Antibody Microarray Analyses of Signal Transduction Protein Expression and Phosphorylation during Porcine Oocyte Maturation Steven Pelech,*,†,§ Lucie Jelinkova,# Andrej Susor,# Hong Zhang,† Xiaoqing Shi,§ Antonin Pavlok,# Michal Kubelka,# and Hana Kovarova# Kinexus Bioinformatics Corporation, Suite 1, 8755 Ash Street, Vancouver, BC, Canada V6P 6T3, Department of Medicine, University of British Columbia, Vancouver, BC, Canada, and Department of Reproductive and Developmental Biology, Institute of Animal Physiology and Genetics, Rumburska 89, Libechov, Czech Republic Received January 31, 2008
Kinex antibody microarray analyses was used to investigate the regulation of 188 protein kinases, 24 protein phosphatases, and 170 other regulatory proteins during meiotic maturation of immature germinal vesicle (GV+) pig oocytes to maturing oocytes that had completed meiosis I (MI), and fully mature oocytes arrested at metaphase of meiosis II (MII). Increases in apparent protein levels of protein kinases accounted for most of the detected changes during the GV to MI transition, whereas reduced protein kinase levels and increased protein phosphorylation characterized the MI to MII transition. During the MI to MII period, many of the MI-associated increased levels of the proteins and phosphosites were completely or partially reversed. The regulation of these proteins were also examined in parallel during the meiotic maturation of bovine, frog, and sea star oocytes with the Kinex antibody microarray. Western blotting analyses confirmed altered expression levels of Bub1A, IRAK4, MST2, PP4C, and Rsk2, and the phosphorylation site changes in the kinases Erk5 (T218 + Y220), FAK (S722), GSK3-beta (Y216), MEK1 (S217 + S221) and PKR1 (T451), and nucleophosmin/B23 (S4) during pig oocyte maturation. Keywords: antibody microarray • pig • frog • oocyte maturation • meiosis • protein kinases • phosphorylation • phosphatases
Introduction The conversion of an immature oocyte into a fertilizable egg is one of the most highly conserved steps in eukaryotes that undergo sexual reproduction. There are striking parallels in the regulation of this fundamental process from echinoderms (e.g., sea stars) and amphibians (e.g., frogs) to mammals (e.g., mice and pigs). 1–5 Although there are significant differences, there are many similarities between meiotic and mitotic cell cycle progression. In diverse species, the reductive cell divisions that occur during meiosis and following fertilization are characterized by the absence of large increases in RNA, protein, and lipid synthesis. These meiotic and mitotic cell cycles are precisely orchestrated through the transient production of regulatory proteins such as cyclins and the phosphorylation of proteins catalyzed by protein kinases. However, our knowledge of the underlying mechanisms that mediate the conversion of a mammalian oocyte into a fertilizable egg is still quite rudimentary. This is in part due to the very limited number of oocytes that are produced by mammals in their lifetime and, until recently, insufficiently sensitive procedures for their analysis. * To whom correspondence should be addressed. E-mail, spelech@ kinexus.ca.; phone, 604-323-2547, extension, 10; fax, 604-323-2548. † Kinexus Bioinformatics Corporation. § Department of Medicine, University of British Columbia. # Department of Reproductive and Developmental Biology, Institute of Animal Physiology and Genetics.
2860 Journal of Proteome Research 2008, 7, 2860–2871 Published on Web 05/17/2008
Oocytes from many species are naturally arrested at prophase of MI, near the G2-M border of the cell cycle. Hormonal induction of meiotic maturation is first evident with the breakdown of the nuclear envelope (also known as germinal vesicle (GV) breakdown), and this is accompanied by a burst in total cellular protein phosphorylation catalyzed by a plethora of protein kinases. In one of the best characterized oocyte model systems, the African clawed frog Xenopus laevis oocyte, these kinases include Aurora,6,7 CDK1,10–12 CDK2,13 CDK7/ MO15,14 Chk2/Cds1,15 casein kinase 2 (CK2),16 ERK2,9,12,17–22 Haspin,23,24 JNK,25,26 MEK1,27–29 MEK2,30 Mos,13,31,32 p38γ MAPK,33 PKB/Akt,34 Plx1,35 Raf,36–40 andRSK1andRSK2.9,20,38,41–43 cAMP-dependent protein kinase (PKA)34 and xPAK44 are protein kinases that become inhibited during frog meiotic maturation. Many of these kinases and others (e.g., calmodulin-dependent kinase 2, phosphatidylinositol 3-kinase) are similarly regulated in other oocytes such as from the sea star,45–54 clam,55 mouse,56–60 rat,61 pig,62–71 sheep,72 goat,73 and cow.66,62,74–77 In frog oocytes, several of these kinases are integrated into common signaling pathways, such as the Raf1/RafB/Mos f MEK1/2 f ERK1/2 f RSK1/RSK2 f Bub1 cascade. The mitogen-activated protein kinase (MAPK) ERK2 is the protein that displays the most marked increase in tyrosine phosphorylation at GV breakdown, in concert with the tyrosine dephosphorylation of CDK1 in frog oocytes.17 This dephosphorylation of CDK1 at Tyr-15 is catalyzed by the dual specificity phosphatase CDC25, which is itself activated by phosphoryla10.1021/pr800082a CCC: $40.75
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tion at an unknown site by Plx-1 or at Ser-205 by p38γ MAPK.17 CDC25 is also inhibited by phosphorylation at an unknown site by Chk1 or Chk2/Cds1, 15,78 and at Ser-287 by PKA33,79–81 and RSK1/2. 41 Tyr-15 in CDK1 is phosphorylated by the protein kinases Wee1 and Myt1 in X. laevis oocytes, although Myt1 appears to be the principal Thr-14 kinase in this model system.32 Myt1 undergoes inhibitory autophosphorylation,82 and it can be phosphorylated and inhibited by Plx1, after it is prephosphorylated by CDK1.83 Likewise, Wee1B can be phosphorylated and inhibited by PKA during frog oocyte maturation.81,84,85 The phosphorylation of relatively few other proteins during oocyte maturation have been described, but they include CPEB,86 cyclin B,39,87 elongation factor 1-gamma,88 Emi2/Xerp1,89–91 histone H3 at Thr-3,37 maskin,92 MCAK,93 and separase.94,95 Several protein kinases have been implicated in maintaining the arrest of immature oocytes at the G2/M border in prophase of meiosis I. In the frog, these include PKA,96 Chk2/Cds1,15 CDK7/MO15,14 and xPAK.44 xPAK blocks the activation of Plx1.44 PKA acts to inhibit progesterone-induced synthesis of Mos.97 Inhibition of PKA by the inhibitor H-89 is insufficient to induce X. laevis oocyte maturation, but it potentiates progesterone-induced GV breakdown.98 In the mouse oocyte, PKCR/β activation with 1-oleoyl 2-acetyl glycerol or mezerein inhibits GVBD, whereas PKC inhibitors such as staurosporin99 and Calphostin C100 accelerate the onset of GV breakdown. Likewise, PKC activation prevents rat101 and sea star46 oocyte maturation. In contrast, PKC activation by phorbol ester102 or microinjection of PKCζ103 causes the maturation of X. laevis oocytes. As described above, many insights have been learned from studies of oocyte maturation in amphibians and invertebrates and have often been shown to have relevance to mammals, when tested individually. However, there have not been broader-based studies to evaluate whether the aforementioned proteins would emerge from an unbiased analysis of hundreds of cell signaling proteins and whether other players contribute to the regulation of meiosis. The recent availability of antibody microarrays, which can use as little as 50 µg of cell lysate protein, has now allowed this to become feasible for scarce populations of cells such as mammalian oocytes. Herein, we describe the regulation of more than 60 proteins by protein turnover, phosphorylation and/or altered protein-protein interaction during meiotic maturation of oocytes from pigs and other species.
