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Keywords:

  • phosphorylation;
  • Xenopus;
  • embryo;
  • proteomics;
  • mass spectrometry

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES
  8. Supporting Information

Phosphorylation is universally used for controlling protein function, but knowledge of the phosphoproteome in vertebrate embryos has been limited. However, recent technical advances make it possible to define an organism's phosphoproteome at a more comprehensive level. Xenopus laevis offers established advantages for analyzing the regulation of protein function by phosphorylation. Functionally unbiased, comprehensive information about the Xenopus phosphoproteome would provide a powerful guide for future studies of phosphorylation in a developmental context. To this end, we performed a phosphoproteomic analysis of Xenopus oocytes, eggs, and embryos using recently developed mass spectrometry methods. We identified 1,441 phosphorylation sites present on 654 different Xenopus proteins, including hundreds of previously unknown phosphorylation sites. This approach identified several phosphorylation sites described in the literature and/or evolutionarily conserved in other organisms, validating the data's quality. These data will serve as a powerful resource for the exploration of phosphorylation and protein function within a developmental context. Developmental Dynamics 238:1433–1443, 2009. © 2009 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES
  8. Supporting Information

Many proteins are regulated by the reversible phosphorylation of specific serine, threonine, or tyrosine residues, including proteins that control the diverse and complex cellular processes that govern vertebrate development. For example, during Xenopus embryogenesis, the β-catenin protein preferentially accumulates in the nuclei of cells destined to form the dorsal and anterior structures of the embryo. Regulated phosphorylation of specific serine residues at the N-terminus of the β-catenin protein in response to Wnt signaling are required for this spatially controlled accumulation of β-catenin (Amit et al.,2002; Liu et al.,2002). Phosphorylation of CPEB (cytoplasmic polyadenylation element binding), a protein that binds to the 3′ untranslated regions of maternal mRNAs, leads to the translational activation of these stored mRNAs in response to the appropriate developmental cues (Hake and Richter,1994; Mendez et al.,2000; Sarkissian et al.,2004). A myriad of other examples exist that establish phosphorylation as a universal mechanism for controlling the functions or levels of proteins and the activity of key cellular pathways.

Xenopus embryos have served as a powerful model for examining the role of protein phosphorylation in the pathways and processes governing early vertebrate development. However, the studies of many important phosphorylation events described in the literature were guided initially by discoveries made in other systems. That is, although phosphorylation plays clear and important roles during the development of Xenopus embryos, only a small number of the phosphorylation events examined in this model organism have been identified directly from studies of this organism. Given the probability that many more phosphorylation events critical to vertebrate development remain undiscovered, a large and functionally unbiased data set identifying both phosphoproteins and the precise sites of their phosphorylation would provide a powerful foundation for future functional studies. Here, we begin to define the breadth and potential temporal and spatial dynamics of protein phosphorylation within the context of developing Xenopus embryos using recent advances in mass spectrometry and protein preparation that enable the accurate identification of phosphopeptides.

The methodological advances that facilitate the identification of specific phosphopeptides from complex mixtures include the use of metal-ion chromatography to enrich phosphorylated peptides and improvements in peptide fragmentation. In particular, the development of an electron-based dissociation method, electron capture dissociation (ECD; Zubarev et al.,1998), and more recently the related electron transfer dissociation (ETD; Coon et al.,2004,2005a,b; Syka et al.,2004) has allowed efficient cleavage of peptide backbone bonds without regard to phosphorylation and that leave phosphorylated residues intact (Good and Coon,2006; Chi et al.,2007; Good et al.,2007; Khidekel et al.,2007; Lecchi et al.,2007; Molina et al.,2007; Swaney et al.,2007; Phanstiel et al.,2008). Thus, the new methods allow both the phosphopeptide and the precise site(s) of phosphorylation to be identified.

We used ETD to identify 1,441 different phosphorylation sites present on 654 different proteins from Xenopus oocytes, eggs, and embryos. Additional analysis of gastrula stage embryos identified phosphorylation sites on an additional 541 proteins. The Xenopus phosphoproteins were involved in a diverse array of cellular functions. In particular, many of the identified phosphoproteins are known regulators of embryonic development including translational regulatory proteins, signaling proteins and transcription factors. Our results provide a data-rich foundation for exploiting the experimental advantages of Xenopus to examine regulatory phosphorylation events that direct vertebrate embryonic development and cellular function(s).

