Barnard DC et al. (2005), Differential phosphorylation controls Maskin as...

XB-ART-1488

Mol Cell Biol 2005 Sep 01;2517:7605-15. doi: 10.1128/MCB.25.17.7605-7615.2005.

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Differential phosphorylation controls Maskin association with eukaryotic translation initiation factor 4E and localization on the mitotic apparatus.

Barnard DC , Cao Q , Richter JD .

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Several cytoplasmic polyadenylation element (CPE)-containing mRNAs that are repressed in Xenopus oocytes become active during meiotic maturation. A group of factors that are anchored to the CPE are responsible for this repression and activation. Two of the most important are CPEB, which binds directly to the CPE, and Maskin, which associates with CPEB. In oocytes, Maskin also binds eukaryotic translation initiation factor 4E (eIF4E), an interaction that excludes eIF4G and prevents formation of the eIF4F initiation complex. When the oocytes are stimulated to reenter the meiotic divisions (maturation), CPEB promotes cytoplasmic polyadenylation. The newly elongated poly(A) tail becomes bound by poly(A) binding protein (PABP), which in turn binds eIF4G and helps it displace Maskin from eIF4E, thereby inducing translation. Here we show that Maskin undergoes several phosphorylation events during oocyte maturation, some of which are important for its dissociation from eIF4E and translational activation of CPE-containing mRNA. These sites are T58, S152, S311, S343, S453, and S638 and are phosphorylated by cdk1. Mutation of these sites to alanine alleviates the cdk1-induced dissociation of Maskin from eIF4E. Prior to maturation, Maskin is phosphorylated on S626 by protein kinase A. While this modification has no detectable effect on translation during oocyte maturation, it is critical for this protein to localize on the mitotic apparatus in somatic cells. These results show that Maskin activity and localization is controlled by differential phosphorylation.

