Ringo/Cyclin-dependentKinaseandMitogen-activated ...logical cell cycle progression. Cell cycle...

Ringo/Cyclin-dependent Kinase and Mitogen-activatedProtein Kinase Signaling Pathways Regulate the Activity ofthe Cell Fate Determinant Musashi to Promote Cell CycleRe-entry in Xenopus Oocytes*□S

Received for publication, September 2, 2011, and in revised form, December 29, 2011 Published, JBC Papers in Press, January 3, 2012, DOI 10.1074/jbc.M111.300681

Karthik Arumugam‡1,2, Melanie C. MacNicol§¶1, Yiying Wang‡3, Chad E. Cragle�, Alan J. Tackett**, Linda L. Hardy§,and Angus M. MacNicol‡§‡‡4

From the ‡Department of Physiology and Biophysics, §Department of Neurobiology and Developmental Sciences, ¶Center forTranslational Neuroscience, �Interdisciplinary BioSciences Graduate Program, **Department of Biochemistry and MolecularBiology, and ‡‡Winthrop P. Rockefeller Cancer Institute, University of Arkansas for Medical Sciences, Little Rock, Arkansas 722205

Background: The mechanisms that regulate the activity of the mRNA translational regulator, Musashi, are unknown.Results:Musashi is activated by Ringo/cyclin-dependent kinase and MAP kinase signaling.Conclusion: Musashi-directed mRNA translation induces MAP kinase signaling and establishes a positive feedback loop toamplify Musashi activity.Significance:Musashi activation to promote translation of target mRNAs presents a potential target for the control of patho-logical cell cycle progression.

Cell cycle re-entry during vertebrate oocyte maturation ismediated through translational activation of select targetmRNAs, culminating in the activationofmitogen-activatedpro-tein kinase and cyclin B/cyclin-dependent kinase (CDK) signal-ing. The temporal order of targetedmRNA translation is crucialfor cell cycle progression and is determined by the timing ofactivation of distinct mRNA-binding proteins. We havepreviously shown in oocytes from Xenopus laevis that themRNA-binding proteinMusashi targets translational activationof early class mRNAs including the mRNA encoding the Mosproto-oncogene. However, the molecular mechanism by whichMusashi function is activated is unknown. We report here thatactivation of Musashi1 is mediated by Ringo/CDK signaling,revealing a novel role for early Ringo/CDK function. Interest-ingly, Musashi1 activation is subsequently sustained throughmitogen-activated protein kinase signaling, the downstreameffector of Mos mRNA translation, thus establishing a positivefeedback loop to amplify Musashi function. The identified reg-ulatory sites are present in mammalian Musashi proteins, andour data suggest that phosphorylation may represent an evolu-tionarily conserved mechanism to control Musashi-dependenttarget mRNA translation.

Vertebrate oocytes are stored in an immature, growth-ar-rested state at the G2/M transition, and their maturation andfertilization competence requires cell cycle re-entry and pro-gression to metaphase of meiosis II (1, 2). Oocyte cell cyclere-entry occurs in the absence of active gene transcription, andthe proteins required for this process are synthesized from pre-existing mRNAs in a specific temporal pattern that is predom-inantly directed through regulatory sequences within themRNA 3�-untranslated regions (3, 4). In the oocytes of the frogXenopus laevis, multiple mRNA-binding proteins have beenimplicated in sequence-specific mRNA translational control,including the developmental regulator Pumilio, the stem cellself-renewal factor Musashi, and the cytoplasmic polyadenyl-ation element-binding protein (CPEB)5 (5–8). However, themechanism by which the function of thesemRNA translationalcontrol proteins is coordinated to produce a temporally inte-grated pattern of protein expression is not fully understood(9, 10).The timing of progesterone-stimulated translational activa-

tion of targeted Xenopusmaternal mRNAs during oocyte mat-uration can be classified as “early” (before germinal vesicle(nuclear) breakdown (GVBD), e.g. Mos) or “late” (coincidentwith, or after, GVBD e.g. cyclin B1) (11). Furthermore, late classmRNA translational activation is dependent upon prior trans-lation of early class mRNAs (11, 12). We have previously dem-onstrated that activation of Musashi function is required tomediate early class mRNA translation before GVBD and com-pletion of meiosis I (5). Musashi-dependent translation of theearly class Mos mRNA results in MAP kinase activation andsubsequent CPEB-mediated, late class mRNA translation (5,

* This work was supported, in whole or in part, by National Institutes of HealthGrants RO1 HD35688 (to A. M. M.) and RR020146 (to M. C. M.).

□S This article contains supplemental methodologies and Figs. 1–3.1 Both authors contributed equally to this work.2 Present address: Center for Genomic Regulation, Dept. of Differentiation

and Cancer, C/Dr. Aiguader 88, 08003 Barcelona, Spain.3 Present address: Division of Genetic and Reproductive Toxicology, National

Center for Toxicological Research, 3900 NCTR Rd., Jefferson, AR 72079.4 To whom correspondence should be addressed: Dept. of Neurobiology and

Developmental Science, University of Arkansas for Medical Sciences, 4301W Markham, Slot 814, Little Rock, AR 72205. Tel.: 501-686-8164; Fax: 501-686-6517; E-mail: [email protected].