RESULTS AND DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES
  8. Supporting Information

Xenopus Phosphopeptide Analysis With Tandem Mass Spectrometry

To identify Xenopus phosphoproteins and phosphorylation sites, proteins from oocytes, eggs, and embryos were analyzed (Fig. 1A). Proteins were extracted from oocytes, eggs, stage 7 and stage 10.5 embryos (Fig. 1B) and treated with trypsin to generate peptides. The peptides were modified to create their corresponding methyl ester derivatives (Ficarro et al.,2002). This peptide modification serves to eliminate nonspecific binding during subsequent steps. Phosphopeptides were isolated by immobilized metal affinity chromatography (IMAC; Ficarro et al.,2002; Ndassa et al.,2006) and separated by reversed-phase chromatography before elution into the mass spectrometer by electrospray ionization (Martin et al.,2000). The mass spectrometer was set to continuously cycle through the acquisition of a full MS spectrum followed by subsequent ETD-MS/MS scans of the ten most abundant phosphopeptide cations observed. The resulting spectra were analyzed using the open mass spectrometry search algorithm (OMSSA; Geer et al.,2004) to identify the relevant phosphopeptides. Identified peptides with an e-value greater than zero were validated with a target-decoy searching method (Käll et al.,2008) and identified phosphopeptides with less than 5% false discovery rate were included in this current analysis. We report all hits at this false discovery rate to include potentially relevant regulatory proteins, and we believe it is warranted given the potential for functional analysis and validation in Xenopus. In addition, we have partitioned the data such that phosphopeptides with less than 0.5% false discovery rate have been indicated (Supp. Table S1, which is available online).

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Figure 1. Phosphoproteomic analysis of Xenopus oocytes, eggs, and embryos: A: Diagram depicting the stages of Xenopus laevis development examined. B: Diagram of the phosphoproteomic strategy used to analyze proteins from Xenopus laevis oocytes, eggs, and embryos. C: Freon extraction of protein samples preferentially removes vitellogenin proteins. Increasing amounts of protein samples from Xenopus eggs were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis before (lanes 1 to 3) and after Freon extraction (lanes 4 to 6). Two of the major vitellogenin proteins, lipovitellin 2B (31 kDa) and lipovitellin I (120 kDa), were removed upon treatment with Freon.

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In our initial analyses, the predominant phosphoproteins identified were vitellogenins; the abundant phosphoproteins stored in the cytoplasm of oocytes, eggs, and embryos for nutritive purposes during development. The presence of the vitellogenins prevented the efficient identification of other phosphoproteins. Therefore, we added another step to our protocol. Specifically, before trypsin treatment, the vitellogenins were removed using Freon (Fig. 1C; Evans and Kay,1991).

Identification of Phosphoproteins From Xenopus Oocytes, Eggs, and Embryos

Protein samples from Xenopus oocytes, eggs, stage 7 and stage 10.5 embryos were analyzed to identify 1,441 sites of phosphorylation from 1,133 phosphopeptides derived from 654 proteins (Fig. 1B; Table 1, Supp. Table S1). As predicted from proteomic studies in other systems (Olsen et al.,2006), the most prominent phosphorylation events occurred on serine (76% frequency) followed by threonine (21% frequency). Tyrosine phosphorylation events were rare, constituting only 2% of the sites identified (Table 1). These percentages did not change significantly over the course of development. Most of the phosphopeptides possessed a single phosphorylated residue (Table 1). However, 22% of the peptides contained at least two and as many as six phosphorylated residues (Tables 1–3). Such multi-site phosphorylations often occur in regulatory domains of proteins that must integrate multiple signaling inputs (Gingras et al.,1999; Liu et al.,2002; Zeng et al.,2005). For example, we observed that a peptide derived from the adenomatous polyposis coli (APC) protein was simultaneously phosphorylated on six closely spaced residues (Table 3). APC is a component of the Wnt signaling pathway and activation of Wnt signaling leads to the phosphorylation of APC on multiple residues (Brocardo and Henderson,2008).

Table 1. Phosphorylation Events Sorted by Stage
 OocyteEggBlastulaGastrulaAll stages
  1. Identification of Xenopus phosphoproteins from oocytes, eggs, and embryos. Summary of data from the phosphoproteomic analysis of Xenopus oocytes, eggs, stage 7 embryos, and stage 10.5 embryos. All the results for individual proteins are found in Supp. Table S1.

STY2983504623311441
S80%73%77%75%76%
T18%24%21%22%21%
Y2%2%2%3%2%
Unique peptides2412723602601133
Phosphorylation Events on each peptide     
 1193208280204885
 246566442208
 3310181344
 410012
Unique proteins195224281199654
Table 2. Identification of Xenopus Phosphoproteins From Manipulated Gastrula Stage Embryos
 OrganizerNonorganizerLiCl treatmentβ-Catenin morpholinoTotals
  1. Summary of data from the phosphoproteomic analysis of gastrula stage embryo fragments (organizer and nonorganizer) and manipulated embryos (LiCl treated and β-catenin morpholino injected). All the results for individual proteins are found in Supp. Table S1.