Materials and Methods Preparation of Porcine and Bovine Oocytes. Ovaries, collected from slaughtered pigs and cows, were transported in physiological saline at 20 °C to the laboratory. The ovaries were briefly washed for 20 s in 70% ethanol and then twice in physiological saline. The pig oocytes were obtained by aspiration of antral follicles about 2-5 mm in diameter, and bovine oocytes were from follicles about 3-6 mm in diameter. Only oocytes surrounded by compact cumuli were used for culture. Oocytes were cultured in droplets of TCM 199 medium (Sevapharma, Czech Rep.) supplemented with 10% fetal calf serum, 5 IU PG 600 (Intervet International, NE), and antibiotics at 38.5 °C in an atmosphere of 5% CO2.104 The samples of pig oocytes were collected at 0 h (GV), 28 h (MI), and 44 h (MII) during in vitro oocyte maturation, whereas the bovine oocytes were collected at 0 h (GV) and 24 h (MII). At the end of culture, the cumulus cells and corona radiata of the oocytes were removed
research articles by mechanical stripping using vortexing. Denuded oocytes were then washed using physiological saline, and after the last wash, the oocytes were stored at -80 °C until use. Morphological evaluation of oocytes was used to verify GV, MI, or MII stage of in vitro maturation and quality of the oocytes collected for antibody microarray and immunoblotting analyses. The oocytes were mounted on microscope slides with vaseline, covered with a cover glass, and fixed in ethanol/acetic acid 3:1 for 24 h. Staining was performed with 2% orcein in 50% aqueous-acetic acid and 1% sodium citrate. The slides were then placed in 40% acetic acid and observed with a phase-contrast NU Zeiss microscope (Jena, Germany). The collection of the oocytes used for the proteomic study was based on the criteria that at least 85% of oocytes reached an appropriate maturation stage. Frog Oocyte Preparation and Treatment. Stage VI X. laevis oocytes were isolated from human gonadrotropin-injected frogs (Xenopus One, Ann Arbor, MI) and treated with 1 µg/mL progesterone as previously described.9 After 7 h treatment, those progesterone-treated oocytes that displayed germinal vesicle breakdown (∼80% of treated population) and untreated oocytes were immediately harvested for homogenization. Sea Star Oocyte Preparation and Treatment. Pisaster ochraceus sea stars were collected from beaches in the greater Vancouver area. For the preparation of immature and mature oocytes, the cells were isolated from ovaries and treated with 10 µM 1-methyladenine in natural seawater as described.46,47 Maturation was marked by GV breakdown with the disappearance of the nucleus, and this typically occurred within 90 min after 1-methyladenine addition for at least 80% of the oocyte population. Preparation of Oocyte Lysates. The cells were washed twice and lysates were generated by sonication in homogenization buffer [0.5% Nonidet P-40, 20 mM 3-(N-morpholino) propane sulfonic acid (MOPS), 2 mM EGTA, 5 mM EDTA, 30 mM sodium fluoride, 40 mM β-glycerophosphate, 20 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonylfluoride, 3 mM benzamidine, 5 µM pepstatin A, and 10 µM leupeptin, pH 7.2]. Following ultracentrifugation (100 000g, 30 min, 4 °C), the supernatant (detergent-solubilized lysate) was stored at -80 °C. Protein concentration was assessed by the Bradford assay.105 Antibody Microarray Analyses. The Kinex Antibody Microarray KAM-1.0 analyses were performed with detergentsolubilized pig, cow, frog, and sea star oocyte lysate proteins as described previously106 and on the Kinexus Internet Web site at www.kinexus.ca. Briefly, 50 µg of oocyte lysate proteins were labeled with a fluorescent dye at a concentration of 2 mg/ mL, and unincorporated dye molecules were removed by ultrafiltration. Purified labeled proteins from two samples were incubated separately on opposite sides of a Kinex KAM-1.0 antibody microarray. This microarray features two identical fields of antibody grids, each field containing 604 antibodies printed in duplicate. The pan-specific and phosphosite-specific antibodies were printed at concentrations of approximately 100 µg/mL with 10 nL per spot. The internal variation for spot printing between chips within the same print run was less than 4%. After probing, arrays were scanned using a ScanArray scanner (Perkin-Elmer, Wellesley, MA) with a resolution of 10 µm, and resulting images were quantified using ImaGene software (BioDiscovery, El Segundo, CA). Values provided in the figures are the means of the recorded measurements from each antibody spot pair, and the difference in these measureJournal of Proteome Research • Vol. 7, No. 7, 2008 2861
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Figure 1. Protein expression and phosphorylation changes during porcine oocyte meiotic maturation as revealed by antibody microarray analysis. The percentage of antibody spots that exhibited changes in signal intensity of greater than or equal to 25%, 33%, 50%, and 75% during the GV to MI period (A) and MI to MII period (B) are shown. For this analysis, only those antibodies that had a percent range of less than or equal to 25% of their mean intensity signal from duplicate measurements were included in the calculations used to generate this figure. This corresponded to 515 (297 pan-specific; 218 phosphosite-specific) and 522 (298 pan-specific; 224 phosphositespecific) antibodies in panels A and B, respectively. Expression and phosphorylation changes are shown with circles and triangles, respectively. Increases and decreases are indicated with filled and open symbols, respectively.
ments divided by 2 are the ranges from the means that are shown as error bars. Western Blot Analyses. Custom Kinetworks KCPS multiimmunoblotting analyses were performed using 300 µg of detergent-solubilized pig and frog oocyte lysate proteins as described previously107 and at the Kinexus Internet Web site at www.kinexus.ca. The Kinetworks analysis involves resolution of a proteins in a single lysate sample by SDS-PAGE and subsequent immunoblotting overnight at 4 °C with panels of up to 3 primary phosphosite-specific antibodies per channel in a 20-lane Immunetics multiblotter. Phosphosite antibodies were sourced from Invitrogen (Carlsbad, CA), Cell Signaling Technologies (Beverly, MA), and Millipore (Temecula, CA). The antibody mixtures were carefully selected to avoid overlapping cross-reactivity with target proteins. The membranes were later rinsed with TBST buffer (50 mM Tris base, 150 mM NaCl, and 0.5% Triton X-100 (v/v), pH 7.4) and then incubated with the relevant horseradish peroxidase conjugated secondary antibodies for 45 min at room temperature. The immunoblots were developed with enhanced chemiluminescence (ECL) Plus reagent (Amersham, Arlington Heights, IL), and signals were captured by a Fluor-S MultiImager and quantified using Quantity One software (Bio-Rad, Hercules, CA). Background was less than 100 cpm for these analyses.
Results Meiosis Related Changes in Cell Signaling Protein Expression and Phosphorylation in Porcine Oocytes by Antibody Microarray Analysis. The Kinex KAM-1.0 antibody microarray was used to perform an unbiased characterization of nearly 400 distinct protein kinases and other signaling proteins that are regulated during meiotic maturation of porcine oocytes. This microarray featured 604 antibodies (347 pan-specific and 257 phosphosite-specific) from 20 different commercial suppliers. It tracked at least 188 different protein kinases, 24 protein phosphatases, and 170 other regulatory proteins. All of the antibodies were previously demonstrated by Kinexus to perform well for Western blotting applications for their protein 2862
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and phosphorylation site targets. For clarity, all of the numbering of the phosphosites in this report refer to the human cognates. When fully grown pig oocytes are removed from their follicles, they can resume meiosis and mature spontaneously under in vitro conditions in the presence of follicle stimulating hormone. Lysate proteins were prepared from freshly dissected immature germinal vesicle (GV) containing oocytes arrested at prophase of MI, oocytes that had completed metaphase stage of MI 28 h after removal from their follicles and incubation in vitro, and mature oocytes arrested at MII after 44 h incubation in vitro. The MI oocytes were characterized by GV breakdown and formation of the first meiotic spindle. The MII oocytes featured the extruded typical first polar body. The lysate samples of these oocyte populations were separately dyelabeled prior to affinity capture with the Kinex antibody microarray, with two samples analyzed per chip. Each antibody was tested in duplicate for each sample, and for each sample with the 604 antibodies, the average percent range and median percent range from the average of the duplicates were typically 12% and 6%, respectively. Consequently, a 25% change in the intensity of the recorded signal from each antibody spot pair between two samples was deemed to be a potential alteration in protein turnover, phosphorylation and/or protein-protein interaction between these samples. For the purposes of simplification of presentation in the Results section, it is assumed that the pan-specific antibodies tracked changes in protein levels, and the phosphosite antibodies monitored the phosphorylation status of specific phosphosites in target proteins. When the first (GV to MI) and second (MI to MII) periods of meiotic maturation were analyzed, approximately 28% and 40%, respectively, of the antibodies revealed changes of 25% or greater in protein expression and/or phosphorylation (Supplemental Table 1, Supporting Information). Figure 1 shows how these changes were distributed, and the decline in the percentage of antibodies that revealed changes of greater than 32%, 49%, and 74%. For example, 10.5% (54 of 515 × 100%) of the antibodies appeared to show increases of protein expression of 25% or more during the GV to MI transition, which
Altered Protein Levels and Phosphorylation during Meiosis
Figure 2. Distribution of protein expression and phosphorylation changes of greater than or equal to 25% during porcine oocyte meiotic maturation in the GV+ to MI (A, C, E, G, and I) and MI to MII (B, D, F, H, and J) periods. The data shown in Figure 1 were analyzed with respect to the specific expression of protein kinases (A and B), protein phosphatases (C and D) and other proteins (E and F) with pan-specific antibodies, and the phosphorylation of serine and threonine phosphosites (G and H) and tyrosine phosphosites (I and J) with phosphosite-specific antibodies. The total number of antibodies represented in each type of analysis is shown in white text. The percent distribution of increased changes greater than or equal to 25% (black), decreased changes greater than or equal to -25% (white), and no changes (i.e., less than or equal to 24%; gray) are illustrated and provided numerically.