5 The abbreviations used are: CPEB, cytoplasmic polyadenylation element-binding protein; GVBD, germinal vesicle (nuclear) breakdown; MPF,M-phase promoting factor; MBE, Musashi binding element; MAP, mitogen-activated protein; CDK, cyclin-dependent kinase.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 13, pp. 10639 –10649, March 23, 2012© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

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13). The sequential function ofMusashi- and CPEB-dependenttranslational control promotes and maintains M-phase pro-moting factor activity (MPF, cyclin B/CDK) and cell cycle pro-gression up to metaphase of meiosis II (9). However, the mech-anism by which the initial progesterone-stimulated triggeractivates Musashi function is unknown.An immediate early response to progesterone stimulation is

the inactivation of Pumilio as a repressor protein and transla-tion of the Pumilio target mRNA encoding Ringo/Speedy, anatypical activator of cyclin-dependent kinases 1 and 2 (Cdk1and Cdk2) (8, 14–16). Ringo/CDK has been recently shown tomediate direct phosphorylation and inhibition of the Myt1kinase (an inhibitor of MPF) to facilitate activation of MPF andGVBD (17). Amolecular link between Pumilio inactivation andactivation of Musashi has not been established, and indeed ithas not been clear if translation of the Ringo mRNA impingedupon Musashi activation or if Ringo and Musashi acted in sep-arate pathways leading to MPF activation and GVBD.In this studywedetermined if translation of theRingomRNA

mediates Musashi activation in response to progesterone stim-ulation. Utilizing tandemmass spectrometry, we found that anevolutionarily conserved serine at position 322 (Ser-322) in theXenopusMusashi1 protein undergoes progesterone-dependentphosphorylation coincident with the initiation of Musashi-de-pendent mRNA translational activation. We found that pre-venting phosphorylation of Ser-322 delayed cell cycle re-entryand progression through oocyte maturation. A second con-served site, Ser-297 also contributes to Musashi activation, asabrogation of both Ser-322 and Ser-297 phosphorylation inhib-ited progesterone-stimulated cell cycle reentry. Conversely,Musashi phospho-mimetic mutants of Ser-297 and Ser-322accelerate and potentiate cell cycle reentry. We demonstratethat Musashi phosphorylation and activation is initially medi-ated through Ringo/CDK signaling and is subsequently ampli-fied through MAP kinase signaling as the oocytes progressthrough the meiotic cell cycle. We further show that mamma-lian Musashi1 also undergoes regulated phosphorylation andthat a phospho-mimetic mutant of mammalian Musashi1 pro-motes translation of a Musashi target mRNA in NIH3T3 cells.

EXPERIMENTAL PROCEDURES

Plasmid Construction—Detailed methodologies employedare described in the supplemental material.Oocyte Culture and Microinjections—Xenopus oocytes were

isolated and cultured as described previously (18). Oocyteswere induced to mature with 2 �g/ml progesterone (19). Therate of GVBD was scored morphologically by observing theappearance of a white spot on the animal pole. Because oocytesfrom different frogs mature at different rates in response toprogesterone, the culture times were standardized betweenexperiments to the time taken for 50% of oocytes to undergoGVBD. Where indicated, oocytes were pretreated with 50 �M

MEK inhibitor, UO126 (Promega), or DMSO vehicle for 30min. Animal protocols were approved by the University ofArkansas for Medical Sciences Institutional Animal Care andUse committee in accordance with Federal regulations.Luciferase Reporter Assays—Mammalian NIH3T3 cells were

transiently co-transfectedwith plasmids encodingRenilla lucif-

erase, firefly luciferase fused to a 3�-UTR containing a Musashibinding element (MBE) designated Fluc-MBE, and either theGST moiety alone, a GST-tagged mammalian Musashi1(mMsi), or GST-tagged mammalian Musashi1 S337E, essen-tially as described previously (20). 24 h after transfection thecells were lysed, and samples were prepared for both proteinand total RNA analyses. For each protein sample luciferaseactivity wasmeasured in triplicate with the dual luciferase assaysystem, and the values were normalized (20). Values are shownrelative to the firefly luciferase plasmid co-transfected with theGST moiety, arbitrarily set to 100%. Error bars represent S.E.from three independent experiments. Semiquantitative PCRwas used to assess the relative levels of Fluc-MBE reportermRNA expression under each experimental transfectioncondition.Mass Spectrometry—Oocytes were microinjected RNA en-

coding a GST-tagged form of Xenopus Musashi1, and after an18-h culture period to allow expression of the introducedMusashi protein, oocytes were split into two pools and eitherleft untreated (time matched control) or stimulated with pro-gesterone (treated). Oocyte lysate was prepared when proges-terone oocytes had fully matured. The ectopic Musashi1 pro-tein was recovered over glutathione-Sepharose beads, resolvedby SDS-PAGE, and visualized by Coomassie-staining (supple-mental Fig. 1). Under these conditions, the GST-Musashi1 pro-tein appeared to undergo a progesterone-dependent mobilityshift. Control and treated GST-Musashi1 protein bands wereexcised, in-gel-digested with 100 ng of GluC, and prepared forMALDI mass spectrometric analysis (21, 22). MALDI massspectra were collected with a PerkinElmerSciex prOTOF,whereas MS2 and MS3 spectra were collected with a ThermovMALDI-LTQ. Phosphorylation of the GST-Musashi1 fusionprotein was mapped to Ser-524 on peptide 514–549 by moni-toring for a neutral loss of 98 Da in MS2 and sequencing of theneutral loss ion inMS3 (23). This site corresponds to Ser-322 ofthe native Musashi1 protein.Polyadenylation Assays—cDNAs for polyadenylation assays

were synthesized usingRNA ligation-coupled PCR as describedpreviously (24). The increase in PCR product length is specifi-cally due to extension of the poly(A) tail (24, 25). The primersfor GST reporter mRNAs, endogenous Mos, Wee1, and cyclinA1, have been described previously (24).Antisense Oligodeoxynucleotide Injections—Antisense oli-

godeoxynucleotides targeting endogenous Musashi1 andMusashi2 mRNAs, Ringo mRNA, or a randomized control oli-gonucleotide sequence 5�-TAGAGAAGATAATCGTCATC-TTA-3� were employed as previously described (13, 14).Oocytes were incubated at 18 °C for 16 h followed by injectionof mRNA encoding GST rescue proteins with or without pro-gesterone treatment as indicated.Antibodies—Antisera for phosphorylation-specific Musashi1

Ser-322were generated by immunizing rabbitswith the peptideVSSYISAAS(phospho)PAPSTGF (Proteintech Group Inc.).The antibodies were affinity-purified through a peptide affinitycolumn and used at 1:1000. Abcam antibodies to Musashi1were used at 1:1000. Sigma tubulin antibodies were used at1:20,000. The phospho-specific Cdc2 antibody (Cell Signaling)was used at 1:1000 and detects the inhibitory Tyr15 phos-