STY1473618846092001
S1262816464701523
T1875186121400
Y35521878
Unique peptides993335884971517
Unique proteins852724393571153
Table 3. Multiphosphorylated Peptides
Xenopus proteinAccession no.PhosphositesPhosphopeptide
Adenomatous polyposis coli (APC)AAB41671S2523, S2525, S2527HDIsRsHsESPSR
Adenomatous polyposis coli (APC)AAB41671S2804, T2806, T2808, T2809, S2811, S2814KRDsKtEttDsSGsQSPK
B-Raf (MGC86346)AAZ06667S355, S357sSsAPNVHINTIEPVNIDDLIQDP
Casein kinase epsilonNP001084228S405, S408ISAsQAsVPFDHLGK
CDK7AAI30135S170, T176SFGsPNRIYtHQVVTR
NDRG2Q7ZY73S324, T326, S328LSRsRtAsLSSEGNR
NDRG3Q6GQL1T329, S331SRtHsASSSGSMEIPR
PDCD4AAH56125S42, S43RTssRDSAR
RSK2AAH80017S366, S372DsPGIPAsANAHQLFR
4E-BP2AAH68624T33, T37TIPISDDDQLPHDYCTtPGGtLFS
4E-BP2AAH68624S61, T66, S79RTsPLAQtPPRRLPDIPGVTsPNTVVE
4E-BP2AAH68624T66, T78, S79RTSPLAQtPPRRLPDIPGVtsPNTVVE
Translation elongation factor 2 (eef2)AAH60025T57, T59AGETRFtDtRKDEQER

Some of the earliest mechanisms that regulate embryonic patterning (Weaver and Kimelman,2004; Thisse and Thisse,2005) occur during the gastrula stage of Xenopus development. Our interest in the regulation of these events (Lane and Sheets,2000; Mitchell and Sheets,2001; Lane et al.,2004; Mitchell et al.,2007) motivated us to perform additional analysis of proteins from gastrula stage embryos manipulated to perturb embryonic patterning. In particular, we analyzed phosphoproteins present in organizer and nonorganizer cells manually dissected from gastrula embryos (Table 2). Also, we analyzed the phosphoproteins from embryos treated with LiCl that enhances Wnt signaling and anterior development, and proteins from embryos injected with a β-catenin morpholino that disrupts Wnt signaling (Table 2). From these analyses, we identified an additional 541 Xenopus phosphoproteins (Table 2, Supp. Table S1).

Notably, many of the Xenopus phosphorylation events we identified corresponded to those observed in protein orthologs from mice and humans (Fig. 2; Tables 4, 5). For many of these proteins multiple lines of analysis, including cell-labeling experiments, the use of phospho-specific antibodies and mutation of the specific amino acids were used to demonstrate that these specific residues were phosphorylated primarily in the context of cultured cells (see Table 5, column 4). For example, serine 369 (S-369) of the Xenopus RSK-2 kinase was identified as a site of phosphorylation in our analysis (Fig. 2). The same residue in the mouse RSK-2 protein is phosphorylated, and this modification triggers RSK-2 activation in response to specific signaling events (Hauge and Frodin,2006; Hauge et al.,2007).

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Figure 2. Evolutionary conservation of Xenopus phosphorylation sites: A subset of the Xenopus phosphoproteins identified in this study (see Tables 4, 5) were compared with their mouse and human orthologs. Here, six modified peptides corresponding to the designated full-length proteins are shown. Identified sites of phosphorylation are lowercase and bold. The phosphorylation sites in the Xenopus proteins identified in this study were also phosphorylation sites in the mouse and human proteins Rsk-2 (Han et al.,2006), Fox-K2 (Dephoure et al.,2008), casein kinase II (β) (Lowery et al.,2007), HnRNP-K (Villen et al.,2007), Pumilio (Villen et al.,2007), and Mek2 (Beausoleil et al.,2006).

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Table 4. Evolutionary Conservation of Xenopus Phosphorylation Sites and Events
Xenopus proteinConserved phosphorylation site(s)aAccession no.
  • a

    A partial list of phosphorylation sites conserved in mouse and human. Phosphorylation site data was obtained from PhosphoSitePlus (www.phosphosite.org).

RPS6S-235, S-236, S-240, T-241, S-242, S244P39017
Api5S-465AAH77529
MCM4S-31P30664
c-RAFS-621AAH72748
MCM2S-139P55861
DHA1S-298, S-300, S-305AAI06671
stmn1S-37AAH54159
CaRHSP1S-36 S-38AAH44047
AldolaseS-35AAB31152
RSK-2S-369AAF15553
CIRP2Y-160AAH54250
casein kinase II (β)S-209NP_001084126
hnRNP-KS-257AAH44711.1
FOX-K2S-359Q7ZX03
PumilioS-704BAC57980
MEK2S-388NP_001080299.1
Table 5. Validation of Xenopus Phosphorylation Sites From the Analysis of Human and Mouse Proteinsa
Xenopus proteinAccession no.PhosphositesAnalogous human phosphositesAnalogous mouse phosphosites
SiteMethodsSiteMethods
  • a

    Phosphorylation site data were obtained from PhosphoSitePlus (www.phosphosite.org). MS, mass spectrometry; PA, phosphospecific antibody; CL, cell labeling; MT, mutagenesis and functional analysis.

