dependent conformational changes as many othernuclear proteins upon entry into mitosis. In addition, site-directedmutagenesis indicated that the interaction of Pin1 with p54nrb wasmediated by three threonine residues located in the proline-rich carboxy-terminal extremity of the protein. Our results also showed that Pin1binding was favored when at least two of the three threonine residues werephosphorylated, suggesting a regulation mechanism based on multisitephosphorylation.

q 2004 Elsevier Ltd. All rights reserved.

Keywords: mitosis; p54nrb; Pin1; CC-3; MPM-2
*Corresponding author
Introduction

The protein p54nrb is an abundant and highlyconserved nuclear factor that has been implicated ina diversity of reactions occurring in the cellnucleus.1 Initially suspected to be involved in pre-mRNA splicing owing to its extensive homology tohuman splicing factor PSF,2 p54nrb was recentlyimplicated in more generalized functions within thenucleus. In particular, human p54nrb and its mousehomolog NonO were shown to participate, often in

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-terminal domain (ofonal antibody;ulatory factor-1; PP1,lypyrimidine tract-GST, glutathione-S-

ing author:

partnership with PSF and other proteins, intranscriptional control, RNA splicing and editingand DNA unwinding and pairing.1–8 It has alsobeen described as a nuclear matrix-associatedprotein whose expression is reduced in humanbreast estrogen receptor-negative tumor cells.9,10

Recently, p54nrb was found to accumulate in a newnucleoplasmic compartment of human cells,termed paraspeckles, and to relocalize to capstructures at the nucleolar periphery when tran-scription is inhibited.11 Human p54nrb is a 471amino acid residue protein containing two RNP-type RNA recognition motifs and a putative helix-turn-helix domain followed by a highly chargedregion.2 In addition to its nucleic acid bindingproperties, p54nrb has been shown to interact withthe carboxy-terminal domain of the largest subunitof RNA polymerase II, suggesting a role in couplingpre-mRNA synthesis and processing.12 How p54nrb

operating in such diversified nuclear functions is

d.

Figure 1. p54nrb is a CC-3 phosphoantigen. Lysates ofmitotically arrested cells were immunoprecipitated witheither NMT-5, raised against p54nrb, or mAb CC-3 and theresulting fractions were cross-immunoblotted. The firsttwo lanes show the Western blot reactivities of the initialHeLa mitotic lysate.

1164 The Nuclear Protein p54nrb Interacts with Pin1

not known. It has been suggested that p54nrb andPSF mediate different functions depending on theirintranuclear location, which could be regulated byphosphorylation.1 Both proteins have been reportedto copurify with the nuclear envelope under atyrosine-phosphorylated form13 but to our know-ledge, it is not knownwhether p54nrb is phosphoryl-ated on serine or threonine residues. However,posttranslational modifications are suspected tooccur since the recombinant p54nrb proteinexpressed in bacteria binds RNA but not DNA,while the same protein translated in reticulocytelysate can bind DNA, suggesting that a mammaliankinase, or another modification enzyme present inthe reticulocyte lysate, may act as a switch betweenRNA and DNA-binding modes of the protein.4 Inaddition, it was recently shown that a 54/56 kDadoublet associating with the 5 0 splice site in thecontext of large RNA polymerase II–snRNP com-plexes actually represents two forms of the p54nrb

protein differing in their phosphorylation state.8

It is well known that many nuclear processesusually operating in interphase are suspendedduring mitosis. In particular, RNA synthesis andprocessing are globally repressed as many factorsimportant for transcription and splicing are redis-tributed during the transition from interphase tomitosis.14,15 Protein phosphorylation is an import-ant regulator of this transition. During cell division,cells display elevated levels of protein phosphoryl-ation triggered by several serine and threoninekinases at the head of which sits Cdk1/cyclin Bcomplex.16 To characterize these phosphoproteins,different monoclonal antibodies (mAbs) have beenraised against mitotic cell extracts and shown toreact with subsets of proteins that are specificallyphosphorylated when cells divide. The mostcharacterized of these antibodies, mAb MPM-2,reacts with a subset of proteins of the centrosomes,the kinetochores, the mitotic spindle and the mid-body17,18 that correspond to important regulatorsand effectors of mitosis.19–27 In addition, many ofthe MPM-2-reactive phosphoproteins are the tar-gets of the peptidyl-prolyl isomerase Pin1, whichhas been proposed to regulate protein function bycis-trans isomerization of the peptide bond preced-ing proline in a phosphoserine/phosphothreonine-proline dipeptide.28 Another mAb, mAb CC-3,has been produced in our laboratory and selectedfor its phospho-dependent reactivity with severalmitotic antigens29 and two interphase phospho-proteins: a hyperphosphorylated form of RNApolymerase II largest subunit30 and a phosphoryl-ated form of the transcription elongation factorhSpt5.31 In a recent work, we have identified themajor mitotic antigens of mAb CC-3.32 Theyincluded a few proteins involved in transcriptionand pre-mRNA processing and many of them alsoappeared to be novel or already described MPM-2antigens. One of the CC-3 mitotic antigens wasidentified as p54nrb. In the present work, we showthat p54nrb is multiphosphorylated by Cdk1 andpossibly other kinases during mitosis. We also