Regulated Phosphorylation and Activation of Musashi

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phorylation. The phospho-specific MAP kinase antibody (CellSignaling) was used at 1:1000 and detects the activating phos-phorylations at Thr-202/Tyr-204. The GST and Mos antibod-ies (Santa Cruz) were used at 1:1000 and 1:100, respectively.The Xenopus Ringo antibody was a generous gift from Dr.Angel Nebreda and used at 1:1000.Western Blotting—Oocytes were lysed in Nonidet P-40 lysis

buffer containing sodium vanadate and a protease inhibitormixture (Sigma) (26). Where indicated, a portion of the lysatewas transferred to STAT-60 for RNA extraction (19). Proteinlysates were then spun, clarified, and transferred immediatelyto 1� sample buffer (Nupage). The lysates were run on a 10%Nupage gel and transferred to a 0.2-�m-pore-size nitrocellu-lose filter (Protran; Midwest Scientific). The membrane wasblocked with 1% bovine serum albumin (Sigma) in TBST for 60min at room temperature. After incubation with primary anti-body, filters were incubated with horseradish peroxidase-con-jugated secondary antibody using enhanced chemilumines-cence in a Fluorchem 8000 Advanced Imager (Alpha InnotechCorp.).Statistical Analyses—All quantitated data are presented as

the mean � S.E. Statistical significance was assessed by one-way analysis of variance followed by the Bonferroni post hoctest or by Student’s t test when only two groupswere compared.A probability of p � 0.05 was adopted for statisticalsignificance.

RESULTS

Although ectopic expression of Musashi1 rescues early classmRNA translation and oocytematuration inMusashi antisenseoligonucleotide-treated oocytes (13), expression of Musashi isnot sufficient to rescue maturation in the absence of progester-one stimulation (Fig. 1A). This result indicates that Musashitarget mRNAs are not translated in response to Musashi pro-tein synthesis per se but require a progesterone-dependent acti-vation process. To determine whether Musashi is subject toprogesterone-stimulated activating phosphorylation, we uti-lized tandemmass spectrometry to compare Musashi1 proteinisolated from immature and from progesterone-stimulatedoocytes. In the 60%of the protein sequence covered by themassspectrometry analysis, we identified a unique site of phospho-rylation in progesterone-treated samples that was mapped toserine 322 (Ser-322) of the XenopusMusashi1 protein (supple-mental Fig. 1). We note that this serine residue and the imme-diate flanking amino acids (AASP) in Xenopus Musashi1 areconserved in mammalian Musashi1 proteins (Fig. 1B). Thephosphorylation site is also present in Musashi2 proteins,although with some divergence from Musashi1 in the sur-rounding amino acids (Fig. 1B). We generated an antibody to aMusashi1 peptide phosphorylated on the Ser-322 residue, andthrough Western blotting of oocyte lysate we verified thatMusashi1 Ser-322 phosphorylation was specific to progester-one-stimulated oocytes (Fig. 1C). The specificity of the anti-body was demonstrated through expression of a non-phospho-rylatable mutant Musashi1 (where the serine residue waschanged to an alanine, S322A), which was not detected withthe phospho-specific antiserum in progesterone-stimulatedoocytes (Fig. 1C). The phosphorylation of expressed Musashi

Ser-322 occurred early inmaturation before GVBD, coincidentwith the initiation of early class Mos mRNA polyadenylation(Fig. 1D). We confirmed that the phosphorylation of endoge-nous Musashi1 on Ser-322 also increased before GVBD (Fig.1E). These findings temporally position this modification tomediate activation of Musashi translational control function.To determine the role of Ser-322 phosphorylation in control

of XenopusMusashi function, we expressed the non-phospho-rylatable S322A mutant Musashi1 in Musashi antisense oligo-nucleotide-treated oocytes. In contrast to oocytes expressingwild-type Musashi1, oocytes expressing Musashi1 S322A weresignificantly compromised for rescue of progesterone-stimu-lated maturation (Fig. 2A). Conversely, expression of a phos-pho-mimeticmutant protein (Musashi1 S322E) accelerated therate of progesterone-stimulated maturation when comparedwith expression of the wild-type Musashi1 protein (Fig. 2B).This acceleration of cell cycle progression correlated with anincrease in the rate and extent of polyadenylation and transla-tion of the Musashi target mRNA, Mos (Fig. 2, C and D). Weconclude that progesterone-stimulated phosphorylation ofSer-322 contributes to the activation of Musashi function,resulting in target mRNA translation and oocyte maturation.We next sought to identify the progesterone-stimulated sig-

naling pathway responsible for Musashi Ser-322 phosphoryla-tion. The Ser-322 residue is located within a consensus motiffor a proline-directed kinase, such as MAP kinase. However,Mos, the primary MAP kinase activator in oocytes (27–29), isitself regulated through translational activation by Musashi,suggesting that Musashi must be initially activated by a MAPkinase-independent mechanism (5). Consistent with thishypothesis, it has been previously shown that treatment ofoocytes with the MAP kinase signaling inhibitor UO126 doesnot prevent initial polyadenylation or translation of the MosmRNA (5, 30).Alternate progesterone-stimulated, proline-directed kinases

are the cyclin-dependent kinases (CDK1 and CDK2) that areinitially activated through synthesis of the non-cyclin protein,Ringo (14, 16). Ringo synthesis has been reported to precedeand be necessary for progesterone-stimulated translation of theMos mRNA (8), thus positioning Ringo/CDK as a potentialupstream activator of Musashi. Ectopic expression of Ringo issufficient to drive oocyte maturation and translational activa-tion of the Mos mRNA independently of progesterone stimu-lation (8, 14, 16). We observed that inhibition of Musashi func-tion, through expression of a dominant inhibitory form ofMusashi (N-Msi (5)) blocked Ringo-inducedMos mRNA poly-adenylation and delayed oocyte maturation (Fig. 3A). No poly-adenylation of the Mos mRNA occurred even after the delayedmaturation in Ringo injected oocytes (Fig. 3A, right panel).Consistent withMusashi functioning downstream of Ringo, wenote that inhibition of Musashi function with N-Msi did notaffect the upstream process of progesterone-stimulated accu-mulation of Ringo protein (supplemental Fig. 2). Ectopicexpression of Ringowas sufficient to induce phosphorylation ofMusashi on Ser-322 independently of progesterone stimulation(Fig. 3B). Ringo-induced Musashi Ser-322 phosphorylationoccurred before GVBD, consistent with the timing of proges-terone-stimulated Musashi Ser-322 phosphorylation (Fig. 1).