A-RafBAD04840S573S582ms, pa, mtS580ms
B-Raf (MGC86346)AAZ06667S355S363msS346 
B-Raf (MGC86346)AAZ06667S357S365ms, pa, mtS348 
C-RafAAH72748S612S621ms, pa, cl, mtS621ms, pa, cl, mt
CAF-1ABB04523S417S410msS417 
CAM kinase deltaNP_001083858T286T287ms, paT287ms
Casein kinase epsilonNP_001084228S405S405ms, pa, cl, mtS405 
Casein kinase epsilonNP_001084228S408S408ms, pa, cl, mtS408 
b-cateninAAI08765T551T551msT551ms
CDC21/MCM4AAH72870S3S3ms, paS3 
CDC21/MCM4AAH72870S83S88Ms, paS87pa
CDC21/MCM4AAH72870T102T102msT101 
CDC21/MCM4AAH72870T110T110ms, pa, mtT109pa
CDC21/MCM4AAH72870S120S120msT119pa
CDK7AAI30135S170S164msS164ms, mt
CDK7AAI30135T176T170msT170ms, mt
CNOT2AAH73075S166S165msS165 
DDI2 (LOC379186)NP_001079499S188S194ms  
FoxK2Q7ZX03S359S398msS389ms
Giant lethal larvae 1NP_001084898S656S664 S662ms, cl, mt
HnRNP-KAAH44711S257S284ms, pa, cl, mtS284ms
HP1 gamma/CBX3AAO39117S101S93ms, pa, mtS93 
Interleukin binding factor 3NP_001083930S385S384msS384 
NDRG2Q7ZY73S324S328msS328ms
NDRG2Q7ZY73T326T330msT330ms
NDRG2Q7ZY73S328S332msS332ms, pa, cl, mt
NDRG3Q6GQL1T329T329msT329ms
NDRG3Q6GQL1S331S331msS331ms
NDRG3Q6GQL1S334S334msS334ms
NsfliAAH41297S140S140ms, pa, cl, mtS140ms
NsfliAAH41297S176S176msS176ms
Programmed cell death 4AAH56125S42S67ms, pa, mtS67ms, pa, mt
Programmed cell death 4AAH56125S457S457ms, pa, mtS457ms, pa, mt
PumilioBAC57980S704S709msS710ms
RSK2AAH80017S369S369ms, pa, mtS369ms, pa, mt
RSK2AAH80017S375S375msS375 
4E-BP2AAH68624T33T36ms, pa, cl, mtT35ms, pa
4E-BP2AAH68624T37T40ms  
4E-BP2AAH68624T42T45ms, pa, cl, mtT44ms, pa
4E-BP2AAH68624S61S64ms, pa, cl, mtS63ms, pa
4E-BP2AAH68624T66T69ms, pa, cl, mtT68ms, pa
4E-BP2AAH68624T78T81ms  
4E-BP2AAH68624S79S82ms  
4E nuclear import protein (4E-T)AAH77338S372S564msS563ms
4E nuclear import protein (4E-T)AAH77338S395  S586ms
4E nuclear import protein (4E-T)AAH77338T575T769ms  
Translation elongation factor 2 (eef2)AAH60025T57T56ms, pa, clT56ms, pa
Translation elongation factor 2 (eef2)AAH60025T59T58msT58 
TRA-2AAH44990T197T204msT202 

In addition, several residues of the Xenopus 4E-BP2 protein were identified as sites of phosphorylation in our analysis (Tables 3, 5, and Supp. Table S1). 4E-BPs inhibit translation by binding eIF-4E translational initiation factors (Gingras et al.,1999). Studies in cultured cells indicate that the binding of 4E-BPs to initiation factors is controlled by phosphorylation. Our analysis demonstrated that the Xenopus 4E-BP2 was phosphorylated at several different sites, analogous to known phosphorylation sites in human and mouse 4E-BP2 proteins (Table 5). As an independent method to detect 4E-BP2 phosphorylation events, we analyzed 4E-BP2 from oocytes, eggs, stage 7 and stage 10.5 embryos by protein immunoblotting. Analysis with an antibody that recognizes 4E-BP2 when it is phosphorylated at either Thr33 or Thr42 indicated that 4E-BP2 is phosphorylated to varying extents at all stages examined (Fig. 3, lanes 1–4). Analyzing the same filter with an antibody that detects 4E-BP2 regardless of its modification state revealed the presence of nonphosphorylated protein (Fig. 3, lanes 5–8). These results together with the data summarized in Tables 4 and 5 increase confidence in the data obtained with mass spectroscopy.

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Figure 3. Phosphorylation of Xenopus 4E-BP2. Proteins from equivalent numbers of Xenopus oocytes eggs, stage 7 embryos and stage 10+ embryos were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membrane. The membrane was probed with an anti-4E-BP2 antibody that detects 4E-BP2 when it is phosphorylated at Thr-33 or Thr-42. Antibodies were stripped from the filter and the same filter was probed with a second anti-4E-BP2 antibody that detects total 4E-BP2, regardless of its phosphorylation state.