demonstrate that p54nrb interacts with the prolylisomerase Pin1 and that at least two threonineresidues of the three threonine-proline pairs ofthe p54nrb C-terminal extremity should be phos-phorylated for Pin1 binding.

Results

The multi-functional nuclear protein p54nrb is anew CC-3 mitotic antigen

In mitotic cells, mAb CC-3 reacts with numerousantigens in a phospho-dependent fashion. Recently,several CC-3 mitotic antigens were affinity-purifiedfrom HeLa cells and submitted to mass spec-trometry analysis for identification.32 The multi-functional nuclear protein p54nrb was identified as aprotein retained on the CC-3 affinity column.However, as previously reported,12 p54nrb binds tothe RNA polymerase II C-terminal domain (CTD),which is itself a CC-3 antigen.30 This prompted us toverify whether p54 is a bona fide CC-3 antigen or amere co-precipitated CTD partner. To test this,p54nrb was immunoprecipitated from HeLa mitoticextracts using the polyclonal antibody NMT-5,raised against a polypeptide corresponding to themid-region of the protein,10 and immunoblottedwith CC-3. Figure 1 reveals that CC-3 reactswith the NMT-5-precipitated p54nrb protein. Asexpected, the cross-immunoprecipitation experi-ment confirmed that p54nrb could be immuno-precipitated with CC-3. This indicates that p54nrb

is a bona fide CC-3 nuclear antigen and suggests thatthis protein is phosphorylated upon entry of the cellinto mitotis.

Mitotic phosphorylation of p54nrb: generation ofCC-3 and MPM-2 epitopes

A glutathione-S-transferase (GST)-tagged mouserecombinant p54nrb protein and two truncatedderivatives containing respectively p54nrb aminoacids 1–238 (GST-Nter) and 226–473 (GST-Cter),3

The Nuclear Protein p54nrb Interacts with Pin1 1165

were produced in bacteria and submitted to in vitrophosphorylation using a mitotic extract as a sourceof kinases. The GST-p54 protein migrated around80 kDa and was recognized by CC-3 only afterincubation with the mitotic extract (Figure 2(a)). Aminor band migrating faster than GST-p54 was alsorecognized by CC-3 after in vitro phosphorylation(asterisk in Figure 2(a)). This band probablyrepresented a truncated form of the protein corre-sponding to its N-terminal extremity since it wasnot recognized by mAb NMT-1, whose epitope liesat the C terminus of the p54nrb protein,10 and since itreacted with an antibody against the GST-tag,located N-terminal to p54nrb in the fusion protein(data not shown). The RNA polymerase II CTD,containing multiple copies of the CC-3 epitope,served as a positive control.

To map more precisely the CC-3 epitope andto analyze its relationship with that of MPM-2,N-terminal and C-terminal recombinant fragmentsof the protein were incubated with the mitoticextract. Figure 2(c) demonstrates that both phos-phorylated fragments were labelled with CC-3 inWestern blot. Here again, a shorter CC-3 reactivefragment was generated when the GST-p54 and theGST-Nter constructs were incubated with themitotic extract. It can be hypothesized that a CC-3epitope is close to the N-terminal extremity ofp54nrb since the CC-3 positive shorter fragment wasmigrating just slightly slower than a proteincontaining the GST-tag alone and that no CC-3reactivity was elicited upon incubation of the latterwith the mitotic extract (data not shown). Acandidate residue for phosphorylation could bethe threonine at position 15, which is followed by aproline-arginine-lysine stretch (T15-P-R-K) that fits

with the Cdk1 consensus sequence S/T-P-X-K/R.33

Contrary to CC-3, MPM-2 reacts exclusively withthe carboxy-terminal moiety of the p54nrb protein(Figure 2(d)). These findings confirm that CC-3 andMPM-2 epitopes are different, as previously shownfor the RNA polymerase II CTD,32 and suggest thatp54nrb is a mitotic phosphoprotein. Incidentally,Figure 2(b) shows a slight shift in the migrationpattern of p54nrb when samples were prepared frommitotic cells compared to interphase cells, furthersuggesting that p54nrb is phosphorylated in vivowhen cells are dividing (see also Figure 6(b)).