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Conversely, pretreatment of oocytes with Ringo antisense oli-gonucleotides blocked progesterone-induced Musashi Ser-322phosphorylation (Fig. 3C). We conclude that Ringo/CDK isnecessary and sufficient to mediate Musashi activation beforeGVBD.

We determined that Ringo-induced Musashi Ser-322 phos-phorylation specifically required CDK activity, as a mutantRingo (D83A) that fails to bind and activate CDK (31) wasunable to induce Musashi phosphorylation (Fig. 3B). Consis-tent with a role for Ringo/CDK in Musashi phosphorylation

FIGURE 1. Musashi1 undergoes stimulus-dependent phosphorylation. A, Musashi is required to mediate oocyte maturation. Musashi function was atten-uated by injection of antisense DNA oligonucleotides targeting the endogenous Musashi1 and Musashi2 mRNAs as previously described (13). Oocytes werereinjected with water (no rescue) or RNA encoding GST-tagged wild-type Musashi1 and either left untreated (�prog) or stimulated with progesterone (�prog).The ability of the ectopic Musashi to rescue cell cycle progression was assessed (% GVBD). Results from two independent experiments are shown. A GSTWestern blot confirmed expression of the ectopic Musashi1 protein (arrowhead, lower panel). B, conservation of Ser-322 and flanking amino acids in vertebrateMusashi proteins is shown. The amino acid sequence flanking the identified serine phosphorylation site (bold) is conserved in human (hu), mouse (mu), Xenopus(Xl), and zebrafish (dr) Musashi1 and Musashi2 isoforms but not in C. elegans (Ce) or Drosophila (Dm). C, Musashi is phosphorylated on Ser-322 in response toprogesterone stimulation. Musashi (WT) or a non-phosphorylatable mutant Musashi (S332A) were expressed in oocytes as GST fusion proteins and thenstimulated with progesterone. Protein and RNA samples were prepared at the indicated time points. At 5.5 h, 50% of the injected oocytes had completed GVBD,and they were segregated based on whether they had (�) or had not (�) completed GVBD. A Western blot of oocyte lysates with antibody specific to theSer-322 phosphorylated form of Musashi1 demonstrates progesterone-stimulated phosphorylation that is not observed in the S322A mutant Musashi (upperpanel). A GST Western blot shows equivalent levels of the expressed proteins (lower panel). D, the Musashi mRNA target, Mos, is activated coincident withMusashi1 Ser-322 phosphorylation. Total RNA from samples shown in C was analyzed for polyadenylation of the endogenous Mos mRNA. Increased PCRproduct size indicates polyadenylation, and this initiates �3 h after progesterone stimulation (asterisk). E, endogenous Musashi1 was phosphorylated onSer-322 in response to progesterone stimulation. Uninjected oocytes were stimulated with progesterone, and Western blots of protein lysate prepared at theindicated time points were assayed with antisera specific for Ser-322 phosphorylated Musashi1 as described in C. Quantitation of maximum progesterone-induced changes in phospho-Musashi1 levels normalized to tubulin from the same samples revealed a 1.25 � 0.11-fold increase (Student’s t test; p � 0.05, n �4) relative to levels in immature oocytes.




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and activation of Musashi function, inhibition of CDK activitythrough expression of a constitutively active form of the CDKinhibitory kinase Wee1 (Wee107 (19)) prevented progester-one-stimulated Musashi Ser-322 phosphorylation and oocytematuration (Fig. 4A). These results are consistent with our ear-lier study where Wee107 blocked progesterone-induced MosmRNA translation (19). Taken together, these findings positionMusashi function downstream of progesterone-stimulatedRingo/CDK activation.Although our data support a model where Musashi acts

downstream of Ringo and upstream ofMosmRNA translation,a number of previous studies have implicated a role for MAPkinase in mediating Mos mRNA translation (5, 19, 30, 32–34).

To determinewhetherMusashi is activated in response toMAPkinase signaling, we expressed of an activator of MAP kinasesignaling, vRaf (an oncogenic form of Raf-1), in oocytes (35).We observed that vRaf inducedMusashi Ser-322 phosphoryla-tion in the absence of progesterone stimulation (Fig. 4A).Importantly, induction of Musashi Ser-322 phosphorylationthrough vRaf expression was not inhibited in oocytes co-ex-pressing Wee107, indicating that MAP kinase signaling is suf-ficient to induce Musashi Ser-322 phosphorylation independ-ently of CDK activity (Fig. 4A). To determine the contributionof MAP kinase to progesterone-stimulatedMusashi activation,Musashi Ser-322 phosphorylation was examined in oocytestreated with the MAP kinase kinase (MEK) inhibitor, UO126