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Identified Phosphoproteins Represented a Wide Range of Expression Levels

A major challenge of proteomic analysis using mass spectrometry arises because of the extremely large dynamic range of protein levels expressed in specific cells. Many regulatory proteins such as signaling proteins and transcription factors are expressed at low levels. The detection of such important regulatory molecules can be occluded by the presence of much more abundant proteins in a sample. To determine whether our results were heavily biased toward the detection of abundant proteins, we used the Xenopus tropicalis expressed sequence tags (ESTs) resources (http://www.sanger.ac.uk/Projects/X_tropicalis/) (Gilchrist et al.,2004). Xenopus tropicalis ESTs from different stages of development have been organized into clusters in which each cluster represents the ESTs from a single mRNA. The number of ESTs in a cluster provides an estimate of relative mRNA abundance and, therefore, at present and by inference, the best approximate estimate of protein levels. For example, the CIRP2 RNA binding protein is encoded by an abundant mRNA that is highly expressed and the CIRP2 EST cluster contains hundreds of ESTs. In contrast, the EST clusters representing mRNAs expressed at low levels, such as the denticleless mRNA, contain fewer than ten ESTs. Xenopus tropicalis mRNAs and EST clusters were identified for 73 of the phosphoproteins we identified from gastrula embryos (Supp. Tables S2 and S3.). Notably, the mRNAs encoding Xenopus phosphoproteins were predominantly from EST clusters representing the nonabundant and moderately abundant mRNAs (Fig. 4). These data provided evidence that the Xenopus phosphoproteins we identified were encoded by mRNAs with varying levels of expression and were not enriched for the most abundant mRNA class.

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Figure 4. Identified phosphoproteins represented a wide range of expression levels. For each of 73 Xenopus laevis gastrula phosphoproteins (Supp. Tables S1–S3), the corresponding Xenopus tropicalis mRNA was identified along with the associated expressed sequence tags (ESTs). Xenopus tropicalis ESTs for specific mRNAs have been sorted into clusters where the number of ESTs per cluster provides an estimate of mRNA expression levels (http://www.sanger.ac.uk/Projects/X_tropicalis/) (Gilchrist et al.,2004). EST clusters were identified for each of the mRNAs encoding a Xenopus laevis phosphoprotein. The number of ESTs per cluster was compared with the number of phosphoprotein encoded mRNAs that map to each cluster and plotted. The Xenopus phosphoproteins identified in this study corresponded to both the abundantly expressed mRNAs (clusters with large numbers of ESTs) and minimally expressed mRNAs (clusters with few ESTs).

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Xenopus Phosphoproteins Represented a Diverse Array of Functional Categories

Functional annotation revealed that the identified Xenopus phosphoproteins were involved in a diverse array of cellular processes (Fig. 5). In addition, many of the identified phosphoproteins encoded proteins of unknown function (42%). Of note, was the substantial number of proteins that were categorized as “hypothetical.” These data make it clear that many of these proteins are indeed bona fide proteins expressed in living embryos, underscoring a value in this analysis beyond presenting the functionally unbiased information about the Xenopus phosphoproteome.

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Figure 5. Functional annotation of Xenopus phosphoproteins. The function of each phosphoprotein was assigned and grouped using accession numbers. Each fragment on the pie chart represents a functional category. The percentage of phosphoproteins identified in this study that fall into each functional category is indicated. “Unknown proteins” have no assigned function, whereas “Other” proteins have an assigned function that lies outside of the functional categories shown.

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Many of the Xenopus phosphoproteins function to regulate processes that play a central role in vertebrate embryogenesis (Table 6, Supp. Table S3). For example, in oocytes, eggs and early stage embryos normal development depends heavily on posttranscriptional processes such as mRNA translation and mRNA localization. Consistent with the notion that such processes must be robustly regulated in developing embryos, phosphorylation sites were identified on several different Xenopus proteins involved in translation and RNA metabolism (Table 6, Supp. Table S3). For example, the translational regulatory protein embryonic poly (A) binding protein (ePABP) was phosphorylated at multiple sites (Table 6, Supp. Table S3). ePABP binds to mRNA poly (A) tails to stimulate translation and protect mRNAs from degradation (Wilkie et al.,2005). Interestingly, serine 461 of ePAB was phosphorylated in eggs, raising the possibility that phosphorylation may regulate ePABP's function. The identification of this and many other previously unexamined phosphorylation sites sets the stage for future functional studies in a wide variety of research areas relevant to vertebrate embryogenesis and cell function.

Table 6. Phosphorylation of Developmentally Relevant Proteinsa
ProteinAccessionSamplePhos.sites
  • Developmentally relevant Xenopus phosphoproteins.

  • a

    This table shows a partial set of phosphoproteins that are known to function as developmental regulatory proteins in translation, RNA metabolism, signal transduction and transcription. The complete list of this type of proteins is found in Supp. Table S3.