accounted for most of the detected changes during the period of meiotic maturation (Figure 1A). In contrast, there appeared to be much more reduced protein levels and increased protein phosphorylation during the MI to MII transition (Figure 1B). The most marked changes (g50%) were observed with increased protein levels and phosphorylation in both periods. Most of the different proteins displaying expression changes during porcine oocyte meiotic maturation in the GV to MI period were protein kinases, as might be expected since these were specifically targeted by about two-thirds of the panspecific antibodies printed on the Kinex microarray. However, as a group, the protein kinases also appeared to show the greatest percentage of proteins showing both increased and decreased levels during this period (42% of 188 tested kinases) (Figure 2A). This was nearly twice as frequent in comparison with a group of diverse proteins that was representative of proteins in general (22% of 87 other proteins) (Figure 2E). During the GV to MI period, there also appeared to be higher than normal percentage of protein phosphatases that exhibited increased levels (27% of 22 tested phosphatases). In contrast, during the MI to MII period, the relative frequencies of increased numbers of protein level changes in the protein kinases and protein phosphatases appeared to be similar to that seen for other proteins (Figure 2B, D,F). However, when compared to the GV to MI period, the pattern of protein kinase
research articles level differences appeared to be reversed. There appeared to be a 50% higher rate in the number of protein kinases that showed decrease protein levels, and a 31% lower rate in the number of protein kinases that exhibited increase protein levels (Figure 2B). Germinal vesicle breakdown in MI during oocyte maturation in amphibians and echinoderms has been characterized in previous studies as being accompanied by a burst in protein phosphorylation. The 218 or more diverse phosphosite antibodies used to obtain the findings in Figure 1 revealed that more target phosphoproteins were subjected to decreased than increased serine and threonine phosphorylation during the GV to MI period (Figure 2G), while similar numbers of phosphoproteins had either reduced or elevated tyrosine phosphorylation (Figure 2I). Remarkably, during the MI to MII period, there was a 3-fold increase in the percentage of serine/threoninespecific phosphosite antibodies that showed increases in phosphorylation (Figure 2H), and a 2-fold increase in the percentages of tyrosine-specific phosphosite antibodies that showed both increases and decreases in phosphorylation (Figure 2J). This was surprising, because of the apparent reduction in the levels of many protein kinases in general during this period (Figure 2B). With so many apparent changes in protein levels and phosphorylation during porcine oocyte maturation, we focused on those proteins that showed changes in at least one of these parameters of 50% or more during either the GV to MI or the MI to MII periods. Figure 3 provides a summary of the proteins that were the most affected in their apparent expression levels during one or both periods. Twenty-one of the 33 proteins shown are protein kinases and three are protein phosphatases, which roughly matches the ratio of the number of these specific classes of proteins to the total number of proteins analyzed. During the GV to MI period, there were apparent increases (percent change from control (%CFC), control ) GV) in the levels of the protein-serine kinases, Chk1 (+57%), CK1-epsilon (+69%), IRAK4 (+37%), PKC-theta (+51%), PKR1 (+75%), p70 S6 kinase-beta (+33%), Rsk1 (+31%), and Vrk1 (+64%); the protein-tyrosine kinases, ROR2 (+54%) and Tyro10 (+84%); the protein phosphatases, MKP2 (+87%), PP1/C-gamma (+61%), and PTP1C (+68%); and other proteins, caspase 4 (+67%), caspase 9 (+223%), GNB2L1 (+35%), MSH2 (+67%), and p107 Rb-related protein (+51%). In parallel during the GV to MI period, there were decreases in the protein-serine kinases, ASK1 (-31%) and ZIPK (-43%), and the transcription factor STAT4 (-40%). During the MI to MII period, many of the MI-associated increased levels of the aforementioned proteins were completely or partially reversed. In the cases of GNB2L1 (-44%), IRAK4 (-30%), and p70 S6K-beta (-36%), the reduction of apparent protein levels in MII cells was substantially lower than in the GV+ cells (%CFC, C ) GV). This was also observed for the protein kinases BLK (-41%) and JIK/TAO3 (-56%) and the Bcl homologue Bak (-58%). At the same time, there were increased levels of the protein kinases Aurora 2 (+53%), Erk6/ p38-gamma (+58%), PKG1 (+35%), Tlk1 (+63%), TrkA (+28%), and Wee1 (+41%), as well as caspase 5 (+32%) and Trail (+41%) in the MII cells (Figure 3). Figure 4 shows the major phosphorylation changes of 50% or more during either the GV to MI period or the MI to MII period; 16 of the 28 phosphosites shown were in protein kinases and none were in phosphatases. During the GV to MI period, the most marked increases (%CFC, C ) GV) in phosphorylation Journal of Proteome Research • Vol. 7, No. 7, 2008 2863
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Figure 3. Proteins that exhibited expression changes of greater than or equal to 50% during porcine oocyte meiotic maturation. The mean intensity of the pan-specific antibody signals for each target protein ( the range in the duplicates from the means are shown for lysates from GV (white bars), MI (gray bars), and MII (black bars) oocytes. For this analysis, only those antibodies that had a percent range of less than or equal to 25% of their mean intensity signal of duplicate measurements were included in the calculations used to generate this figure. Background signals were subtracted from the spot signals to obtain the values shown.
were observed for the protein kinases MLK3 Thr-277+Ser-281 (+77%), PKC-eta Thr-655 (+51%), PKC-theta Ser-676 (+80%), and other proteins caldesmon Ser-789 (+47%), caveolin 2 Ser36 (+55%), cortactin Tyr-470 (+60%), Shc1 Tyr-349 + Tyr-350 (+121%), and Tau Thr-547 (57%). The most striking decreases in phosphorylation during the GV to MI period were evident for the protein kinases CaMK2-alpha Thr-286 (-33%), CDK1 Thr-161 (-30%); ErbB2 Tyr-1248 (-26%), Erk1/2 Thr-202 + Tyr204/Thr-185 + Tyr-187 (-43%), FAK Ser-732 (-30%), Mnk1 Thr-209 + Thr-214 (-37%), PKB-alpha/Akt1 Thr-308 (-40%), PKC-lambda Thr-555 (-29%), and other proteins B23 Thr-234 (-29%), Dok2 Tyr-142 (-25%), eIF2-alpha Ser-51 (-42%), and eNos Thr-495 (-42%). During the MI to MII period, many of the phosphorylation changes described above were partially or fully reversed (Figure 4). However, the increased phosphorylations of Shc1, PKCtheta, MLK3, and caveolin 2 in MI cells were preserved in MII cells. Furthermore, there were increased (%CFC, C ) GV) phosphorylations of PKC-gamma Thr-655 (+68%), CREB1 Ser133 (+58%), eIF2-alpha Ser-51 (+60%), Erk5 Thr-218 + Tyr220 (+27%), FAK Ser-722 (+60%), FAK Ser-732 (+46%), and Lck 2864
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Figure 4. Proteins that exhibited phosphorylation changes of greater than or equal to 50% during porcine oocyte meiotic maturation. The mean intensity of the phosphosite antibody signals for each target protein ( the range in the duplicates from the means are shown for lysates from GV (white bars), MI (gray bars), and MII (black bars) oocytes. For this analysis, only those antibodies that had a percent range of less than or equal to 25% of their mean intensity signal of duplicate measurements were included in the calculations used to generate this figure. Background signals were subtracted from the spot signals to obtain the values shown.
Tyr-504 (+51%), and decreased phosphorylations of caldesmon Ser-789 (-41%), and histone H1 at the sites targeted by CDK1 (-64%). Comparison of Porcine Meiotic Oocyte Maturation with Other Species. To ascertain which of the protein level and phosphorylation changes in signaling proteins that were uncovered with the Kinex antibody microarray in the pig oocyte system were the most commonly conserved events, similar studies were performed with GV and MII oocytes from cows, immature GV+, and maturing GV- oocytes from sea stars and frogs. The GV- oocytes were homogenized shortly after completion of GV breakdown during MI in the sea star oocytes (∼90 min after 10 µM 1-methyladenine incubation) and frog oocytes (∼7 h after 1 µg/mL progesterone treatment). Figure 5 provides a comparison with the other experimental model systems of the changes in protein expression that were evident in maturing pig oocytes and described in Figure 3. The most consistent changes between the pig and cow oocytes were the increased expressions of Aurora 2 (Aurora A), caspase 1-beta, MSH2, PKG1, Rsk1, Tlk1, Trail and Tyro10, and decreased levels of ICK, IRAK4 and JIK/TAO3. There were also near borderline increases in ASK1 and Wee1, and decreases in the levels of BLK, Bub1A, caspase 4, KHS and MST2 in both systems. The absence of notable changes in the levels of Chk1, PP1/C-gamma, Stat4 and ZIPK between the pig and cow was also common to both.
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Figure 5. Meiotic maturation associated changes in signal transduction protein expression in oocytes from diverse species by antibody microarray analysis. The percent changes from control (%CFC) in pig oocytes where the control was selected as either the GV or MI oocytes is tabulated for the most significantly affected proteins shown in Figure 3 as well as some additional targets. For comparison, similar Kinex KAM-1.0 antibody microarray analyses were also performed with lysates of immature GV oocytes from cow (Bos taurus), frog (X. laevis), and sea star (P. ochraceus). MII bovine eggs, and frog and sea star GVoocytes in MI harvested just after GV breakdown were also tested. Increased %CFC values that are greater than or equal to 25% appear as white text in black boxes. Decreased %CFC values that are greater than or equal to -25% appear as black text in white boxes. %CFC measurements that were less than 25% appear in gray boxes.
