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First published online March 7, 2007
doi: 10.1242/10.1242/jcs.03416

Journal of Cell Science 120, 1093-1103 (2007)
Published by The Company of Biologists 2007
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MAPK interacts with XGef and is required for CPEB activation during meiosis in Xenopus oocytes

Brian T. Keady, Peiwen Kuo, Susana E. Martínez*, Lei Yuan{ddagger} and Laura E. Hake§

Biology Department, Boston College, Chestnut Hill, MA 02467, USA


Figure 1
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  		Fig. 1. Blocking Rho-family GTPase function with Clostridium difficile Toxin B does not interfere with CPEB phosphorylation during early meiosis. Toxin-B-injected oocytes were incubated for 3 hours prior to stimulation of meiosis with progesterone. PD, pulldown; IB, immunoblotting. (A) Protein extracts from oocytes collected post injection or after incubation in progesterone (hr post injection or hr in pg, respectively) at the indicated times (in hours) were treated to activate GTPases and then incubated with GST-PAK-PBD or GST-Rhotekin-RBD. Samples bound to glutathione-beads were analyzed by immunoblotting with anti-Cdc42, anti-Rac1, anti-RhoA and anti-GST antibodies, as indicated. The bottom panel is a representative immunoblot with anti-GST antibody to confirm equivalent loading of GST-PAK-PBD. Similar results were obtained with beads loaded with GST-Rhotekin-RBD. (B) Extracts from parallel sets of oocytes were incubated with His-CPEB and [{gamma}-32P]ATP in an in vitro His-CPEB phosphorylation assay. Ni-bead bound samples were analyzed by SDS-PAGE, Coomassie staining (CS) and autoradiography (32P). (C) Progression to GVBD, monitored by appearance of a white spot at the animal pole, was followed in Toxin-B- and control-injected oocytes, and plotted versus incubation time.

 

Figure 2
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  		Fig. 2. P-T295 of Xenopus Aur-A is not detected until GVBD. (A) Protein extracts from oocytes collected after incubation in progesterone (hr in pg) for the indicated times (in hours) were analyzed by SDS-PAGE and immunoblotting with antibodies against total Aur-A (top panel) and P-T295 Aur-A (P-Aur-A, bottom panel). (B) Protein extracts from oocytes collected at 100% GVBD were immunodepleted with either anti-Aur-A antibody or non-specific IgG. Samples were analyzed by SDS-PAGE and immunoblotting with antibody for either P-T295 Aur-A (P-Aur-A, top panel) or Aur-A (bottom panel). Arrowheads indicate migration of mobility shifted Aur-A;? indicates the band of a protein detected non-specifically by the antibody.

 

Figure 3
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  		Fig. 3. Early phosphorylation of CPEB occurs in the absence of Aur-A activity. (A) His-CPEB phosphorylation assays using Aur-A immunodepleted extracts. Protein extracts from oocytes collected after incubation in progesterone (hr in pg) for the indicated times (in hours) were immunodepleted with non-specific IgG antibody, antibody against total Aur-A or no antibody and used in His-CPEB phosphorylation assays. Immunodepleted samples were analyzed by SDS-PAGE and immunoblotted with anti-Aur-A antibody. Phosphorylated His-CPEB signal intensity was adjusted relative to the quantity of input His-CPEB. Asterisk indicates 100% GVBD oocytes. (B) Samples from A were immunoblotted with antibodies against Aur-A, phosphorylated (active) MAPK (P-MAPK), and unphosphorylated and phosphorylated Cdc2. Asterisk indicates 100% GVBD oocytes. (C) The Aurora kinase inhibitor ZM447439 blocks Aur-A activity in vitro. Increasing amounts of recombinant Aur-A were treated with either ZM447439 or DMSO, then incubated with myelin basic protein (MBP) as a substrate in the presence of [{gamma}-32P]ATP. (D) ZM447439 inhibits endogenous AurA kinase. Affinity-purified Aur-A antibody or NS-IgG immunoprecipitates from GVBD extracts were incubated with MBP and [{gamma}-32P]ATP in the presence of ZM447439 or DMSO. (E) ZM447439 does not block CPEB phosphorylation. Protein extracts from oocytes collected at the indicated times were treated with either 20uM ZM447439 or DMSO and used in His-CPEB phosphorylation assays. Asterisk indicates 100% GVBD oocytes. Input extracts were incubated with histone H1 and [{gamma}-32P]ATP in an H1 kinase assay (pH1, lower panel). (F) ZM447439 does not inhibit GVBD. Oocytes were injected with either 40 nl of 500 µM ZM447439 or DMSO two hours prior to the addition of progesterone. After overnight incubation, the number of oocytes that had achieved GVBD was scored. CS, Coomassie staining; 32P, autoradiography; IB, immunoblotting.