Proteins involved in translation or RNA processing   
4E-BP2AAH68624OocyteT:34 T38
4E-T initiation factor 4E import factor 1AAH77338EggS:395
Cap methyltransferase (xCMT)Q9I8S2BlastulaS:50
EiF4G1AAH89197BlastulaS:45
Embryonic poly A binding protein (ePAB)AAK29408BlastulaS:461
MASKINAAF19726GastrulaS:638
ePABP2AAO33927GastrulaT:71
PumilioAAL14121GastrulaS:211
Vg1 RNA binding proteinAAC18597GastrulaS:192, S:193
Proteins involved in signaling or transcription   
Casein Kinase II (Beta)P28021EggS:109
Erb-b2NP_001089062EggY:440
Eyes absent-3NP_001089994Gastrula bcatS:286
FOXK2Q7ZX03OocyteS:359
ISWI (imitation switch)AAG01537BlastulaS:1024
Mi2 deacetylaseAAD55392EggS:79
Sox3AAH72222Gastrula nonorganizerS:244
Suppressor of fusedAAH73701GastrulaS:291
TreacleAAW56574GastrulaS:700
V-etsAAI55947EggS:239
β-TRCPAAA02810EggS:19

Conclusion

The data presented here provides an information-rich foundation for investigating the role of specific phosphorylation events by exploiting the many established experimental advantages of Xenopus oocytes, eggs, embryos and in vitro extracts. Despite the large number of undocumented phosphorylation sites uncovered in this study, the data presented here remains some distance from saturation. Further developments in mass spectrometry technology that couple high mass measurement accuracies with ETD promise an even deeper exploration of the Xenopus phosphoproteome in the future (Hogan et al.,2005; McAlister et al.,2007,2008; Hubler et al.,2008). Our data clearly illustrate that the study of regulatory phosphorylation in controlling vertebrate development is rich with unexamined questions.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES
  8. Supporting Information

Xenopus laevis Eggs, Oocytes, Embryos and Gastrula Stage Modified Tissues

Xenopus laevis oocytes, eggs, and embryos were generated using standard methods (Lane and Sheets,2000; Mitchell and Sheets,2001; Lane et al.,2004). Two hundred defolliculated oocytes (st. VI), unfertilized eggs, embryos (st. 7 and st. 10+) were flash frozen in liquid nitrogen.

In addition, samples of gastrula stage embryos (st. 10+) subjected to several different manipulations were also collected for analysis. These included LiCl treatment to activate the β-catenin pathway and injection of a morpholino to β-catenin to block β-catenin expression and signaling. For LiCl treatment, 32-cell embryos were exposed to 0.3 M LiCl in 0.1× MMR for 12 min. After LiCl treatment, embryos were washed four times every 15 min with R/10 buffer at pH 7.4. Embryos were allowed to develop to stage 10+ and flash frozen in liquid nitrogen. β-catenin injected embryos were prepared by injecting four- to eight-cell embryos with a β-catenin morpholino (Heasman et al.,2000). These embryos were then allowed to develop to stage 10+, where they were flash frozen in liquid nitrogen. Organizer and nonorganizer tissues were prepared by selective tissue enrichment of stage 10+ embryos using a Gastromaster microsurgery instrument. An equivalent of two hundred embryos for each tissue sample were flash frozen for protein extraction.

Protein Extraction

Tissue was homogenized in 15 mM Tris pH 8, 75 μg PMSF, 50 mM NaF, 1 mM NaOV, and 25 mM β-glycophosphate. An equal volume of 1,1,2-trichlorotrifluoroethane (Freon) was mixed with homogenate, centrifuged and the lower phase containing yolk proteins removed. The sample was mixed with Trizol (400 μl), incubated for 3 min at room temperature and separated by centrifugation. Isopropanol was added to the bottom phase and incubated for 10 min to precipitate the proteins, and centrifuged to pellet the proteins. The pellet was washed three times with 0.3 M guanidine-HCl (in 95% ethanol), washed once with 100% ethanol, and dried to remove residual ethanol. The pellet was resuspended in 6 M guanidine-HCl, 25 mM NH4HCO3, 50 mM NaF, 1 mM NaOV, and 25 mM β-glycophosphate. A buffer exchange using 6 M urea followed by 25 mM ammonium bicarbonate (pH 8.0) was done to lower guanidine hydrochloride concentration to 0.1 M, and the urea concentration to 2 M.