It was more difficult to compare protein expressions differences between the pig and frog and sea star oocytes with respect to early meiotic maturation, in part because of the vast evolutionary differences between these organisms. Nevertheless, the increased expressions of caspase 9 and KHS were the most consistent in all three species, and the increased levels of IRAK4, p107 Rb-related protein, PP1/C-gamma, ROR2, and Vrk1, and decrease in ZIPK in the pig were reproduced in either the frog or sea star systems (Figure 5). Figure 6 provides a comparison with the cow, frog, and sea star oocytes for the major changes in protein phosphorylation that were observed in maturing pig oocytes and presented in
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Figure 6. Meiotic maturation associated changes in signal transduction protein phosphorylation in oocytes from diverse species by antibody microarray analysis. The results were obtained and presented as described in the legend to Figure 5.
Figure 4. The most consistent changes between the pig and cow oocytes were the increased phosphorylations of Erk5 Thr218 + Tyr-220, FAK Ser-722, PKC-gamma Thr-655, PKC-mu/ PKD Ser-738 + Ser-742, PKC-theta Ser-676, and Shc1 Tyr-349 + Tyr-350. There was also a near borderline increase in eIF2alpha Ser-51, and a decrease in the level of caldesmon Ser-789 in both species. The absence of notable changes in the phosphorylations of CDK1 Thr-161 and Tyr-15, cortactin Tyr470, Dok2 Tyr-142, Erk1 Thr-202 + Tyr-204, Erk2 Thr-185 + Tyr-187, GSK3-beta Tyr-216, Mnk1 Thr-209 + Thr-214, and PKR1 Thr-451 was common to both the pig and cow. There was even less consistency between the pig, frog, and sea star oocytes with respect to phosphorylation changes following GV breakdown in MI. The increased phosphorylation of caldesmon pS789 was the most consistent in all three species, and the increased phosphorylation of caveolin 2 Ser36, and cortactin Tyr-470, and decreased phosphorylation of Mnk1 Thr-209 + Thr-214, PKB-alpha/Akt1 Thr-308, and PKClambda Thr-555 in the pig were reproducible in the frog (Figure 5). There were also near borderline reductions in the phosJournal of Proteome Research • Vol. 7, No. 7, 2008 2865
research articles phorylation of eIF2-alpha Ser-51, S6 Ser-235, and Erk5 Thr218 + Tyr-220 between the pig and frog (Figure 6). Validation of Antibody Microarray Results by Immunoblotting. The Kinex antibody microarray analyses of the oocytes from the pig, cow, frog, and sea star uncovered many potential leads for altered protein levels and phosphorylation. However, it should be appreciated that studies that rely on the capture of dye-labeled, nondenatured proteins by antibodies are fraught with limitations. In view of the potential issues of antibody cross-reactivity with nontarget proteins and masked epitopes due to the protein complex formation or protein structural conformations, the Kinex antibody microarray detected changes in protein expression and phosphorylation were further investigated by Western blotting. The immunoblotting approach uses denatured proteins and serves as the gold standard for quantification of specific proteins in crude cell and tissue lysates. To confirm the changes in protein levels in the GV and MII porcine oocytes that were indicated by the Kinex antibody microarray experiments, similar cell lysates were subjected to Kinetworks multi-immunoblotting. This immunoblotting procedure was adopted to maximize the number of signaling proteins that could be tested, since there was very little oocyte material available for these confirmatory studies. As up to 2 or 3 antibodies were tested per lane on the Western blot, there was a small chance that a cross-reactive protein may have comigrated with a target protein and generated a false positive. In addition, it was difficult to assign any cross-reactive proteins to detection with a specific antibody. The immunoblotting of GV and MII pig oocyte protein lysates with pan-specific antibodies from the Kinex microarray for the targets shown in Figure 5 permitted successful detection of 14 of the 16 tested antibodies (Figure 7). JIK/TAO3 and PKG1 could not be visualized on the immunoblots, and Aurora 1, IRAK4 and MST2 yielded very weak signals. When the (24% cutoff was used for detection of changes, it appears that only 5 of the 14 detected proteins (i.e., Bub1A, IRAK4, MST2, PP4C and Rsk2) displayed consistent results between the microarrays and immunoblots for detection of changes in protein levels. Immunoblotting of GV and MII pig oocyte protein lysates with the 21 phosphosite-specific antibodies from the Kinex microarray for the targets shown in Figure 6 permitted successful detection of 18 of tested targets (Figure 8). PKC-mu/ PKD Ser-738 + Ser-742, Rb Thr-826 and PKC-gamma Thr-655 were not observed, and B23 Ser-4 and STAT3 Ser-727 were barely detectable. Again, only about 6 of the 18 detected phosphoproteins (i.e., B23 Ser-4, Erk5 Thr-218 + Tyr-220, FAK Ser-722, GSK3-alpha Tyr-279, MEK1 Ser-217 + Ser-221 and PKR1 Thr-451) showed immunoblotting results that matched the trends seen with the antibody microarray. Nevertheless, the immunoblotting experiments with the porcine oocytes revealed marked changes in the phosphorylation of 16 of these phosphoproteins (Figure 8A). When these antibodies were used to visualize phosphorylation changes following GV breakdown in frog oocytes, almost all of the increases in phosphorylation by immunoblotting with the pig oocytes were reproduced in the maturing frog oocytes. The exceptions were the increased phosphorylations of B23 Thr-234 (the site is missing in the frog cognate protein), Erk1 Thr-202 + Tyr-204 (Erk1 is not present in frog oocytes), and CDK1 Tyr-15 (which may relate to the difference in cell cycle transition) (Figure 8B). Almost none of the frog oocyte protein phosphorylation results by Western blotting (Figure 8B) matched the antibody microarray findings 2866
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Figure 7. Kinetworks multi-immunoblotting analyses of lysates from pig oocytes for protein expression levels. Representative immunoblots are shown of lysates from immature, GV pig oocytes and MII pig eggs probed with various pan-specific antibodies for protein kinases and other signal transduction proteins. The migration positions in Daltons of the detected proteins based on molecular weight marker proteins are indicated as well as the percent change in intensity of the detected proteins in MII oocytes as compared to immature, GV oocytes.
generated with the similar frog oocyte lysates (Figure 6). Collectively, these findings demonstrated that only about a third (5 of 13) of the changes in protein expression and phosphorylation greater than 24% inferred from antibody microarray analysis in the pig system could be validated independently by immunoblotting.
Discussion Gene microarray studies have been widely used in diverse model systems to indirectly explore potential changes in protein levels based on mRNA measurements. While gene expression profiling can provide some insight into cell- and tissue-specific levels of proteins, more often than not, mRNA levels poorly correlate with their protein products. In the special case of oocytes, these cells are loaded with maternal mRNA in preparation for the reductive cell divisions that occur after fertilization, so gene microarray analysis is especially problematic. This is compounded by the fact that most proteins require posttranslational covalent modifications such as phosphorylation in order to become fully functional. This is catalyzed by around 500 different protein kinases in mammals, and there is growing evidence that indicates that there may be over a million phosphosites in mammalian phosphoproteomes (S. Pelech, unpublished data). Therefore, phosphosite profiling should be an effective strategy to elucidate much more about the regulation of important fundamental cellular processes such as cell cycle progression. The availability of phosphosite-specific antibodies has been a tremendous boon to studies of cell
Altered Protein Levels and Phosphorylation during Meiosis
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Figure 8. Kinetworks multi-immunoblotting analyses of lysates from pig and frog oocytes for protein phosphorylation. (A) Representative immunoblots are shown of lysates from immature, GV pig oocytes blocked in prophase of MI and metaphase II arrested pig eggs in MII probed with various phosphosite-specific antibodies. The migration positions in Daltons of the detected phosphoproteins based on molecular weight marker proteins are indicated as well as the percent change in intensity of the detected phosphoproteins in MII oocytes as compared to GV oocytes. In some cases, no phosphorylation bands were detected in the MI oocyte samples, so it was not possible to accurate measure the percent change in intensity. (B) Representative immunoblots of lysates from immature frog oocytes or those harvested after 7 h treatment with 1 µM progesterone (GVBD) are shown. The average percent change in intensity of the detected phosphoproteins in GV breakdown oocytes as compared to GV+ immature oocytes is provided for cells harvested from 3 to 6 different frogs and immunoblotted separately.