Phosphorylated p54nrb is a target for the prolyl-isomerase Pin1

It has been shown that many MPM-2 and CC-3antigens are bound to the important mitoticregulator Pin1.30,34 To verify if Pin1 could interactwith p54nrb in vitro, blot overlay experiments werecarried out using recombinant Pin1 as a probe. Thep54nrb protein immunoprecipitated from eithermitotic or interphase cells was electrophoresedand blotted onto nitrocellulose before renaturationand incubation with the Pin1 probe. The resultsindicated that Pin1 could bind to p54nrb immuno-precipitated from mitotic (thick arrow in Figure 3)but not from interphase cells. In addition, anunexpected band with an apparent Mr of 100 kDawas recognized by the overlaid Pin1 (arrowhead inFigure 3) and most probably corresponds to anunknown protein co-immunoprecipitated withp54nrb (see Discussion). Minor bands in bothsamples correspond to the rabbit immunoglobulinheavy chains of the NMT-5 antibody (thin arrows)as revealed by their reactivity with the secondary

Figure 2. In vitro phosphorylationof p54nrb by a mitotic extract.(a) Recombinant p54nrb (GST-p54;80 kDa) and RNA polymerase IIcarboxy-terminal domain (CTD)used as a phosphorylation controlwere reactive with mAb CC-3 afterin vitro phosphorylation using amitotic cell extract (ME) as a sourceof kinases. A total mitotic cell homo-genate (TMH)was used as a positiveWesternblot control. (b)Aslight shiftwas observed in the migration ofp54nrb when samples were preparedfrom whole mitotic cells (TMH)compared to unsynchronized cells(TIH), suggesting that a phosphoryl-ation event occured in vivo. (c) and(d)CC-3 epitopes couldbegeneratedin both GST-Cter and GST-Nterfusion proteins whereas MPM-2epitope(s) were confined to theC-terminal extremity. The bandsmarked by an asterisk probablycorrespond to N-terminal truncatedforms of both GST-p54nrb and GST-Nter proteins (see the text).

Figure 3. Interaction between Pin1 and p54nrb immuno-precipitated from mitotic cells. The p54nrb protein wasimmunoprecipitated from mitotic or asynchronous cellswith antibody NMT-5 and submitted to a blot overlayexperiment using recombinant Pin1 as a probe. Pin1recognizes p54nrb (thick arrow) and an unidentified100 kDa protein (arrowhead) after immunoprecipitationfrom a lysate from mitotic cells but not from asynchro-nous cells. The thin arrows represent the Ig heavy chainsof the NMT-5 antibody used in the immunoprecipitationassay, as revealed by a Western blot using the secondaryantibody alone (middle panel). The efficiency of theimmunoprecipitation in both cell extracts was monitoredby a Western blot using NMT-1 as a primary antibody(lower panel).

Figure 4. The p54nrb protein can be pulled down frommitotic cell lysates by Pin1. Western blot analysis of theeluates from a Pin1 affinity column loaded either with amitotic or an asynchronous cell extract. The p54nrb proteinfrommitotic but not from interphase cells could be pulleddown by the Pin1 beads, as revealed by antibody NMT-5.

Figure 5. In vitro interaction between Pin1 andphosphorylated p54nrb. Blot overlay experiment usingrecombinant Pin1 protein as a probe. Fusion proteinswere phosphorylated or not with the mitotic extract,submitted to electrophoresis, transferred to nitrocellulosefilters and partially renatured before being overlaid withthe probe. The GST-p54nrb and the GST-Cter constructsstrongly interact with the Pin1 probe only when phos-phorylatedwith themitotic extract, whereas the GST-Nterconstruct hardly reacts. No reaction occured with the GSTprotein produced by the vector.


antibody alone (Figure 3, middle panel). Theefficiency of the immunoprecipitation was moni-tored by NMT-1 Western blotting (Figure 3, lowerpanel), confirming that p54nrb obtained from inter-phase cells was not a Pin1 substrate.

To demonstrate that the Pin1–p54nrb interactionoccurs in a more physiological context, we investi-gated whether p54nrb present in crude mitotic orinterphase cell extracts could be affinity purified ona Pin1-coupled matrix. Since the native p54nrb

possesses a stretch of histidine residues in itsN-terminal domain that precludes the use of aNi2C-chelate affinity matrix, a GST-Pin1 fusionprotein was produced and crosslinked to gluta-thione. The column was loaded with extracts frommitotic or asynchronous cells and the boundproteins were analyzed by Western blotting.Figure 4 shows that p54nrb from mitotic but notinterphase cell extracts was retained on the GST-Pin1 column. No reactive proteins were bound on acolumn made from the GST protein alone (data notshown). These results indicate that Pin1 binds to themultifunctional protein p54nrb in mitosis.