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Species referenced: Xenopus laevis
Genes referenced: cdk1 cpeb1 eif4a2 eif4e eif4g1 mos pabpc4 prss1 ptpa tacc3
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	Fig. 1. Maskin is phosphorylated during oocyte maturation. A. Protein from oocytes that were incubated with progesterone (Prog) for various times was immunoblotted and probed for Maskin (top panel). The arrows denote the Maskin electrophoretic mobility shift that occurred 6 h after progesterone addition, which was coincident with germinal vesicle breakdown. B. Oocytes were incubated with [32P]orthophosphate overnight before some of them were stimulated with progesterone. Maskin was immunoprecipitated and analyzed by SDS-PAGE and autoradiography. The lower band represents a nonspecific protein that does not react with Maskin antibody on Western blots. The right portion of the panel shows a Coomassie (Coom.) blue-stained SDS gel loaded with E. coli-expressed Maskin. C. The Maskin band in panel B (8 h in progesterone) was blotted onto a polyvinylidene difluoride membrane, digested with trypsin, and analyzed by two-dimensional phosphopeptide mapping (middle panel). Mature oocyte (egg) extracts were supplemented with E. coli-expressed Maskin (as in panel B, right) and [32P-Î³]ATP; Maskin was then isolated and processed for two-dimensional phosphopeptide mapping as above. The circled regions in the two panels show areas with similar phosphopeptides. TLC refers to thin-layer chromatography, and TLE refers to thin-layer electrophoresis.
	Fig. 2. Time course of Maskin phosphorylation. A. Oocytes, some of which were injected with mos antisense or sense oligonucleotides, were incubated with progesterone (Prog) for up to 8 h. They were then examined for H1 kinase activity, which is an indicator of MPF (cdk1/cyclin B) activation. Note that injected mos antisense oligonucleotide (oligo) prevented MPF activation. B. E. coli-expressed Maskin and [32P]ATP were added to extracts derived from noninjected oocytes or oocytes that were injected with mos antisense oligonucleotide. Following an incubation period, Maskin was processed for two-dimensional phosphopeptide mapping. The circled areas denote Maskin phoshopeptides that were present throughout oocyte maturation, irrespective of whether MPF was activated.
	Fig. 3. Identification of maturation-specific Maskin phosphoamino acids. A. E. coli-expressed Maskin was phosphorylated in egg extracts, and the resulting phosphopeptides were identified arbitrarily. B. Several Maskin deletion proteins were added to egg extracts and processed for two-dimensional phosphopeptide mapping. The phoshopeptides that were lost when each deletion mutant was analyzed are noted. C. Maskin was phosphorylated in vitro with cdk1/cyclin B1 (MPF) and processed for two-dimensional phosphopeptide mapping. D. Maskin was phosphorylated in vitro with MAP kinase and processed for phosphopeptide mapping. E to I. Phosphorylation of Maskin containing an S343A, T58A, S638A, S152A, or S638A and S453A mutations in egg extracts. J. Phosphorylation of Maskin containing T58A, S152A, S311A, S343A, S453A, and S638A mutations (A6) in egg extracts. K. Representation of a Maskin phosphopeptide map showing the positions of peptides containing phosphorylated amino acids. Phosphopeptides 1 and 2 were not identified in these assays; phosphopeptide 2 was also detected in oocytes without progesterone stimulation (see Fig. â€‹Fig.4).4). L. Positions of phosphorylated residues of Maskin relative to the eIF4E binding domain and the coiled-coil domain. TLC, thin-layer chromatography; TLE, thin-layer electrophoresis.
	Fig. 4. Maskin is phosphorylated by PKA in oocyte extracts. A. E. coli-expressed Maskin was phosphorylated in egg extracts in the presence or absence of PKI, a specific inhibitor of PKA, and then processed for two-dimensional phosphopeptide mapping. The circled areas show phosphopeptides that were eliminated by PKI. B. Maskin was phosphorylated in oocyte extracts or by pure PKA and processed as described previously. The phosphopeptides from these two reactions were also mixed and analyzed on a two-dimensional phosphopeptide map. C. Wild-type or S626D Maskin proteins were phosphorylated in egg extracts and analyzed as described above. The circled area shows phosphopeptides that were eliminated when the mutant Maskin was used. The right portion shows all the Maskin phosphoamino acids in relation to the eIF4E binding and coiled-coil domains. TLC, thin-layer chromatography; TLE, thin-layer electrophoresis
	Fig. 5. Phosphorylation controls Maskin association with eIF4E and translation of CPE-containing mRNA. A. mRNAs encoding wild-type (WT) Maskin, A6 Maskin (T58A, S152A, S311A, S343A, S453A, S638A), and eIF4E were translated in reticulocyte lysates in the presence of [35S]methionine. The lysates also contained 0.2 mM GTP and in some cases 0.25 mM 7mGTP (i.e., free cap), cdk1/cyclin B1, or the phosphatase PP2A. One-twentieth of each lysate was analyzed for Maskin and eIF4E synthesis (load), while the rest was applied to cap columns. The material that was retained on the columns was analyzed by SDS-PAGE and autoradiography. B. mRNAs encoding WT or A6 Maskin proteins were injected into oocytes, which were then stimulated with progesterone (Prog) for various times. Protein from the oocytes was extracted, Western blotted, and probed for Maskin and cyclin B1. C. E. coli-expressed WT and A6 Maskin proteins were injected into oocytes, some of which were treated with progesterone. At the indicated times, the oocytes were harvested; the protein was extracted and probed on Western blots for Maskin and cyclin B1.
	Fig. 6. Maskin and CPEB are associated with mitotic spindles and centrosomes. Xenopus egg extracts were supplemented with rhodamine-labeled tubulin and demembranated frog sperm DNA. Following incubation, the spindles were fixed and centrifuged onto glass coverslips, where they were immunostained for Maskin or CPEB and were costained with DAPI to detect DNA.
	Fig. 7. Serine 626 directs Maskin to the mitotic apparatus. A. Xenopus tissue culture cells (XTC) were immunostained for Maskin and tubulin and costained with DAPI. HeLa cells were also transfected with plasmids encoding GFP fused to wild-type or S626A mutant Maskin molecules. The cells were immunostained for tubulin and costained with DAPI. B. Limited sequence alignment among Xenopus Maskin, human and mouse TACC3, and Drosophila DTACC denoting a conserved serine corresponding to Maskin serine 626. C. HeLa cells were transfected with the same constructs, as well as GFP-Maskin A6, as noted for panel A (bottom). Antibody directed against GFP was use to coimmunoprecipitate MAP-215, a protein that links TACC family proteins to microtubules; this protein and the Maskin fusion proteins were detected by Western blotting.
	Fig. 8. Phosphorylation of Maskin. During oogenesis, when oocytes are arrested at the diplotene stage of prophase I, PKA phosphorylates (P) Maskin residue S626. When the oocytes are stimulated by progesterone to reenter the meiotic divisions (oocyte maturation), Aurora A is activated (23, 32) and phosphorylates CPEB S174; this event stimulates polyadenylation (1). The newly elongated poly(A) tail is bound by poly(A) binding protein (PABP), which in turn binds eIF4G and helps it displace Maskin from eIF4E, thereby initiating translation (2). cdk1, whose activity is detected after that of Aurora A (23), phosphorylates Maskin residues T58, S152, S311, S343, S453, and S638. These phosphorylations help Maskin dissociate from eIF4E. The S626 phosphorylation regulates the association of Maskin with the mitotic apparatus during embryogenesis and in adult somatic cells (13). Green, red, and blue Pâ€™s indicate phosphorylation catalyzed by PKA, cdk1, and Aurora A, respectively.

References [+] :

Barnard, Symplekin and xGLD-2 are required for CPEB-mediated cytoplasmic polyadenylation. 2004, Pubmed, Xenbase