Phosphopeptide Enrichment

Extracted proteins were prepared for proteolysis by reducing with 500 μM dithiothreitol (DTT) for 1 hr, 1.2 mM iodo-acetate for 45 min in the dark followed with 1.25 mM DTT. The proteins were then treated with sequencing grade trypsin for 1 hr at 37°C and quenched with glacial acetic acid (pH 3.0). The sample was desalted using a C18 column, and the peptide containing eluent brought to dryness using a vacuum centrifuge. Esterification of peptides was completed by adding 40 μl of thionyl chloride to 1 ml of anhydrous methanol and adding this solution to the dried peptides for 1 hr with mixing at room temperature. The modified peptides were brought to dryness again, and resuspended in a reconstitution buffer of equal parts methanol, acetonitrile, and water, with a 0.01% acetic acid concentration. Modified peptides were loaded onto a 560-μm I.D. × 8-cm fused-silica column packed with Poros MC particles (Applied Biosystems) activated with 100 mM FeCl3. Nonphosphorylated peptides were removed by rinsing the column with the reconstitution buffer. Captured phosphopeptides were then eluted onto an nHPLC precolumn (360 μm × 75 μm, packed with 10–30 μm C8 particles) using 15 μL of 50 mM phosphate buffer, pH 8.0.

LC-MS/MS

The precolumn was connected to an nHPLC analytical column (360 μm o.d. × 50 μm i.d.) packed with 5 cm of 5-μm C8 reversed-phase packing material and equipped with an integrated, electrospray emitter tip (Martin et al.,2000). Peptides were eluted into a Thermo LTQ mass spectrometer modified to perform ETD (Coon et al.,2004; Syka et al.,2004). Peptides were eluted at a flow rate of 60 nl/min with the following gradient: 0–7% B in 5 min, 7–45% B in 70 min, 45–100% in 15 min, 100–0% B in 5 min, (A = 0.1 M aqueous acetic acid, B = 70% acetonitrile in 0.1 M aqueous acetic acid). The mass spectrometer was set to continuously cycle through the acquisition of a MS1 spectra from which the 10 most abundant precursors were automatically selected for tandem MS interrogation by means of ETD (Haas et al.,2006).

LiCl-treated and β-catenin-morpholino–injected embryos were prepared and analyzed in a consistent manner with protocol above, with the exception that the LiCl treated samples were analyzed by ETD only, and the β-catenin-morpholino–injected samples were analyzed by ETD and collision activated dissociation on a hybrid linear ion trap-orbitrap mass spectrometer (McAlister et al.,2007).

Data Analysis

All tandem mass spectra were searched against the Xenopus laevis subsection of the NCBI database using the Open Mass Spectrometry Search Algorithm (OMSSA). Static modifications of 58.01 Da on cysteine (carbamidomethylation), and 14.02 Da on glutamic acid, aspartic acid, and the peptide c-terminus to account for the o-methyl ester modification. Dynamic modifications of +79.9663 Da at serine, threonine, and tyrosine residues for phosphorylation, and +16 Da at methionine for oxidation were also used. Confidence values were assessed by a target-decoy search strategy (Käll et al.,2008) to identify phosphopeptides in two groups (<0.5% and <5% false positive rate). Identified peptides were grouped into 12 functional categories according to their predicted parent protein according to accession number using a functional annotation database clustering program called Database for Annotation Visualization and Integrated Discovery (DAVID) available at ( http://david.abcc.ncifcrf.gov/home.jsp).

Immunoblotting

Total protein from equivalent numbers of Xenopus oocytes eggs, stage 7 embryos and stage 10+ embryos were loaded onto each lane and electrophoresed on 12.5% SDS-polyacrylamide gels. Proteins were transferred to PVDF membrane, the membrane was blocked and probed with an anti-4E-BP2 antibody that detects 4E-BP2 when it is phosphorylated at Thr-33 or Thr-44 (Cell Signaling, phospho-4E-BP1 [Thr37/46] #2855). The membrane was incubated with goat anti-rabbit antibodies coupled to horseradish peroxidase. Antibody binding was detected using a chemiluminescent substrate and exposure to x-ray film. Antibodies were stripped from the filter by incubation in a solution of 0.2 M Glycine-HCl pH2.5, 0.05% Tween-20, 100 mM 2-mercaptoethanol for 60 min. The same filter was probed with a second anti-4E-BP2 antibody that detects total 4E-BP2, regardless of its phosphorylation state (Santa Cruz). Antibody binding was detected as described above.