signaling, although the costs of these reagents have hampered their more widespread usage in broad-based studies. The development of antibody microarrays should permit the screening of hundreds of pan- and phosphosite-specific antibodies at a fraction of the costs of other quantitative analytical approaches. Furthermore, it is ideally suited for similar situations to the present study where there is a paucity of cells or tissue material. The Kinex antibody microarray appears to be a magnitude more sensitive than Western blotting for detection of proteins with the antibodies used on this array. Despite the commercial availability of antibody microarrays for over 6 years, very few other reports have been published that describe their application in the investigation of intracellular protein regulation.108–113 In this study, we explored the use of the Kinex antibody microarray, which included 347 panand 257 phosphosite-specific antibodies, to evaluate the effectiveness of this methodology to reveal alterations in protein expression and phosphorylation during meiotic maturation of oocytes from four different species. Our aim was to identify proteins that are highly conserved in their regulation during oocyte maturation, which may play fundamental roles in this
process. In the case of the pig and frog oocyte systems, we followed up with immunoblotting studies, and determined that only about a third of the changes (greater than 24%) in either protein levels or phosphorylation inferred by the antibody microarray analysis could be validated. This is actually very consistent with our experience with several other model systems, including EGF treatment of human tumor cell lines.106 We have found that typically 20% of the changes in protein expression or phosphorylation inferred by the antibody microarrays cannot be confirmed as subsequent immunoblotting validation studies fail to show any immunoreactive proteins on the immunoblots. It appears that the antibody microarray technique is at least a magnitude more sensitive than Western blotting. The high rate of false positives evident from the antibody microarray analysis may be generated from a number of confounding factors. With the diversity of protein expression, proteolysis, and phosphorylation that occurs in different cell types, the risk of nonspecific protein cross-reactivities is very high with antibodies. Unlike immunoblotting following SDSPAGE, the antibody microarray methodology requires the use Journal of Proteome Research • Vol. 7, No. 7, 2008 2867
research articles of nondenatured proteins, most of which may reside within protein complexes in cell lysates. Consequently, the dye-bound protein that may be captured by an antibody microarray may include additional proteins besides those that are directly targetedbytheimmobilizedantibodies.Changesinprotein-protein interactions may be detected as apparent differences in the levels of the target proteins. With covalent modification of proteins, such as phosphorylation, it is also feasible that epitopes may become exposed or masked as a consequence of conformational changes in these proteins. Finally, it should be appreciated that changes in phosphorylation signal strength on the microarrays, as with immunoblots, may reflect changes in protein mass as opposed to the stoichiometry of protein phosphorylation. Despite the aforementioned caveats, the low cost and sample requirements for antibody microarray analysis make this powerful technology very attractive for broad-based proteomics to discover potentially important players in signaling pathways. However, follow-up validation by immunoblotting is absolutely critical in view of the low reliability of antibody microarrays. Consequently, the other proteins in Figures 5 and 6 that demonstrated altered protein expression and phosphorylation during meiotic maturation should be viewed only as leads for further confirmation. However, once the validity of an antibody microarray for specific proteins and phosphosites is established for an experimental model system, then it can be used with high accuracy and confidence for those proteins. In view of the unreliability of antibody microarray results upon careful inspection, how then should the data in Figures 1 and 2 of this study really be considered? At the very least, the antibodies have been very sensitive probes for detection of perturbations of target proteins. Apparent changes in protein expression or phosphorylation may also reflect differences in protein-protein complex formation, which is also important in cellular regulation. It seems likely though that the phosphosite antibodies on the microarray in this study uncovered underlying changes in protein phosphorylation, even if they were not necessarily in the targeted phosphosites. Phosphosite antibodies are usually double affinity purified against both the phosphorylated and dephosphorylated forms of the immunizing peptide with the phosphosite so that the phosphate moiety is a critical part of the antibody recognition. Therefore, the interpretations of the global phosphorylation changes in the GV to MI and MI to MII periods of pig oocyte maturation in Figure 2 are probably still correct. Another serious concern that has been raised from our work is the large number of false negatives that were generated by the antibody microarray. This would be the number of antibodies that failed to show changes greater than 24% by the antibody microarray analysis, but did in fact reveal large alterations in protein expression or phosphorylation by Western blotting. In this study, this corresponded to 20 of 23 antibodies that are shown in Figures 7 and 8. We have typically observed about an 85% false negative rate in previous studies with other experimental model systems (S. Pelech; H. Zhang, data not published). As the antibody microarray approach can produce false negatives, an alternative strategy to detect additional protein expression and phosphorylation changes is the multiimmunoblotting technique that we have previously described.107 However, we have also determined in these experiments that more than 80% of the antibodies that fail to show a change by antibody microarray analysis also do not demonstrate a difference by immunoblotting. Consequently, the 2868
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Pelech et al. microarray analysis is still a very efficient method to hone in on possible alterations in target proteins, although it will miss many important regulatory events. Some of the best characterized phosphoprotein changes with frog oocyte maturation near the onset of GV breakdown are the increased phosphorylations of MEK1, ERK2, RSK1 and ribosomal S6 protein, which were not evident by the antibody microarray analysis (Figure 6), but were very prominent by immunoblotting (Figure 8B). Evidence has accumulated from very diverse oocyte systems including mouse,114–117 pig,62,68,118 rat,101 cow,119,120 goat,73 as well as sea stars121 that ERK1 and ERK2 are not essential for the initiation of GV breakdown prior to meiosis I, but they play important roles in preventing DNA synthesis and maintaining M phase arrest prior to meiosis II. When these MAPKs are inactivated with MEK1-specific inhibitors such as PD98159 or UO126, cyclin B is degraded, and oocytes re-enter interphase and form a pro-nucleus. In the case of the frog,37,122–124 however, initial activation of CDK1 by progesterone is triggered via production of Mos, and the sequential activation of the MEK1 f ERK2 f RSK1/2 kinase cascade. Mos can also induce CDK1 activation independently of ERK2 via phosphorylation of Myt1 in Xenopus.80 Additional CDK1 molecules are recruited through the CDK1-mediated inactivation of Cds1, apparently by phosphorylation and activation of Plx1.83 (A similar CDK1 activation loop is mediated through phosphorylation of the Plx1 homologues Polo-like kinase 1 (Plk1) in sea star,52 mouse,58 and pig.70) Still, in the maturing frog oocyte, ERK2 and RSK1/2 are necessary to inhibit the anaphase promoting complex, which normally mediates degradation of cyclins and progression into S phase.13,31,125 Xenopus ERK2 phosphorylates Mos at Ser-3, which may stabilize Mos for completion of meiosis.41 There is growing evidence that chromosome condensation during maturation is not necessarily linked to GV breakdown. In studies with cycloheximide-treated pig oocytes, in which CDK1 was not activated and GV breakdown was blocked, chromosome condensation still proceeded normally.126 Likewise, 100 µM butyrolactone I treatment of bovine oocytes inhibited activation of CDK1, MAPK and GV breakdown, but not chromosome