FIGURE 2. Musashi1 Ser-322 phosphorylation facilitates oocyte maturation and target mRNA translational activation. A, inhibition of Musashi1 Ser-322phosphorylation attenuates oocyte maturation. Oocytes were injected with antisense oligonucleotides to ablate endogenous Musashi function and subse-quently reinjected with water (no rescue), GST-tagged wild-type Musashi1 (Msi WT), or the non-phosphorylatable mutant Musashi (Msi S322A) and scored forprogesterone-dependent maturation when 50% of Msi WT expressing oocytes reached GVBD. Error bars represent S.E. from four independent experiments(p � 0.01, Student’s t test). The Msi WT and S322A proteins were expressed to equivalent levels in the rescue assay as assessed by GST Western blotting (lowerpanel). A Western blot of the same lysates with tubulin antiserum confirmed equivalent protein loading. B, mutational mimicry of Musashi1 Ser-322 phosphor-ylation accelerates oocyte maturation. Oocytes were injected with Musashi antisense oligonucleotides as described in A and subsequently reinjected withwater (No rescue), GST wild-type Musashi1 (Msi WT), or the phosphomimetic mutant Musashi (Msi S322E), and progesterone-dependent maturation was scoredwhen 50% of Musashi S322E-expressing oocytes reached GVBD. Error bars represent S.E. from three independent experiments (p � 0.05, Student’s t test). TheMsi WT and S322E proteins were expressed to equivalent levels in the rescue assay as assessed by GST Western blot (lower panel). Western blot of the samelysates with MAP kinase antiserum confirmed equivalent protein loading. C, mutational mimicry of Musashi1 Ser-322 phosphorylation accelerates translationalactivation of the Mos mRNA. Total RNA was isolated from oocytes expressing GST Msi WT or the GST Msi S332E mutant protein described in B. Samples wereprepared when 50% of Msi S322E oocytes reached GVBD and segregated based on whether they had or had not completed GVBD. Samples from time-matchedMsi WT and water-injected (No rescue) were also prepared, and endogenous Mos mRNA polyadenylation was assessed. D, mutational mimicry of MusashiSer-322 phosphorylation enhances Mos protein accumulation. Protein lysates were isolated from oocytes expressing GST Msi WT or the GST Msi S322E protein,and Mos protein levels were determined by Western blot. Samples were prepared when 50% of the injected oocytes reached GVBD and segregated based onwhether they had or had not completed GVBD. Note the Msi S322E-expressing oocytes display higher Mos protein levels despite being harvested 30 min beforeMsi WT oocytes. Imm, immature oocytes.




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(36). Significant inhibition of MAP kinase signaling resulted inattenuation of progesterone-stimulatedMusashi Ser-322 phos-phorylation (Fig. 4B). However, a low level of Musashi Ser-322phosphorylation was reproducibly retained in UO126-treatedoocytes (Fig. 4B), supporting a model wherein both a MAPkinase-independent process (e.g. Ringo/CDK signaling) as wellas MAP kinase-dependent signaling contribute to full proges-terone-stimulated Musashi Ser-322 phosphorylation and acti-vation (Fig. 4C).The residual function of the Musashi S322A mutant protein

in the rescue assay (Fig. 2A) suggested that one or more addi-tional modifications of the Musashi1 protein occurred inresponse to progesterone stimulation. We examined theMusashi1 protein sequence not covered by the mass spectrom-etry analyses and sought to identify additional CDK and MAPkinase serine/proline or threonine/proline target motifs. Onlyone such site was conserved in all vertebrate Musashi1 andMusashi2 isoforms and corresponded to Ser-297 of the Xeno-pus Musashi1 protein (Fig. 5A). This site is also conserved inCaenorhabditis elegans Musashi (as a threonine/proline pair).Mutational substitution of both Ser-297 and Ser-322 to alanineresidues (Musashi1 S297A/S322A) completely abrogated res-cue of progesterone-stimulated cell cycle progression inMusashi antisense-treated oocytes (Fig. 5B). The effect of thedouble mutant upon rescue of Musashi function was greater

than the effect of the single S322A mutant (see Fig. 2A). Nopolyadenylation of the endogenous Mos mRNA was observedin the Musashi1 S297A/S322A-expressing oocytes (Fig. 5C),indicating that phosphorylation of Musashi1 is linked to initia-tion of target mRNA polyadenylation. Conversely, expressionof the phospho-mimetic Musashi1 S297E/S322E doublemutant accelerated progesterone-stimulated oocyte matura-tion compared with expression of the wild-type Musashi pro-tein (Fig. 5D). Together, our data suggest that phosphorylationof Ser-297 as well as phosphorylation of Ser-322 is necessaryfor activation of Musashi1 in response to progesteronestimulation.We have previously shown that expressed mammalian

Musashi1 can functionally compensate for loss of endogenousMusashi function in Xenopus oocytes and can direct stimulus-dependent translational activation of target mRNAs (20). Con-sistent with the conservation of the phosphorylation site andflanking amino acids (Fig. 1B), we observed that expressedmammalian Musashi1 undergoes Ser-337 phosphorylation inresponse to progesterone stimulation of oocytes (Fig. 6A, mam-malian Musashi Ser-337 is equivalent to Xenopus MusashiSer-322).To further pursue the functional consequences of phosphor-

ylation of mammalian Musashi1 on this conserved site, weemployed a mammalian NIH3T3 reporter assay system to