EST Cluster Comparison

Seventy-three Xenopus laevis gastrula phosphoproteins were randomly chosen for analysis from Supp. Tables S2, S3 and S4; APC-binding protein (EB2) (NP_001083449), Ataxin2 (AAH97692), β-catenin (AAI08765), Brg1 (AAX08100), BRM (AAQ02780), Interleukin Enhancer Binding Factor 3 (NP_001083930), Casein Kinase II (Beta) (P28021), Cnot2 (AAH73075), Denticleless (AAH73015), ELAV (NP_001081035), enhancer of zeste-2 (EZH2) (AAK30208), Eyes absent-3 (NP_001089994), FOXD3a (Q9DEN4), FoxN3a (NP_001090178), Fzd7 (AAH42228), ID2 (BAA76634), ISWI (imitation switch) (AAG01537), Jumonji (AAH88951), Lethal giant larvae 2 (AAH72924), Lin-28 (AAH42225), Lunitic fringe (AAI29635), MASKIN (AAF19726.1), Mastermind1 (NP_001080927), Mi2 deacetylase (AAD55392.1), mRNA-4 p56 (FRGY2) (P21574), Poly (A) binding protein-1 (AAO33927), Programmed cell death 4 (AAH56125), Pumilio (AAL14121), RAP55 (AAH42251), Ski2 (NP_001084112), Sox3 (AAH72222), Tra-2 (AAH44990), β-TRCP (AAA02810), Treacle (AAW56574), V-ets (AAI55947), Vg1 RNA binding protein (AAC18597), Xeek1 (AAC59904), zinc finger protein 36 (AAH84221), zinc finger transcription factor SALL4 (AAH95923), Api5 (AAH77529), death associated protein (AAI33257), deoxyuridine triphosphatase 3 (NP_0001091360), echinoderm microtubule associated protein 4 (AAI10955), eukaryotic translation elongation factor 2 (EF-2) (AAH44327), eukaryotic translation initiation factor 4 gamma (EIF4G) (AAH89197), eukaryotic initiation factor 4E binding protein 2 (4E-BP2) (AAI33809), heat shock protein (90 kDa) (NP_001086624), high mobility group AT-hook 2 (AAI24963), histone binding protein N1/N2 (CAA28419), insulin-like growth factor 2 mRNA binding protein 1 (AAH57700), NDRG (NP_001080389), NOL1/NOP2/Sun domain 2 (Nsun2) (AAH68818), Nsfl1 (p47) (AAH41297), Nucleoplasmin (CAA28460), PAICS (AAH41276), pyruvate dehydrogenase alpha (PDHA) (AAI06671), programmed cell death 4 (AAH56125), RAD23 (AAH44115), Ribosomal protein L15 (AAH46569), Ribosomal protein L34 (AAH99259), Ribosomal protein S6 (AAH54151), serpin (AAA49703), Ribosomal protein L28 (AAH53798), stathmin 1/oncoprotein 18 (NP_001080672), eukaryotic translation initiation factor 4H (EiF4H) (NP_001083502), MCM4 (AAH83031), nuclear autoantigenic sperm protein (NASP1) (NP_001089907), nucleolin (AAI57416), nucleoporin (CAB53357), Ribosomal protein L24 (AAH78474), Ribosomal protein S1a (CAA40592), Ribosomal protein L13 (AAH75140), Upstream of NRAS (NP_001090026), CIRP2 (AAH54250), eukaryotic translation initiation factor 4E nuclear import factor 1 (4E-T) (NP_001086710). For each Xenopus laevis phosphoprotein, the analogous Xenopus tropicalis mRNA was identified along with the associated ESTs. The Xenopus tropicalis ESTs for specific mRNAs have been sorted into clusters where the number of ESTs per cluster provides an estimate of mRNA expression levels (http://www.sanger.ac.uk/Projects/X_tropicalis/) (Gilchrist et al.,2004) EST clusters were identified for each mRNA corresponding to a Xenopus laevis phosphoprotein. The number of ESTs per cluster was compared with the number of phosphoprotein encoded mRNAs that map to each cluster and plotted.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES
  8. Supporting Information

We thank C.A. Fox and Y. Zhang for comments on the manuscript. J.J.C. was funded by the National Science Foundation, J.J.C. and M.D.S. were funded by the NIH, and D.L.S. gratefully acknowledges support from an NIH pre-doctoral fellowship, the Genomic Sciences Training Program.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES
  8. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
DVDY_21941_sm_SupplTabS1.xls375KSupp. Table S1. Xenopus phosphorylation events. The total results from the Xenopus phosphoproteomic analysis as summarized in Table 1. In addition, this table also contains results from the analysis of gastrula stage embryos manipulated in different ways, such as LiCl treatment or embryos injected at the two-cell stage with β-catenin morpholino.
DVDY_21941_sm_SupplTabS2.xls15KSupp. Table S2. Phosphorylation events found at all stage of development examined. The results from the Xenopus phosphoproteomic analysis (Supp. Table S1) were examined to identify phosphorylated sites present in at least three of the four stages of development analyzed. The first column contains the peptide fragment identified with the phosphorylation site in lowercase. Also shown are the start and end points of the peptide relative to the full-length protein it was derived from (the accession number is also listed along with the protein name or Unigene identifier if the protein was unidentified in Xenopus laevis).
DVDY_21941_sm_SupplTabS3.xls32KSupp.Table S3. Phosphorylation of proteins relevant to embryonic development. The results from the Xenopus phosphoproteomic analysis (Supp. Table S1) were examined to identify phosphorylated sites present on proteins of interest during the four stages of development analyzed. The first column contains the peptide fragment identified with the phosphorylation site in lowercase. Also shown are the start and end points of the peptide relative to the full-length protein it was derived from (the accession number is also listed along with the protein name or Unigene identifier if the protein was unidentified in Xenopus laevis).

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