condensation.127 In maturing Xenopus oocytes, ERK2 and RSK are required for Ser-10 phosphorylation of histone H3; however, this is also achieved by PKA, which blocks maturation and does not cause chromosome condensation.128 Thus, there is a poor correlation between histone H3 Ser-10 phosphorylation and chromosome condensation.129 By serendipity, we recently discovered a 40-kDa phosphoprotein that cross-reacted with a phospho-MEK1 antibody in the human HeLa and HCT116 tumor cell lines.130 We identified this protein as B23 nucleophosmin and demonstrated that it underwent enhanced phosphorylation on Ser-4 during M phase.130 When we mutated this serine residue to a glutamic acid to mimic its constitutive phosphorylation and transfected the construct into HeLa cells, there was a marked stimulation of centrosome duplication.130 In the present study, B23 Ser-4 phosphorylation was also enhanced in the pig and frog oocytes during meiotic maturation (Figure 8), so it is feasible that this is targeted by Plk1/Plx1 as we established previously in HeLa and HCT116 cells.130 We also observed a marked increase in Stat3 S727 phosphorylation during both pig and frog oocyte maturation by immunoblotting (Figure 8). Phosphorylation at this site by CDK1 was enhanced in nocodazole-treated HeLa cells, and this phosphorylation event appears to contribute the inhibition of
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Altered Protein Levels and Phosphorylation during Meiosis 131
cell division at M phase. During oocyte maturation, it is likely that enhanced CDK1 and ERK1/2 activity both contribute to the phosphorylation of Stat3 S727. An increase in phosphorylation of GSK3-alpha at the activating Tyr-279 site was observed by Western blotting in the maturing frog oocytes, but not in the pig oocytes (Figure 8). Phosphorylation of this activation site in GSK3 is catalyzed by MEK1, and as phosphorylation of the MEK1 Ser-217 and Ser-S221 activation sites was much more robust in the frog oocyte than the pig oocyte, it may have been less responsive in the pig to input from MEK1. The increased phosphorylation of FAK Ser-722 seen in both the pig and frog oocytes could have been due to activation of GSK3, since this kinase has been previously implicated in phosphorylation of this site and inactivation of FAK.132 Among the other phosphorylation differences between the pig and frog oocyte systems was the phosphorylation of CDK1 Tyr-15 and eIF2-alpha Ser-51. It is possible that apparent opposite effects on the phosphorylation of these sites between the pig and frog may be due to differences in the timing of these events in meiosis. This may also be true for some of the other phosphorylation differences that we observed in Figure 8 such as for GSK3-alpha Tyr-279 and PKR1 Thr-451. Interestingly, PKR1 is one of the main kinases that is thought to be responsible for phosphorylation of eIF2-alpha at Ser-51, which would act to inhibit protein synthesis.133 PKR1 is fully activated following its autophosphorylation, and Thr-451 is one of the critical activation sites.134 However, in the frog oocyte near the time of GV breakdown, PKR Thr-451 phosphorylation was reduced, while the phosphorylation of eIF2-alpha Ser-51 became increased (Figure 8B). Apparently, yet other protein kinases appear to participate in the regulation of meiotic events that still remain to be uncovered. Future studies will identify additional protein kinases and phosphoprotein substrates that may contribute to the control of meiotic maturation of mammalian oocytes, and the interaction of these components in signaling networks that orchestrate cell cycle control. The major goal for the future will be further validation of the potential protein targets that will be selected from these studies, which may be useful as biomarkers of oocyte quality. This knowledge not only might be beneficial for basic science for improvement of oocyte culture conditions, which are still far from optimal, but also it may have implications for reproductive biotechnology which may have utility in other species, including humans. Abbreviations: ASK1, apoptosis signal regulating proteinserine kinase MAP3K5; Aurora 2 (AuroraA), AurB protein-serine kinase; B23, nucleophosmin; Bak, Bcl2 homologous antagonist/ killer BCK2L7; BLK, B lymphoid tyrosine kinase; Bub1A, budding uninhibited by benzimidazoles 1-related protein-serine kinase; CaMK2R, calcium/calmodulin-dependent proteinserine kinase 2 alpha; CASP4/5/9, caspases 4, 5 and 9; CDK1/ 2, cyclin-dependent protein-serine kinases 1 and 2; Chk1, checkpoint protein-serine kinase 1; CK1ε, casein protein-serine kinase 1 epsilon; CREB1, cAMP response element binding protein 1; Dok2, docking protein 2; eIF2R, eukaryotic translation initiation factor 2 alpha; eNos, nitric-oxide synthase, endothelial; ErbB2, Neu/HER2 receptor-tyrosine kinase; Erk1/2/5/6, extracellular regulated protein-serine kinases 1, 2, 5 and 6 (p38gamma, MAPK12); FAK, focal adhesion protein-tyrosine kinase; GV, germinal vesicle ; GNB2L1, guanine nucleotide-binding protein beta (receptor for activated C kinase 1); GSK3R/β, glycogen synthase protein-serine kinase 3 alpha and beta; ICK, MAK-related intestinal cell protein-serine kinase; IRAK4, in-
terleukin 1 receptor-associated kinase 4; JIK, STE20-like proteinserine kinase TAO3; JNK2, c-Jun N-terminus protein-serine kinase 2; KHS, MEKKK5 protein-serine kinase homologous to SPS1/STE20; Lck, lymphocyte-specific protein-tyrosine kinase; MAP kinase, mitogen-activated protein kinase; MI, metaphase of Meiosis I; MII, metaphase of Meiosis II, MEK1/3, MAP kinase protein-serine kinase 1 (MAP2K1) and 3 (MAP2K3); MKP2, VH2 MAP kinase phosphatase 2; MLK3, mixed-lineage protein-serine kinase 3; Mnk1, MAP kinase-interacting protein-serine kinase 1; MSH2, DNA mismatch repair protein mutS homologue 2, colon cancer, nonpolyposis type 1; MST2, mammalian STE20like protein-serine kinase 2; p107, retinoblastoma proteinrelated p107 PRB1; PKBR, Akt1 protein-serine kinase B alpha; PKCγ/η/λ/θ/µ, protein-serine kinase C gamma, eta, lambda (iota), theta and mu (PKD); PKG1, cGMP-dependent protein kinase type 1; PKR1, double-stranded RNA dependent proteinserine kinase; PP1/Cγ, protein-serine phosphatase 1, catalytic subunit, gamma isoform; PP4C, protein-serine phosphatase 4, catalytic subunit; PTP1C, SHP1 protein-tyrosine phosphatase 1C; Rb, retinoblastoma-associated protein 1; ROR2, ROR2 neurotrophic receptor-tyrosine kinase; Rsk1/2, ribosomal S6 protein-serine kinases 1 and 2; S6Kβ, p70 ribosomal proteinserine S6 kinase beta; Shc1, SH2 domain-containing transforming protein 1; STAT3/4, Signal transducer and activator of transcription 4 and 5; Tlk1, Tousled-like protein-serine kinase 1; Trail, tumor necrosis factor-related apoptosis-inducing ligand; TrkA, nerve growth factor receptor-tyrosine kinase; Tyro10, DDR2 neurotrophic receptor-tyrosine kinase of discoidin domain receptor family, member 2 precursor; Vrk1, vaccinia related protein-serine kinase 1; ZIPK, death associated protein-serine kinase 3/ZIP kinase.
Acknowledgment. This work was supported by a grant from the Canadian Institutes of Health Research to S. Pelech, Grant Agency of Academy of Sciences of the Czech Republic (No. 1QS500450568) to H. Kovarova, Czech Science Foundation (No. 204/06/1297) to M. Kubelka, and Institutional Research Concept IAPG No. AV0Z50450515. Supporting Information Available: Supplemental Table 1, Kinex KAM-1.0 antibody microarray analysis of GV, MI and MII porcine oocytes. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Sadler, K. C.; Ruderman, J. V. Dev. Biol. 1998, 1, 25–38. (2) Yamashita, M. Semin. Cell Dev. Biol. 1998, 9, 569–579. (3) Heikinheimo, O.; Gibbons, W. E. Mol. Hum. Reprod. 1998, 4, 745– 756. (4) Karaiskou, A.; Dupre, A.; Haccard, O.; Jessus, C. Biol. Cell. 2001, 93, 35–46. (5) Castro, A.; Peter, M.; Lorca, T.; Mandart, E. Biol. Cell. 2001, 93, 15–25. (6) Castro, A.; Mandart, E.; Lorca, T.; Galas, S. J. Biol. Chem. 2003, 278, 2236–2241. (7) Ma, C.; Cummings, C.; Liu, X. J. Mol. Cell. Biol. 2003, 23, 1703– 1716. (8) Maton, G.; Thibier, C.; Castro, A.; Lorca, T.; Prigent, C.; Jessus, C. J. Biol. Chem. 2003, 278, 21439–21449. (9) Cicirelli, M. F.; Pelech, S. L.; Krebs, E. G. J. Biol. Chem. 1988, 263, 2009–2019. (10) Huang, C.-Y. F.; Ferrell, J. E. EMBO J. 1996, 15, 2169–2173. (11) Furuno, N.; Ogawa, Y.; Iwashita, J.; Nakajo, N.; Sagata, N. EMBO J. 1997, 16, 3860–3865. (12) Fisher, D. L.; Brassac, T.; Galas, S.; Doree, M. Development 1999, 126, 4537–4546. (13) Furuno, N.; Nishizawa, M.; Okazaki, K.; Tanaka, H.; Iwashita, J.; Nakajo, N.; Ogawa, Y.; Sagata, N. EMBO J. 1994, 13, 2399–2410.