FIGURE 3. Musashi function is necessary for Ringo-induced early class mRNA translational activation. A, inhibition of Musashi blocks Ringo-inducedtranslational activation of the Mos mRNA. Immature oocytes were injected with RNA encoding the dominant inhibitory Musashi (N-Msi) or with water,incubated to allow expression of the N-Msi protein, and subsequently reinjected with RNA encoding Ringo. Total RNA was prepared at various times after RingoRNA injection, and progression through maturation (GVBD) was assessed. The time required for 50% of the oocyte population to reach GVBD was significantlydelayed in N-Msi-expressing oocytes (7 versus 4 h). Polyadenylation of the endogenous Mos mRNA was assessed as described in the legend to Fig. 2C. In theN-Msi expressing oocytes, Ringo did not induce polyadenylation of the Mos mRNA, and deadenylation of the Mos mRNA was observed. B, Ringo/CDK inducesMusashi Ser-322 phosphorylation. Immature oocytes were injected with RNA encoding GST tagged Musashi1 and incubated overnight. The oocytes were thenleft untreated (Imm) or re-injected with RNA encoding Ringo or with inactive Ringo D83A, which does not activate CDK activity, as indicated, and time-matchedprotein lysates were prepared when 50% of Ringo-injected oocytes completed GVBD. Ringo-injected oocytes were segregated based on whether they had (�)or had not completed GVBD (�). Ringo D83A-injected oocytes did not mature. Western blotting was performed with appropriate antisera to analyze phos-phorylation of Musashi1 Ser-322, phosphorylation (activation) of MAP kinase, ectopic Ringo protein expression, and expression of GST-Musashi1 protein asindicated. C, Ringo is required for progesterone-stimulated Musashi Ser-322 phosphorylation. Immature oocytes were co-injected with RNA encoding GST-Musashi1 and either control antisense oligonucleotides (Con AS) or antisense oligonucleotides targeting the endogenous Ringo mRNA (Ringo AS). The injectedoocytes were then left untreated (Imm) or stimulated with progesterone (�prog). Oocytes were collected when the 50% of the control population had reachedGVBD (3.5 h) and were segregated along with time-matched Ringo AS injected oocytes, and protein lysates were analyzed for Musashi1 Ser-322 phosphory-lation and GST-Musashi expression by Western blot. No maturation of Ringo AS injected oocytes was observed at the time points analyzed.




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assess Musashi-dependent mRNA translational repression.NIH3T3 cells lack endogenous Musashi1 expression (37), butMusashi-dependent mRNA repression can be reconstitutedthrough ectopic expression ofMusashi protein and appropriatereporter mRNA constructs (20, 37). We observe increasedtranslation (decreased repression) of a MBE-containingreporter mRNA in NIH3T3 cells expressing a phospho-mi-meticmurineMusashi1 S337E protein compared with reportertranslation in NIH3T3 cells expressing wild-type murineMusashi1 (Fig. 6B). Because wild-typeMusashi1 andMusashi1Ser-337 were expressed to similar levels (Fig. 6C) and thereporter mRNA was expressed to equivalent levels in eachtransfected sample (Fig. 6D), the differences in translationalrepression reflect inherent differences in activity between thewild-type andmutantMusashi1 proteins. This finding suggeststhat phosphorylation of the Ser-337 site in mammalianMusashi1 represents a conserved regulatory mechanism topromote Musashi target mRNA translation in response toextracellular stimuli.

DISCUSSIONIn this study we present evidence that Musashi functions

downstream of progesterone-stimulated Ringo/CDK activa-

tion and upstream of MAP kinase and MPF (cyclin B/CDK)signaling in the promotion of oocyte cell cycle reentry (Fig.4C). Our findings further suggest the action of a signal amplifi-cation step, where progesterone-stimulated translation of theMusashi-dependent Mos mRNA and subsequent activation ofMAP kinase generates a positive feedback loop in which MAPkinase serves to induce additional Musashi activation. We pro-pose that Musashi activation is mediated through phosphory-lation of Musashi1 on Ser-297 and Ser-322. The sequentialmechanism ofMusashi activation by Ringo/CDK signaling andthen MAP kinase signaling is consistent with earlier observa-tions that translation of the Mos mRNA was initiated correctlyunder conditions of MAP kinase inhibition but then showedattenuated amplification (30). The dual mechanisms ofMusashi activation fit with current concepts of the mechanismof biological cell fate switch where a weak, initiating signal trig-gers a robust cellular response to effect an “all-or-none” cell fatetransition (38).Activation of Musashi presents a novel role for Ringo/CDK

signaling that compliments its role in attenuating the activityof the MPF inhibitor, Myt1, to promote MPF-induced cellcycle reentry (17). It remains to be determined if Ringo/CDK

FIGURE 4. Ringo/CDK and MAP kinase signaling pathways direct Musashi phosphorylation on Ser-322. A, MAP kinase signaling induces Musashi1 Ser-322phosphorylation independently of CDK. Immature oocytes were injected with RNA encoding GST-tagged Musashi1 in the presence or absence of RNA encoding theCDK inhibitor Wee107 and incubated overnight. Oocytes were then split into pools and left untreated, injected with RNA encoding vRaf, or stimulated with proges-terone as indicated. Progesterone- and vRaf-stimulated oocytes were segregated when the populations reached 50% GVBD (� and � GVBD, respectively). Proteinlysates were analyzed by Western blot for Musashi1 phosphorylation on Ser-322, MAP kinase phosphorylation (indicative of activation), Cdc2 phosphorylation(indicative of inactive cyclin B/CDK), and relative GST-Musashi1 expression. B, progesterone-stimulated Musashi1 Ser-322 phosphorylation is mediated by both MAPkinase-dependent and MAP kinase-independent signaling. Immature oocytes were injected with RNA encoding GST-Musashi1 and incubated overnight, then treatedwith UO126 to inhibit MAP kinase signaling (�) or DMSO vehicle control (�) for 30 min before progesterone addition. Time-matched protein lysates were preparedwhen 50% of the control oocytes reached GVBD. Control oocytes were segregated based on whether they had (�) or had not (�) completed GVBD. Lysates wereanalyzed by Western blot for phosphorylation of Musashi1 on Ser-322, phosphorylation (activation) of MAP kinase, total MAP kinase, and expression of GST-Musashi1in the same samples. C, shown is a schematic illustrating relevant progesterone-dependent signaling events impinging upon early class Musashi-mediated, mRNAtranslation before MPF (cyclin B/CDK) activation and oocyte GVBD. The points of experimental manipulation are shown along with the deduced Musashi amplificationloops. For the sake of clarity, the complex roles of CPEB are not indicated in the diagram (“Discussion”).