To determine more precisely the region of p54nrb

involved in this interaction, the GST-p54 constructand its two fragments were phosphorylated or notby the mitotic extract before being submitted to ablot overlay experiment using Pin1 as a probe. Asshown in Figure 5, a strong Pin1 reaction wasobserved on the phosphorylated GST-Cter fragment

but the GST-Nter was hardly bound by the Pin1probe. To control the blot overlay experiment and toshow that Pin1 interaction was driven by its WWdomain, the W34A-Pin1 protein, mutated in thisdomain, was overlaid on an equivalent blot and noreaction was observed (data not shown).

Cdk1 is involved in mitotic phosphorylation ofp54nrb and in Pin1 interaction

In an effort to identify the kinases involved in themitotic phosphorylation of p54nrb, we found thatthe addition of the cyclin-dependent protein kinaseinhibitor roscovitine to the mitotic extract decreasedsignificantly the phosphorylation of the GST-Cterin vitro, but not that of the GST-Nter fusion protein(Figure 6(a)). To corroborate the role for Cdk(s)in vivo, the effect of roscovitine was investigated onthe electrophoretic migration of p54nrb. As shownpreviously, p54nrb migrated as a slower band inextracts from cells arrested in mitosis by nocodazolecompared to asynchronous cells (compare the mostleft and the most right lanes of Figure 6(b)). Whenadded to the culture medium for the last period of

Figure 6. Cdk1 is involved inmitotic phosphorylation of p54nrb

and Pin1 binding. (a) The additionof the Cdk inhibitor roscovitine tothe in vitro phosphorylation assaydecreased significantly the phos-phorylation of the GST-Cter butslightly affected that of the GST-Nter fusion protein. (b) The in vivoeffects of roscovitine were evalu-ated by adding the inhibitor to theculture medium for the last one orthree hours of the incubationperiod with nocodazole to syn-chronize the cells. The mobilityshift of the p54nrb band, noticedbetween nocodazole-synchronizedand asynchronous cells (leftmostand rightmost lanes, respectively)was attenuated when roscovitinewas added at the end of thenocodazole incubation period.(c) The efficiency of cyclin B1depletion by antibody Ab-3, asrevealed by Western blot. (d) Thecyclin B1-enriched immunopreci-pitated fraction (IP) was as able asthe mitotic extract (ME) to generatethe CC-3 epitopes on the differentconstructs whereas the cyclin B1-

depleted mitotic extract (DME) could only do so on the GST-Nter protein. (e) The same fractions as in (d) were overlaidwith Pin1, showing that Pin1 specific binding to full length p54nrb (GST-p54) and to the GST-Cter fusion protein occurredafter phosphorylation by either the whole mitotic extract (ME) or the cyclin B1-enriched immunoprecipitated fraction(IP).


the nocodazole treatment, roscovitine induced aprogressive decrease of the upper band togetherwith an increase of the lower band (Figure 6(b)).

To identify the kinase(s) of the mitotic extract ableto phosphorylate p54nrb, we immunodepleted Cdk1from the extract by incubation with an anti-cyclinB1 antibody. The efficiency of immunodepletionwas high, as can be judged by immunoblotting(Figure 6(c)). The kinase activity of the immuno-precipitated fraction (IP in Figure 6(d)) and ofthe depleted mitotic extract (DME) on the GST-p54nrb constructs were compared to the unfractio-nated mitotic extract by CC-3 immunoblotting(Figure 6(d)). It can be seen that phosphorylationof both the GST-p54nrb and the GST-Cter constructswas considerably reduced by Cdk1 depletion andthat the cyclin B1 immunoprecipitate was asefficient as the whole mitotic extract in generatingthe CC-3 epitope. In contrast, not only the cyclin B1immunoprecipitate but also the depleted extractcould phosphorylate the GST-Nter fusion protein,suggesting that different kinases are involved in thegeneration of the CC-3 phosphoepitope of the N-terminal extremity of p54nrb. To verify if Pin1 couldrecognize all the phosphorylated fragments ofp54nrb, the same samples were subjected to blotoverlay using recombinant Pin1 as a probe.Figure 6(e) clearly shows that Pin1 binding isexclusive to the C-terminal extremity of p54nrb,after phosphorylation by either the whole mitotic

extract or the immunopurified Cdk1. Altogether,these in vitro and in vivo experiments stronglysuggest that Cdk1 is the mitotic kinase thatphosphorylates the Pin1 binding sites of the C-terminal extremity of p54nrb.