Journal of Proteome Research • Vol. 7, No. 7, 2008 2869
research articles (14) Shuttleworth, J.; Godfrey, R.; Colman, A. EMBO J. 1990, 10, 3233– 3240. (15) Gotoh, T.; Ohsumi, K.; Matsui, T.; Takisawa, H.; Kishimoto, T. J. Cell Sci. 2001, 114, 3397–3406. (16) Mulner-Lorillon, O.; Marot, J.; Cayla, X.; Pouhle, R.; Belle, R. Eur. J. Biochem. 1998, 171, 107–117. (17) Posada, J.; Sanghera, J.; Pelech, S.; Aebersold, R.; Cooper, J. A. Mol. Cell. Biol. 1991, 11, 2517–2528. (18) Sanghera, J. S.; McNabb, C. K.; Tonks, N.; Pelech, S. L. Biochim. Biophys. Acta 1991, 1095, 153–160. (19) Palmer, A.; Nebreda, A. R. Prog. Cell Cycle Res. 2000, 4, 131–143. (20) Gross, S. D.; Schwab, M. S.; Taieb, F. E.; Lewellyn, A. L.; Qian, Y. W.; Maller, J. L. Curr. Biol. 2000, 10, 430–438. (21) Minshull, J.; Sun, H.; Tonks, N. K.; Murray, A. W. Cell 1994, 79, 475–486. (22) Polverino, A.; Frost, J.; Yang, P.; Hutchison, M.; Neiman, A. M.; Cobb, M. H.; Marcus, S. J. Biol. Chem. 1995, 270, 26067–26070. (23) Dai, J.; Higgins, J. M. Cell Cycle 2005, 4, 665–668. (24) Dai, J.; Sultan, S.; Taylor, S. S.; Higgins, J. M. Genes Dev. 2005, 19, 472–488. (25) Bagowski, C. P.; Xiong, W.; Ferrell, J. E., Jr J. Biol. Chem. 2001, 276, 1459–1465. (26) Browaeys-Poly, E.; Fafeur, V.; Vilain, J. P.; Cailliau, K. Biochim. Biophys. Acta 2005, 1743, 1–4. (27) Kosako, H.; Gotoh, Y.; Nishida, E. EMBO J. 1993, 12, 787–794. (28) Kosako, H.; Gotoh, Y.; Nishida, E. EMBO J. 1994, 13, 2131–2138. (29) Shirakabe, K.; Gotoh, Y.; Nishida, E. J. Biol. Chem. 1992, 267, 16685–16690. (30) Yashar, B. M.; Kelley, C.; Yee, K.; Errede, B.; Zon, L. I. Mol. Cell. Biol. 1993, 13, 5738–5748. (31) Matten, W. T.; Copeland, T. D.; Ahn, N. G.; Vande Woude, G. F. Dev. Biol. 1996, 179, 485–492. (32) Peter, M.; Labbe, J. C.; Doree, M.; Mandart, E. Development 2002, 129, 2129–2139. (33) Perdiguero, E.; Pillaire, M. J.; Bodart, J. F.; Hennersdorf, F.; Frodin, M.; Duesbery, N. S.; Alonso, G.; Nebreda, A. R. EMBO J. 2003, 22, 5746–5756. (34) Andersen, C. B.; Sakaue, H.; Nedachi, T.; Kovacina, K. S.; Clayberger, C.; Conti, M.; Roth, R. A. Biochem. J. 2003, 369, 227– 238. (35) Karaiskou, A.; Lepretre, A. C.; Pahlavan, G.; Du Pasquier, D.; Ozon, R.; Jessus, C. Development 2004, 131, 1543–15552. (36) Fabian, J. R.; Morrison, D. K.; Daar, I. O. J. Cell Biol. 1993, 122, 645–652. (37) Shibuya, E. K.; Polverino, A. J.; Chang, E.; Wigler, M.; Ruderman, J. V. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 9831–9835. (38) Baert, F.; Bodart, J. F.; Bocquet-Muchembled, B.; LescuyerRousseau, A.; Vilain, J. P. J. Biol. Chem. 2003, 278, 49714–49720. (39) Haccard, O.; Jessus, C. EMBO Rep. 2006, 7, 321–325. (40) Gaffre, M.; Dupre, A.; Valuckaite, R.; Suziedelis, K.; Jessus, C.; Haccard, O. Oncogene 2006, 25, 5155–5162. (41) Chun, J.; Chau, A. S.; Maingat, F. G.; Edmonds, S. D.; Ostergaard, H. L.; Shibuya, E. K. Cell Cycle 2005, 4, 148–154. (42) Gavin, A. C.; Ni Ainle, A.; Chierici, E.; Jones, M.; Nebreda, A. R. Mol. Biol. Cell 1999, 10, 2971–2986. (43) Schwab, M. S.; Roberts, B. T.; Gross, S. D.; Tunquist, B. J.; Taieb, F. E.; Lewellyn, A. L.; Maller, J. L. Curr. Biol. 2001, 11, 141–150. (44) Faure, S.; Vigneron, S.; Galas, S.; Brassac, T.; Delsert, C.; Morin, N. J. Biol. Chem. 1999, 274, 3573–3579. (45) Charlton, L.; Clark-Lewis, I.; Aebersold, R.; Pelech, S. L. J. Cell Biochem. 1997, 75, 310–326. (46) Meijer, L.; Pelech, S. L.; Krebs, E. G. Biochemistry 1987, 26, 7968– 7974. (47) Morrison, D. L.; Yee, A.; Paddon, H. B.; Vilimek, D.; Aebersold, R.; Pelech, S. L. J. Biol. Chem. 2000, 275, 34236–34244. (48) Pelech, S. L.; Charest, D. L. MAP kinase-dependent pathways in cell cycle control. In Progress in Cell Cycle Research; Meijer, L., Guidet, S., Tung, L., Eds.; Plenum Press: New York, 1995, pp 3353. (49) Sanghera, J. S.; Charlton, L.; Paddon, H. B.; Pelech, S. L. Biochem. J. 1992, 283, 829–837. (50) Hiraoka, D.; Hori-Oshima, S.; Fukuhara, T.; Tachibana, K.; Okumur, E.; Kishimoto, T. Dev. Biol. 2004, 276, 330–336. (51) Mori, M.; Hara, M.; Tachibana, K.; Kishimoto, T. Development 2006, 133, 1823–1830. (52) Okano-Uchida, T.; Okumura, E.; Iwashita, M.; Yoshida, H.; Tachibana, K.; Kishimoto, T. EMBO J. 2003, 22, 5633–5642. (53) Okumura, E.; Fukuhara, T.; Yoshida, H.; Hanada, S. S.; Kozutsumi, R.; Mori, M.; Tachibana, K.; Kishimoto, T. Nat. Cell Biol. 2002, 4, 111–116.
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Pelech et al. (54) Tachibana, K.; Machida, T.; Nomura, Y.; Kishimoto, T. EMBO J. 1997, 16, 4333–4339. (55) Durocher, Y.; Guerrier, P. Int. J. Dev. Biol. 1996, 40, 557–566. (56) Kalab, P.; Kubiak, J. Z.; Verlhac, M. H.; Colledge, W. H.; Maro, B. Development 1996, 122, 1957–1964. (57) Pahlavan, G.; Polanski, Z.; Kalab, P.; Golsteyn, R.; Nigg, E. A.; Maro, B. Dev. Biol. 2000, 220, 392–400. (58) Tong, C.; Fan, H. Y.; Lian, L.; Li, S. W.; Chen, D. Y.; Schatten, H.; Sun, Q. Y. Biol. Reprod. 2002, 67, 546–554. (59) Viveiros, M. M.; Hirao, Y.; Eppig, J. J. Dev. Biol. 2001, 235, 330– 342. (60) Yao, L. J.; Zhong, Z. S.; Zhang, L. S.; Chen, D. Y.; Schatten, H.; Sun, Q. Y. Biol. Reprod. 2004, 70, 1392–1399. (61) Tan, X.; Chen, D. Y.; Yang, Z.; Wang, Y. C.; Li, M.; Schatten, H.; Sun, Q. Y. Zygote 2001, 9, 269–276. (62) Wehrend, A.; Meinecke, B. Anim. Reprod. Sci. 2001, 66, 175–184. (63) Tatemoto, H.; Muto, N. Zygote 2001, 9, 15–23. (64) Sugiura, K.; Naito, K.; Iwamori, N.; Kagii, H.; Goto, S.; Ohashi, S.; Naruoka, H.; Yada, E.; Yamanouchi, K.; Tojo, H. Zygote 2002, 10, 31–36. (65) Shimada, M.; Terada, T. Mol. Reprod. Dev. 2002, 62, 124–131. (66) Fan, H. Y.; Huo, L. J.; Meng, X. Q.; Zhong, Z. S.; Hou, Y.; Chen, D. Y.; Sun, Q. Y. Biol. Reprod. 2003, 69, 1552–1564. (67) Ito, J.; Shimada, M.; Terada, T. Biol. Reprod. 2004, 70, 797–804. (68) Ohashi, S.; Naito, K.; Sugiura, K.; Iwamori, N.; Goto, S.; Naruoka, H.; Tojo, H. Biol. Reprod. 2003, 68, 604–609. (69) Shimada, M.; Ito, J.; Yamashita, Y.; Okazaki, T.; Isobe, N. J. Endocrinol. 2003, 179, 25–34. (70) Anger, M.; Klima, J.; Kubelka, M.; Prochazka, R.; Motlik, J.; Schultz, R. M. Mol. Reprod. Dev. 2004, 69, 11–16. (71) Villa-Diaz, L. G.; Miyano, T. Biol. Reprod. 2004, 71, 691–696. (72) Lee, J. H.; Campbell, K. H. Biol. Reprod. 