 

Figure 4
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  		Fig. 4. MAPK activation and protein synthesis are required for early CPEB phosphorylation. (A) U0126 blocks CPEB phosphorylation. Oocytes were cultured in the presence of U0126, cycloheximide (cyclohex), or no additive (control) for 1 hour prior to the addition of progesterone (pg). Extracts were prepared from oocytes collected at the indicated times (hr in pg) and used in His-CPEB phosphorylation assays. Samples were analyzed by immunoblotting with anti-P-MAPK and MAPK antibodies (lower two panels). (B) Inactivation of MAPK reduces CPEB phosphorylation. Protein extracts from oocytes collected at the indicated times (hr in pg) were treated first with either Mkp1 or buffer, and then His-CPEB and [{gamma}-32P]ATP were added. Samples were analyzed as in A. Asterisk indicates 100% GVBD oocytes. (C) The extracts used in B were used in an H1 kinase assay. Asterisk indicates 100% GVBD oocytes. CS, Coomassie staining; 32P, autoradiography; IB, immunoblotting.

 

Figure 5
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  		Fig. 5. CPEB is phosphorylated by endogenous MAPK during early meiosis. (A) Protein extracts from oocytes collected at the indicated times were used in IP assays with either anti-P-MAPK antibody or non-specific rabbit antibody (NS). His-CPEB tethered to Ni beads were added to the immunoprecipitates and incubated in the presence of [{gamma}-32P]ATP. Ni-bead bound samples were analyzed by SDS-PAGE, Coomassie staining and autoradiography. Asterisks indicate 100% GVBD oocytes. (B) Bead-bound samples from A were immunoblotted with anti-MAPK antibody to confirm efficiency of immunoprecipitation. Asterisks indicate 100% GVBD oocytes. (C) In vitro phosphorylation of His-CPEB using recombinant MAPK. His-CPEB or BSA were incubated with the indicated units of MAPK, together with [{gamma}-32P]ATP. Samples were analyzed by SDS-PAGE, Coomassie staining (CS) and autoradiography (32P). (D) Schema of His-CPEB protein showing positions of MAPK phosphorylation sites determined by LC-MS/MS phosphopeptide mapping after using either recombinant MAPK from experiments described in C or staged oocyte extracts (bottom three lines) as the kinase source. RRM, RNA-recognition motif; ZF, zinc-finger motif; *, S174; PEST, degradation-targeting domain rich in proline, glutamate, serine and threonine. (E) Table of average charge-to-mass values for ionized peptides (b and y) generated after secondary fragmentation of the CPEB tryptic peptide LDSR. (Left) His-CPEB phosphorylated in vitro using MAPK (as shown in D). (Right) His-CPEB phosphorylated by incubation with extracts from early-maturing oocytes. A difference of 80 in the mass-to-charge values of the S174 phosphorylated versus non-phosphorylated peptide ions indicates the presence of the phosphate group. *, S174; CS, Coomassie staining; 32P, autoradiography; IB, immunoblotting; IP, immunoprecipitation.

 

Figure 6
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  		Fig. 6. Phosphorylation of CPEB in early meiosis correlates with transient, low level MAPK activation, which is independent of Mos synthesis. (A) Oocytes were injected with either Mos sense oligonucleotides (Mos-S) or Mos antisense oligonucleotides (Mos-AS). After 1 hour, progesterone was added and oocytes were collected at times indicated (hr in pg). Protein extracts were analyzed by SDS-PAGE and immunoblotted with anti-P-MAPK and anti-MAPK antibodies. (B) In vitro phosphorylation assay of His-CPEB. Oocytes were cultured in the presence of DMSO or U0126 for 1 hour prior to progesterone addition. Extracts from 90 oocytes selected at the indicated times (hr in pg) were used in His-CPEB phosphorylation assays. CS, Coomassie staining; 32P, autoradiography.

 

Figure 7
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  		Fig. 7. XGef is present in a complex with MAPK. (A) Endogenous XGef associates with a U0126 sensitive kinase that can phosphorylate CPEB. Some oocytes were treated with U0126 for 1 hour prior to progesterone addition; protein extracts were prepared from oocytes collected at the indicated times (hr in pg). Endogenous protein complexes were immunoprecipitated with anti-XGef antibody or non-specific IgG. Either recombinant His-CPEB tethered to Ni beads or blank beads alone were added to the immunoprecipitated complexes and incubated in the presence of [{gamma}-32P]ATP. Ni-bead-bound samples were analyzed by SDS-PAGE, Coomassie staining and autoradiography. (B) Immunoblot analysis for the samples used in A with anti-P-MAPK and anti-MAPK antibodies. Asterisk indicates 100% GVBD oocytes. (C) Extracts from A were used in an H1 kinase assay. Asterisk indicates 100% GVBD oocytes. (D) MAPK is present in HA-XGef immunoprecipitates. Protein extracts from prophase oocytes (–pg, not exposed to progesterone) and early meiotic oocytes (+pg, exposed to progesterone for 3 hours), expressing HA-XGef or not were used in immunoprecipitations with anti-HA antibody conjugated to Protein-G-agarose. Immunoprecipitates were analyzed by SDS-PAGE and immunoblotted with anti-MAPK (top panel) and anti-XGef antibodies (middle panel). Input samples were analyzed with MAPK antibody (bottom panel). CS, Coomassie staining; 32P, autoradiography; IB, immunoblotting; IP, immunoprecipitation.

 

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© The Company of Biologists Ltd 2007