Pin1 binding to p54nrb requires more than onephosphorylated site

The carboxy terminus of p54nrb contains aproline-rich domain harboring three threonine-proline pairs that could represent Pin1 interactionsites. To explore this possibility, the mouse GST-Cterconstruct was submitted to site-directed muta-genesis to substitute the threonine residues inpositions 412, 430 and 452 by alanine, eitherindividually or in pairs (Figure 7(a)). The resultingC-ter peptides were phosphorylated in vitro by themitotic extract and submitted to a Pin1 blot overlayexperiment. As shown in Figure 7(b), the Pin1 probedetected all the p54nrb peptides mutated on a singlethreonine residue but none containing two mutatedthreonine residues. The CC-3 immunoreactivity ofthe various mutated proteins confirmed that theassay allowed the phosphorylation of every mutantform of the GST-Cter fusion protein (Figure 7(c)).The results obtained using this solid phase assaysuggest that at least two threonine residues of thethree T-P pairs of the p54nrb C-terminal extremityshould be phosphorylated for Pin1 binding.

Figure 7. Pin1 binding to p54nrb

requires more than one phosphory-lated site. (a) A schematic represen-tation of the different mutationsproduced on the C-terminal frag-ment of p54nrb. Three threonineresidues located at possible Pin1interaction sites (T-P pairs at posi-tions 412, 430 and 452) of the Cterminus of mouse p54nrb weresubstituted for alanine residueseither individually (single muta-tions, right panel) or in pairs(double mutations, left panel). Themutated GST-Cter proteins werephosphorylated with the mitoticextract and either submitted to anoverlay experiment using recombi-nant Pin1 as a probe (b) or to a CC-3Western blot to monitor the phos-phorylation status of the fusionproteins (c). Pin1 interaction withthe C-terminal extremity of p54nrb

was observed with any of the threeproteins mutated at a single site,whereas no reactivity was detectedwith the double-mutated con-structs, containing a single phos-phorylated threonine.


Discussion

The multi-functional nuclear protein p54nrb isphosphorylated in mitosis

Considerable evidence now indicates that manyof the processes occuring in the cell nucleus aretightly coupled35,36 and for this reason, it wasexpected that individual proteins could be involvedin multiple nuclear events. Several studies haveidentified PSF and p54nrb as such multifunctionalproteins. These proteins are involved, either indi-vidually or in combination, in a variety of nuclearprocesses into which their nucleic acid bindingproperties are mainly required and it has beenproposed that their different functions might beregulated by phosphorylation.1 PSF has beenshown to be differentially phosphorylated whethercells are dividing, blocked in G1/S or apoptotic37

and a recent study indicated that it is associatedwith PKC and serves as a substrate for this kinase.38

In addition, PSF has been picked up as a putativeprotein phosphatase PP1 target molecule in a two-hybrid screen,39 reinforcing the idea that it isinvolved in a phosphorylation-dependent nuclearmechanism. The evidence that p54nrb activity is

regulated by phosphorylation is more scarce. It hasbeen identified, together with PSF, as a member of asubset of tyrosine-phosphorylated proteins associ-ated with the nuclear envelope13 but a directdemonstration that p54nrb is serine or threoninephosphorylated was lacking. Yet, there are severalexperimental indications that this protein is sub-mitted to posttranslational modifications. Forinstance, recombinant p54nrb purified from bacteriawas not able to bind DNAwhereas the same proteinexpressed in a reticulocyte lysate could do so.4

Another indication comes from the observation thatboth bacterially expressed PSF and p54nrb could notbind to an RNA polymerase II CTD affinity column,unlike the corresponding proteins purified fromhuman cell extracts.12 Finally, a recent workreported that slow and fast-migrating forms ofp54nrb associate with the 5 0 splice sites within largecomplexes present in HeLa cell nuclear extracts.8

In our search for mitotic regulators and effectors,we affinity-purified several cellular phospho-proteins using mAb CC-3, shown to recognizemultiple antigens from mitotic cells in a phospho-dependent fashion.29 One of the proteins retainedon the affinity matrix was p54nrb and we provided,in the present study, several lines of evidence that


this protein is actually serine and/or threonine-phosphorylated during mitosis. First, p54nrb is abona fide CC-3 phosphoantigen, as demonstrated bythe Western blot CC-3 reactivity of the p54nrb