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directly phosphorylates Musashi1 or if an intermediarykinase is involved. Nonetheless, the serine/proline motif ofthe phosphorylation sites and the early timing of regulatoryMusashi phosphorylation are compatible with Musashi1being a direct target of Ringo/CDK. Interestingly, cyclinB/CDK (i.e.MPF) has been previously shown tomediateMos

mRNA translation via a positive feedback amplification loop(12, 19, 32). It is unlikely that cyclin B/CDK mediates earlyMusashi Ser-322 phosphorylation, however, as cyclinB/CDK activation normally occurs after the initiation ofMosmRNA translation (25) and cyclin B/CDK induction of MosmRNA translation requires MAP kinase (19). It is thus likely

FIGURE 5. Phosphorylation of Ser-322 and Ser-297 mediates Musashi activation. A, shown is conservation of Ser-297 and flanking amino acids in verte-brate Musashi proteins. Schematic alignment of Ser-297 (bold) and flanking amino acids in a range of organisms (see the legend to Fig. 1B) is shown. B,inhibition of Musashi1 Ser-297 and Ser-322 phosphorylation attenuates oocyte maturation. Oocytes were injected with antisense oligonucleotides to ablateendogenous Musashi function and subsequently reinjected with water (No rescue), GST-tagged wild-type Musashi1 (Msi WT), or the non-phosphorylatabledouble mutant Musashi (Msi S297A/S322A) and scored for progesterone-dependent maturation when 50% of Msi WT expressing oocytes reached GVBD. Errorbars represent S.E. from four independent experiments (p � 0.01, Student’s t test). The Msi WT and Msi S297A/S322A proteins were expressed to equivalentlevels in the rescue assay as assessed by GST Western blotting (lower panel). C, inhibition of Musashi1 Ser-297 and Ser-322 phosphorylation attenuates MosmRNA polyadenylation. Oocytes were treated with Musashi antisense oligonucleotides and subsequently reinjected as described in B. Total RNA samples wereprepared when 50% of Msi WT oocytes reached GVBD and segregated based on whether they had not or had completed GVBD (� and �, respectively).Samples from time-matched water injected (No rescue) as well as Msi S297A/S322A-injected oocytes were also prepared, and endogenous Mos mRNApolyadenylation was assessed. Uninjected oocyte samples were also analyzed as a positive control for progesterone-induced Mos mRNA polyadenylation. Anincrease in size of the PCR product in progesterone-treated oocytes is indicative of polyadenylation. Imm, immature oocyte. D, mutational mimicry of Musashi1Ser-297 and Ser-322 phosphorylation accelerates oocyte maturation. Oocytes were injected with Musashi antisense oligonucleotides as described in A andsubsequently reinjected with water (No rescue), GST wild-type Musashi1 (Msi WT), or the phosphomimetic double mutant Musashi (Msi S297E/S322E), andprogesterone-dependent maturation was scored when 50% of Musashi S297E/S322E-expressing oocytes reached GVBD. Error bars represent S.E. from threeindependent experiments (p � 0.05, Student’s t test). The Msi WT and Msi S297E/S322E proteins were expressed to equivalent levels in the rescue assay asassessed by GST Western blot (lower panel).




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that Ringo/CDK and cyclin B/CDK display differential sub-strate specificity toward Musashi1 (17).Ringo/CDK has been previously proposed to induce Mos

mRNA translation through activation of the CPEB (8). CPEB,however, functions relatively late inmaturation at or after com-pletion of GVBD and is not part of the trigger mechanismrequired for initialMosmRNA translation (13, 25).Weproposethat Ringo activates CPEB indirectly through Musashi-depen-dent activation of early Mos mRNA translation and the subse-quent MAP kinase signaling that is required for CPEB activa-tion (39). This indirect model for Ringo-induced CPEBactivation is consistent with the temporal control of mRNAtranslation being enforced by a hierarchy of sequential mRNAtranslational regulatory pathways (9). In this model progester-one stimulates de-repression of the Ringo mRNA through tar-geted inhibition of Pumilio repressor activity. Although it wasinitially proposed that Ringo mRNA repression was mediated

by Pumilio2 (8, 40), recent evidence suggests that Pumilio1mayalso serve this function (41). Translation of Ringo then inducesRingo/CDK activity, leading to the initial activation ofMusashiandMusashi-dependent mRNA translation, as reported in thisstudy. Musashi-dependent mRNA translation then mediatessubsequent CPEB activation and translation of late class, CPE-dependent mRNAs (5, 13). The coordinated action of thesemRNA translational regulators serves to promote andmaintainMPF activity and cell cycle progression.We note that deadenylation of theMosmRNAwas observed

in Ringo-injected oocytes co-expressing the inhibitory N-Msi(Fig. 3A). Curiously, although Ringo-induced Mos mRNA pol-yadenylation was also attenuated in Musashi1 S297A/S322A-expressing oocytes, no deadenylation was observed (supple-mental Fig. 3). The truncated N-Msi lacks the C-terminaldomain of Musashi1 and so may not interact with poly(A)polymerase to assemble an “activation” complex on target

FIGURE 6. Phospho-mimetic mammalian Musashi1 displays abrogated target mRNA repression in NIH3T3 cells. A, expressed murine Musashi1 isphosphorylated on Ser-337 in progesterone-stimulated oocytes. Immature Xenopus oocytes were injected with RNA encoding GST-tagged murine Musashi1(mMsi1) and were stimulated with progesterone (prog) or left untreated (Imm). When 50% of the progesterone-stimulated oocyte population completed GVBD,oocytes were segregated (� or � GVBD) and analyzed by Western blot with phospho-Ser-322 Musashi-specific antiserum and with GST antiserum to showlevels of the expressed protein (GST mMsi1). The progesterone-dependent appearance of Ser-337-phosphorylated mammalian Musashi1 is coincident with agel mobility shift of the expressed Musashi protein. B, phospho-mimetic Musashi1 S337E has reduced ability to repress translation of target mRNAs in NIH3T3cells. MBE-regulated firefly luciferase reporter mRNA was co-transfected with plasmids encoding GST (Control), wild-type murine Musashi1, or murineMusashi1 S337E, and after 48 h cell incubation luciferase activity was assessed and normalized to co-expressed Renilla luciferase (20). Luciferase activity in cellsexpressing wild-type mMusashi1 and mMusashi1 S337E was compared with luciferase activity in cell expressing the GST tag alone (set at 100%). Error barsrepresent S.E. from three independent experiments (p � 0.001, Student’s t test). C, GST and tubulin Western blot of lysates in B show equivalent proteinexpression. D, the levels of Firefly luciferase reporter mRNA under the control of a Musashi-binding element containing 3� UTR (Fluc-MBE) were determinedusing semiquantitative PCR. Total RNA was prepared from the indicated co-transfection conditons, and Fluc-MBE reporter mRNA was PCR-amplified fordifferent cycle numbers as indicated. The PCR products were visualized after separation through a 2% agarose gel. No significant differences in stability of theFluc-MBE construct (arrowhead) were detected in cells expressing the GST moiety alone (Control), GST wild-type Musashi1, or GST Musashi1 S337E. m, indicatesDNA marker lane.