2006, 74, 691–698. (73) Dedieu, T.; Gall, L.; Crozet, N.; Sevellec, C.; Ruffini, S. Mol. Reprod. Dev. 1996, 45, 351–358. (74) Bilodeau-Goeseels, S. Theriogenology 2003, 60, 1679–1690. (75) Tomek, W.; Smiljakovic, T. Reproduction 2005, 130, 423–430. (76) Vigneron, C.; Perreau, C.; Dalbies-Tran, R.; Joly, C.; Humblot, P.; Uzbekova, S.; Mermillod, P. Mol. Reprod. Dev. 2004, 69, 457–465. (77) Vigneron, C.; Perreau, C.; Dupont, J.; Uzbekova, S.; Prigent, C.; Mermillod, P. Mol. Reprod. Dev. 2004, 69, 466–474. (78) Nakajo, N.; Oe, T.; Uto, K.; Sagata, N. Dev. Biol. 1999, 207, 432– 444. (79) Duckworth, B. C.; Weaver, J. S.; Ruderman, J. V. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 16794–16799. (80) Eyers, P. A.; Liu, J.; Hayashi, N. R.; Lewellyn, A. L.; Gautier, J.; Maller, J. L. J. Biol. Chem. 2005, 280, 24339–24346. (81) Han, S. J.; Conti, M. Cell Cycle 2006, 5, 227–231. (82) Kristjansdottir, K.; Safi, A.; Shah, C.; Rudolph, J. Cell Cycle 2006, 5, 421–427. (83) Inoue, D.; Sagata, N. EMBO J. 2005, 24, 1057–1067. (84) Han, S. J.; Chen, R.; Paronetto, M. P.; Conti, M. Curr. Biol. 2005, 15, 1670–1676. (85) Wang, J.; Cao, W. L.; Liu, X. J. Cell Cycle 2006, 5, 213–217. (86) Martinez, S. E.; Yuan, L.; Lacza, C.; Ransom, H.; Mahon, G. M.; Whitehead, I. P.; Hake, L. E. Mol. Biol. Cell 2005, 16, 1152–1164. (87) Takakura, I.; Naito, K.; Iwamori, N.; Yamashita, M.; Kume, S.; Tojo, H. J. Reprod. Dev. 2005, 51, 617–626. (88) Mulner-Lorillon, O.; Cormier, P.; Cavadore, J. C.; Morales, J.; Pouhle, R.; Belle, R. Exp. Cell Res. 1992, 202, 549–551. (89) Rauh, N. R.; Schmidt, A.; Bormann, J.; Nigg, E. A.; Mayer, T. U. Nature 2005, 437, 1048–1052. (90) Tung, J. J.; Hansen, D. V.; Ban, K. H.; Loktev, A. V.; Summers, M. K.; Adler, J. R.; Jackson, P. K. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 4318–4323. (91) Tung, J. J.; Jackson, P. K. Cell Cycle 2005, 4, 478–482. (92) Pascreau, G.; Delcros, J. G.; Cremet, J. Y.; Prigent, C.; ArlotBonnemains, Y. J. Biol. Chem. 2005, 280, 13415–13423. (93) Ohi, R.; Sapra, T.; Howard, J.; Mitchison, T. J. Mol. Biol. Cell 2004, 15, 2895–2906. (94) Fan, H. Y.; Sun, Q. Y.; Zou, H. Cell Cycle 2006, 5, 198–204. (95) Stemmann, O.; Gorr, I. H.; Boos, D. Cell Cycle 2006, 5, 11–13. (96) Qian, Y.; Erikson, E.; Taieb, F. E.; Maller, J. L. Mol. Biol. Cell 2001, 12, 1791–1799. (97) Matten, W.; Daar, I.; Vande Woude, G. F. Mol. Cell. Biol. 1994, 14, 4419–4426. (98) Noh, S. J.; Han, J. K. J. Exp. Zool. 1998, 280, 45–56. (99) Lefevre, B.; Pesty, A.; Koziak, K.; Testart, J. J. Exp. Zool. 1992, 264, 206–213. (100) Sun, Q. Y.; Lu, Q.; Breitbart, H.; Chen, D. Y. Reprod. Fertil. Dev. 1999, 11, 81–86.
research articles
Altered Protein Levels and Phosphorylation during Meiosis (101) Lu, Q.; Smith, G. D.; Chen, D. Y.; Yang, Z.; Han, Z. M.; Schatten, H.; Sun, Q. Y. Biol. Reprod. 2001, 64, 1444–1450. (102) Stith, B. J.; Maller, J. L. Exp. Cell Res. 1987, 169, 514–523. (103) Carnero, A.; Liyanage, M.; Stabel, S.; Lacal, J. C. Oncogene 1995, 11, 1541–1547. (104) Pavlok, A.; Lapathitis, G.; Cech, S.; Kubelka, M.; Lopatarova, M.; Holy, L.; Klı´ma, J.; Motlı´k, J.; Havlicek, V. Mol. Reprod. Dev. 2005, 71, 197–208. (105) Bradford, M. M. Anal. Biochem. 1976, 72, 248–254. (106) Pelech, S. L.; Zhang, H. In Principles of Molecular Oncology, 3rd ed.; Bronchud, M. H., Ed.; Humana Press, Inc.: Totowa, NJ, 2008. (107) Pelech, S.; Sutter, C.; Zhang, H. Kinetworks protein kinase multiblot analysis. In Methods in Molecular Biology. Cancer Cell Signaling: Methods and Protocols; Terrian, D., Ed.; Humana Press: Totowa, NJ, 2003; Vol. 218, pp 99-111. (108) Knezevic, V.; Leethanakul, C.; Bichsel, V. E.; Worth, J. M.; Prabhu, V. V.; Gutkind, J. S.; Liotta, L. A.; Munson, P. J.; Petricoin, E. F.; Krizman, D. B. Proteomics 2001, 1, 1271–1278. (109) Hudelist, G.; Pacher-Zavisin, M.; Singer, C. F.; Holper, T.; Kubista, E.; Schreiber, M.; Manavi, M.; Bilban, M.; Czerwenka, K. Breast Cancer Res. Treat. 2004, 86, 281–291. (110) Ahn, E. H.; Kang, D. K.; Chang, S. I.; Kang, C. S.; Han, M. H.; Kang, I. C. Proteomics 2006, 6, 1104–1109. (111) Smith, L.; Watson, M. B.; O’Kane, S. L.; Drew, P. J.; Lind, M. J.; Cawkwell, L. Mol. Cancer Ther. 2006, 5, 2115–2120. (112) Wingren, C.; Borrebaeck, C. A. OMICS 2006, 10, 411–427. (113) King, M. L.; Murphy, L. L. J. Exp. Ther. Oncol. 2007, 6, 147–155. (114) Araki, K.; Naito, K.; Haraguchi, S.; Suzuki, R.; Yokoyama, M.; Inoue, M.; Aizawa, S.; Toyoda, Y.; Sato, E. Biol. Reprod. 1996, 55, 1315–1324. (115) Faerge, I.; Terry, B.; Kalous, J.; Wahl, P.; Lessl, M.; Ottesen, J. L.; Hyttel, P.; Grondahl, C. Biol. Reprod. 2001, 65, 1751–1758. (116) Su, Y. Q.; Rubinstein, S.; Luria, A.; Lax, Y.; Breitbart, H. Biol. Reprod. 2001, 65, 358–365. (117) Su, Y. Q.; Wigglesworth, K.; Pendola, F. L.; O’Brien, M. J.; Eppig, J. J. Endocrinology 2002, 143, 2221–2232.
(118) Lee, J.; Miyano, T.; Moor, R. M. Zygote 2000, 8, 119–125. (119) Gordo, A. C.; He, C. L.; Smith, S.; Fissore, R. A. Mol. Reprod. Dev. 2001, 59, 106–114. (120) Motlik, J.; Pavlok, A.; Kubelka, M.; Kalous, J.; Kalab, P. Theriogenology 1998, 49, 461–469. (121) Picard, A.; Galas, S.; Peaucellier, G.; Doree, M. EMBO J. 1996, 15, 3590–3598. (122) Bodart, J. F.; Gutierrez, D. V.; Nebreda, A. R.; Buckner, B. D.; Resau, J. R.; Duesbery, N. S. Dev. Biol. 2002, 245, 348–461. (123) Choi, T.; Rulong, S.; Resau, J.; Fukasawa, K.; Matten, W.; Kuriyama, R.; Mansour, S.; Ahn, N.; Vande Woude, G. F. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 4730–4735. (124) Haccard, O.; Lewellyn, A.; Hartley, R. S.; Erikson, E.; Maller, J. L. Dev. Biol. 1995, 168, 677–682. (125) Maller, J. L.; Gross, S. D.; Schwab, M. S.; Finkielstein, C. V.; Taieb, F. E.; Qian, Y. W. Novartis Found. Symp. 2001, 237, 58–78. (126) Kubelka, M.; Rimkevicova, Z.; Guerrier, P.; Motlik, J. Mol. Reprod. Dev. 1995, 41, 63–69. (127) Kubelka, M.; Motlik, J.; Schultz, R. M.; Pavlok, A. Biol. Reprod. 2000, 62, 292–302. (128) Schmitt, A.; Nebreda, A. R. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 4361–4366. (129) Jelinkova, L.; Kubelka, M. Biol. Reprod. 2006, 74, 905–912. (130) Zhang, H.; Shi, X.; Paddon, H.; Hampong, M.; Dai, W.; Pelech, S. J. Biol. Chem. 2004, 279, 35726–35734. (131) Shi, X.; Zhang, H.; Paddon, H.; Lee, G.; Cao, X.; Pelech, S. Biochemistry 2006, 45, 5857–5867. (132) Bianchi, M.; De Lucchini, S.; Marin, O.; Turner, D. L.; Hanks, S. K.; Villa-Moruzzi, E. Biochem. J. 2005, 391, 359–370. (133) Srivastava, S. P.; Davies, M. V.; Kaufman, R.J. J. Biol. Chem. 1995, 270, 16619–16624. (134) Zhang, F.; Romano, P. R.; Nagamura-Inoue, T.; Tian, B.; Dever, T. E.; Mathews, M. B.; Ozato, K.; Hinnebusch, A. G. J. Biol. Chem. 2001, 276, 24946–24958.
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Journal of Proteome Research • Vol. 7, No. 7, 2008 2871