protein immunoprecipitated from mitotic cellswith the NMT-5 antibody. This demonstrationneeded to be done to rule out the possibility thatp54nrb co-purified with RNA polymerase II on theCC-3 affinity column since Emili and co-workersshowed that p54nrb could bind to the CTD, whichitself is a strong CC-3 phospho-dependent anti-gen.30 Second, the mitotic phosphoepitopes definedby mAbs MPM-2 and CC-3 could be generated onrecombinant p54nrb after incubation with a mitoticextract in the presence of ATP. These experimentsalso suggested that p54nrb was multiphosphoryl-ated, since both the N-terminal and C-terminalrecombinant fragments of the protein were CC-3reactive after phosphorylation with the mitoticextract and since MPM-2 recognized exclusivelythe C-terminal fragment. Third, as shown inFigures 2(b) and 6(b), the p54nrb band fromnocodazole-blocked HeLa cells migrated moreslowly than that of untreated cells, suggesting thatthe mitotic phosphorylation occurs in vivo. Finally,as we will discuss below, the demonstration that thePin1–p54nrb interaction is specific to mitoticallyarrested cells and mediated by Cdk1 furtherconfirms that p54nrb is regulated by phosphoryl-ation when cells enter mitosis.

p54nrb interacts with the prolyl isomerase Pin1

Mitosis phase p54nrb exhibited MPM-2 and CC-3antigenicity and could therefore represent a targetfor the peptidylprolyl isomerase Pin1. This possi-bility was verified by combining solid phasebinding (blot overlay) and pulldown assays. Whenused as a blot overlay probe, Pin1 was shown toreact with p54nrb immunoprecipitated from mitoticbut not from interphase cells (Figure 3). In addition,p54nrb was pulled down by GST-Pin1 beads afterincubation with lysates prepared from mitoticallyarrested but not from asynchronous cells (Figure 4).These results strongly suggest that p54nrb and Pin1directly interact in mitotic cells. In addition, theoverlay experiment presented in Figure 3 revealedan interaction between Pin1 and an unidentified100 kDa protein that was co-immunoprecipitatedwith p54nrb from mitotic cell extracts using theNMT-5 antibody. The possibility that this proteincorresponds to the splicing factor PSF, knownto interact with p54nrb, is currently underinvestigation.

As we were able to generate a CC-3 epitope inboth the N-terminal and the C-terminal recombi-nant fragments of p54nrb by a mitotic extract, it wasof interest to see if Pin1 could bind to bothextremities of p54nrb. Pin1 blot overlay experimentsclearly showed a strong interaction with the C-terminal fragment whereas the N-terminal frag-ment was barely reactive (Figure 5), in agreementwith the MPM-2 immunoreactivity pattern.

Cdk1 generates the Pin1 binding sites of p54nrb

in mitotic cells

As shown on Figure 6(a), the addition of the Cdkinhibitor roscovitine in our in vitro phosphorylationassay strongly decreased the phosphorylation of theC-terminal fragment but not that of the N-terminalfragment of p54nrb, as monitored by CC-3 immuno-reactivity. This observation suggests that a cyclin-dependent protein kinase was responsible for themitotic phosphorylation of the C-terminal moietyof p54nrb and that a different kinase generated theCC-3 phosphoepitope of the N-terminal extremity.The involvement of a Cdk in the in vivo mitoticphosphorylation of p54nrb was further corroboratedby the drastic alteration of its electrophoreticmobility noticed after adding roscovitine to livingcells during the M-phase synchronization pro-cedure in the presence of nocodazole. The identifi-cation of Cdk1 as the mitotic kinase involved in thephosphorylation of the C-terminal end of p54nrb

came from immunodepletion studies of the mitoticextract used as a source of kinases with an anti-cyclin B1 antibody. As shown in Figure 6, theimmunodepletion of Cdk1 abolished both CC-3 andPin1 reactivities on the C-terminal extremity of theprotein and these reactivities were recovered whenthe GST-p54nrb and the GST-CTer fusion proteinswere incubated with the anti-cyclin B1 immuno-precipitated fraction.

The interaction between Pin1 and p54nrb ismodulated by multisite phosphorylation

The examination of the amino acid sequence ofthe C terminus of mouse p54nrb revealed thepresence of three proline residues preceded bythreonine residues susceptible to phosphorylationand Pin1 binding (P box in Figure 7). These T-Ppairs are located at positions 412, 430 and 452 ina C-terminal stretch of the mouse p54nrb polypep-tide, which contains a total of 473 amino acidresidues (compared to positions 410, 428 and 450 inthe 471-aa human ortholog2). To explore thepossibility that they were phosphorylated duringmitosis, the three threonine residues were substi-tuted for alanine, either individually or in pairs, andsubmitted to in vitro mitotic phosphorylation. Whenany two of the three-threonine residues weresimultaneously mutated, CC-3 reactivity couldstill be elicited on the resulting peptide, indicatingthat each threonine residue of the stretch isphosphorylable by the mitotic kinase to generatethe CC-3 epitope. However, Pin1 did not bind toany of these double mutants. In contrast, not one ofthe three single mutations abolished the binding ofPin1 to p54nrb, suggesting that the interactionbetween Pin1 and p54nrb is regulated by multisitephosphorylation. Similar conclusions were drawnby He and co-workers, who showed that the mitoticphosphorylation of NHERF-1 at serine 279 andserine 301 by Cdk1 regulated its association withPin1.40 Intriguingly, in both p54nrb and NHERF-1


proteins, the sites of Pin1 binding were separated bya stretch of 18–22 amino acid residues and a similardistance was found between interaction sites ofother Pin1 targets, including cdc25 (threonine 48and threonine 67)41 and tau (threonine 212 andthreonine 231).42 It would be of interest to verifywhether the cooperativity between interaction sitescould be a general model for the action of Pin1, as ithas been recently proposed.42