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mRNAs and/or fail to protect them from attack by deadeny-lases. A link between the C-terminal domain and target mRNApolyadenylation is consistent with the failure of the Musashi1S297A/S322A mutant protein to mediate early class mRNApolyadenylation (Fig. 5C). Ongoing work in our laboratoryseeks to address the mechanism by which Musashi activatestargetmRNA translation, and these studiesmay provide insightinto this deadenylation phenomenon observed with N-Msi.The conservation of the identified phosphorylation sites in

Musashi2 protein isoforms (Figs. 1B and 5A) suggests that asimilar mechanism may contribute to activation of Musashi2.Indeed,we doobserve a progesterone-dependent retardation inMusashi2 proteinmobility byWestern blot, similar to that seenwith ectopic mammalian Musashi1 (Fig. 6A), suggesting thatMusashi2 does undergo regulated phosphorylation.6 However,due to differences in flanking amino acids, our Musashi1 Ser-322 phospho-specific antisera do not cross-react with theMusashi2 protein, and so we are currently generating antiserato directly explore the role of regulatory phosphorylation in theactivation of Musashi2.Although the Ser-297 site appears to be conserved (albeit as a

threonine) inC. elegansMusashi, no conservation of either Ser-297 or Ser-322 is found in the Drosophila Musashi proteinsequence (42). However, a Drosophila gene encoding RNAbinding protein 6 (RBP6-A and RBP6-C variants, accessionnumbersNM_168718 andNM_001104162) is closely related tovertebrateMusashi proteins, and both the Ser-297 and Ser-322sites as well as the following proline residues are conserved inRBP6. It remains to be determined whether RBP6 is subject toregulatory phosphorylation and indeed the extent to whichRBP6 functions likeMusashi to exert control of asymmetric celldivision in Drosophila.

We note that Musashi antisense-treated oocytes are com-pletely inhibited for progesterone-stimulated oocyte matura-tion, whereas Ringo antisense oligonucleotides only delayGVBD (13, 14). Paradoxically then, elimination of the functionof the downstreameffector protein (Musashi) appears to exert astronger inhibitory phenotype on progesterone-stimulatedoocyte maturation than elimination of its upstream activator(Ringo). It is formally possible that a Ringo-independent,redundant mechanism mediates progesterone-stimulatedMusashi activation before GVBD in the absence of Ringo (43).However, this seems unlikely as Ringo antisense treated oocytesare blocked for early Musashi-dependent Mos mRNA poly-adenylation and translation (8), and we see no evidence ofMusashi Ser-322 phosphorylation before GVBD in Ringo anti-sense-treated oocytes (Fig. 3C). An alternative possibility is thatMusashi may exert as yet uncharacterized effects in immatureoocytes that are required for subsequent progesterone-stimu-lated maturation.In mammalian systems Musashi has been proposed to pro-

mote both physiological stem cell self-renewal and pathologicaltumor growth through the repression of translation of targetmRNA encoding inhibitors of cell cycle progression (44–47).During differentiation of stem cells, Musashi function is

reversed, and target mRNAs are translated, although it is notknown at this time if the increased translation requiresMusashiphosphorylation as we observe in oocytes, or if it is simply aresult of Musashi protein down-regulation as Musashi expres-sion is much lower in differentiated cells (48, 49). However, wehave recently observed that Musashi reporter mRNAs aretranslationally activated in response to differentiation of mam-malian neural stem/progenitor cells and that this translationoccurs in the absence of concurrent Musashi protein down-regulation (20). Although the molecular mechanism by whichMusashi converts from a repressor to an activator of targetmRNA translation under these conditions has not been eluci-dated, our NIH3T3 cell assays suggest that phosphorylation ofmammalian Musashi1 may compromise the ability of the pro-tein to exert repression (Fig. 6B) and thus result in the transla-tion of target mRNAs.In summary, we report for the first time thatMusashi under-

goes regulated phosphorylation to promote target mRNAtranslation. During Xenopus oocyte maturation, this phospho-rylation is initiated by Ringo/CDK signaling and subsequentlyamplified and reinforced by a positive feedback loop mediatedby MAP kinase signaling. Based on our findings described inthis study, we propose that regulated phosphorylation ofMusashi on the identified evolutionarily conserved serine resi-dues may represent a common mechanism to control Musashifunction in diverse organisms and cellular contexts.

Acknowledgments—Mass spectrometry support was provided by theUniversity of Arkansas for Medical Sciences Proteomics Facilityand National Institutes of Health Grants P20RR015569 andP20RR16460. DNA sequencing was provided by the University ofArkansas for Medical Sciences Translational Research Institute sup-ported by National Institutes of Health National Center for ResearchResources Grant UL1 RR029884.

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Tackett, Linda L. Hardy and Angus M. MacNicolKarthik Arumugam, Melanie C. MacNicol, Yiying Wang, Chad E. Cragle, Alan J.

OocytesXenopusCell Cycle Re-entry in Pathways Regulate the Activity of the Cell Fate Determinant Musashi to Promote Ringo/Cyclin-dependent Kinase and Mitogen-activated Protein Kinase Signaling

doi: 10.1074/jbc.M111.300681 originally published online January 3, 20122012, 287:10639-10649.J. Biol. Chem.

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