In summary, we have demonstrated that p54nrb ismultiphosphorylated during mitosis and we haveidentified Cdk1 as the kinase that phosphorylatesthreonine residues at position 412, 430 and 452 of itscarboxy terminus, responsible for its interactionwith the prolyl isomerase Pin1. In addition, Pin1interaction with p54nrb was shown to be favored bythe presence of at least two of the phosphorylatedthreonine residues, suggesting the existence ofa regulation mechanism based on multisitephosphorylation.

Materials and Methods

Cell culture and synchronization

HeLa cells (American Type Culture Collection) werecultured in Iscove’s Modified Dulbecco’s Medium(Gibco) supplemented with 10% FBS (Medicorp) andkept at 37 8C in 5% CO2. Cells were blocked in mitosis bythe addition of nocodazole (0.04 mg/ml) for 16 hours. Insome experiments, roscovitine (25 mM) was added to theculture medium for the last one or three hours of thenocodazole incubation period.

Molecular cloning, mutagenesis and proteinexpression

Constructs for the bacterial expression of mouse p54nrb

and its C andN-terminal fragments were kindly providedby F. Moreau-Gachelin3 and subcloned into a pGex-4T1vector. They encode fusion proteins between GST andp54nrb (GST-p54nrb), and between GST and p54nrb aminoacid residues 1–238 or 226–473 (GST-Nter or GST-Cter,respectively). The plasmids were transformed into pLysSEscherichia coli competent cells (Novagen) for proteinexpression under isopropyl-b-D-thiolgalactopyranosideinduction. The expressed fusion proteins were purified ona glutathione Sepharose column according to the manu-facturer’s instruction manual (Amersham Biosciences).The Pin1 fusion protein was constructed either into pGex-4T1 or pET19b vectors as described.30 The Pin1 mutantW34A, impaired in its binding activity, was kindlyprovided by Dr O. Bensaude. The His-tagged Pin1 fusionproteins were purified using His-binding columns asindicated by the manufacturer (Novagen). Mutants of theC-terminal p54nrb fusion protein were obtained by Thr-Ala substitutions of the three possible Pin1 binding siteseither individually or by pairs (Figure 7(a)). They wereprepared using a site-directed two-step mutagenesis PCRmethod with the following forward mutagenic primers(the mutated codon is underlined): T412A (5 0-CCA GCTGGT GCC CCA GCT CCT C-3 0), T430A (5 0-CT TTG GGATTGGCCCCAACAACTGAAC-3 0) and T452A (5 0-GCAATT GGT GGA GCT CCT CCT GCA TTC-3 0). In the firststep, two distinct PCR reactions were carried out usingone mutagenic primer or its complementary reverse

primer and a primer corresponding to each extremityof the GST-Cter construct. In the second, the resultingPCR products were combined and amplified with theextremity primers.

Antibodies

mAb CC-3 has been produced in our laboratory and itscharacterization has been already described.29,30 mAbMPM-2 was purchased from Upstate Biotechnology.Generation of the mAb NMT-1 and of the rabbitpolyclonal antibody NMT-5 against human p54nrb hasbeen described.10 The anti-Pin1 polyclonal antibody wasobtained by immunizing a rabbit with purified full-lengthrecombinant Pin1 protein.30 The anti-cyclin B1 mAb Ab-3was purchased from NeoMarkers.

Sample preparation, immunoprecipitation andimmunoblotting

For whole cell extracts, mitotic or unsynchronizedHeLa cells were directly solubilized in SDS-sample buffer(63 mM Tris–HCl (pH 6.8), 2.3% (w/v) SDS, 10% (v/v)glycerol and 5% b-mercaptoethanol) and boiled for fiveminutes. For immunoprecipitation experiments, cellswere solubilized in IP buffer (50 mM Tris–HCl (pH 7.5),150 mM NaCl, 5 mM EDTA, 0.2% NaF, 0.8% SDS, 1 mMEGTA, 1! protease inhibitor cocktail (Roche Diagnostics)and 40 nM microcystin) and briefly boiled to allowcomplete lysis. To neutralize the SDS and allow anti-gen–antibody interactions, one volume of Triton buffer(50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA,0.2% NaF, 4% Triton X-100, 1 mM EGTA, 1! proteaseinhibitor cocktail and 40 nM microcystin) was added tothe lysates, which were cleared by centrifugation at16,000 g for five minutes. The cleared supernatants werethen incubated for one hour at room temperature withthe primary antibodies first coated on goat anti-mouseIgG-agarose (Sigma-Aldrich). The beads were extensivelywashed with the Triton buffer before solubilization inthe SDS-sample buffer. For immunoblotting, thesamples were resolved by 10% (w/v) or 15% SDS-PAGE(acrylamide:bisZ30 : 0.15) and electrotransferred to nitro-cellulose membranes. The blots were blocked in 1%blocking reagent (Roche Diagnostics) diluted in Tris-buffered saline (10 mM Tris, (pH 7.6), 150 mM NaCl)containing 0.05% Tween-20 (TBS-T) before probing withthe primary antibodies for one hour at room temperaturein the same buffer. After washing, horseradish peroxy-dase-conjugated anti-mouse or anti-rabbit secondaryantibodies were added, incubated for 45 minutes andwashed in the TBS-T before chemiluminescence detection(Roche Diagnostics). For the blot overlay experiments, thenitrocellulose membranes were first renatured overnightat 4 8C in Blotto (5% lyophilized skim milk dissolved inPBS, pH 7.1) containing 0.2% Tween-20, 1 mM DTT and1 mM PMSF and then incubated with the wild-type Pin1or the W34A Pin1 mutant protein at a final concentrationof 1.5 mg/ml, for two hours at 4 8C. To diminish non-specific background, the membranes were washedseveral times in Blotto before incubation with the anti-Pin1 antibody at a 1 : 10,000 dilution for one hour at roomtemperature and chemiluminescence detection asdescribed above.

In vitro phosphorylation

Themitotic extract used for the in vitro phosphorylationexperiments was prepared from nocodazole-arrested


HeLa cells homogeneized in three to five pellet volumesof extraction buffer (50 mM Tris (pH 7.4), 250 mM NaCl,1 mM EDTA, 50 mM NaF, 1 mM DTT, 0,1% Triton X-100,1! protease inhibitor cocktail and 40 nM microcystin) byseveral passages through a 25G needle before clearing bya 30 minute centrifugation at 16,000g. The phosphoryl-ation assay was made with approximately 1 mg ofrecombinant protein in phosphorylation buffer (50 mMTris, pH 7.5, 10 mM MgCl2, 1 mM EGTA, 2 mM DTT,40 mM b-glycerophosphate, 20 mM r-nitrophenylpho-sphate, 0.1 mM sodium vanadate) supplemented with20 nM microcystin, 0.7 mM ATP and 5 ml of mitoticextract. The phosphorylation reactions (25 ml, totalvolume) were carried out at 30 8C for one hour. In someexperiments, roscovitine was added at a final concen-tration of 25 mM. The phosphorylated GST-tagged fusionproteins were purified on a glutathione Sepharosecolumn before solubilization in SDS-sample buffer. ForCdk1 depletion studies, the mitotic extract was incubatedwith the anti-cyclin B1mAbAb-3 previously immobilizedon goat anti-mouse IgG-agarose beads. Three fractionswere kept for the kination assays: the initial mitoticextract, the cyclin B1 immunoprecipitate and the depletedsupernatant.

Pull-down experiments

Whole mitotic or asynchronous cell homogenates weresolubilized in a lysis buffer (50 mM Tris (pH 7.1), 1%Triton X-100, 150 mM NaCl, 1 mM DTT, 1! proteaseinhibitor cocktail and 40 nM microcystin), incubated 15minutes on ice and passed through a 25G needle. Thelysates were cleared and incubated with GST-taggedPin1 recombinant proteins captured on glutathioneSepharose beads for two hours at 4 8C. The pulled downcomplex was extensively washed with lysis buffer beforesolubilization in SDS-sample buffer.

Acknowledgements

The authors gratefully acknowledge F. Moreau-Gachelin for providing the p54nrb cDNA constructsand SebastienMichaud for helpful discussions. Thisresearch was supported by the Natural Sciences andEngineering Research Council of Canada (grant122625-01 to M.V. and studentships to A.P., A.L.A.and S.B.L.).

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Edited by J. Karn

(Received 29 September 2004; received in revised form 14 December 2004; accepted 15 December 2004)

Documents

The Multifunctional Nuclear Protein p54nrb is Multiphosphorylated in Mitosis and Interacts with the Mitotic Regulator Pin1