Protein Kinase Assays for Measuring MPF and MAPK Activities in Mouse and Rat Oocytes and Early Embryos

Kubiak, Jacek Z.

doi:10.1007/978-1-62703-191-2_5

Cited by 9 publications

(7 citation statements)

References 21 publications

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“…Kinase activities are generally evaluated by radioactively labeled substrates in in vitro assays [26], but can barely be demonstrated at the subcellular level and in a cell type-specific manner. Here, we used FRAP to demonstrate that because of polarized BASL, MPK6 mobility is unequal between two daughter cells.…”

Section: Discussionmentioning

confidence: 99%

Phosphorylation of the Polarity Protein BASL Differentiates Asymmetric Cell Fate through MAPKs and SPCH

2016

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SUMMARY Cell polarization is commonly used for the regulation of stem cell asymmetric division in both animals and plants. Stomatal development in Arabidopsis, a process that produces breathing pores in the epidermis, requires asymmetric cell division to differentiate highly specialized guard cells while maintaining a stem cell population [1, 2]. The BREAKING OF ASYMMETRY IN THE STOMATAL LINEAGE (BASL) protein exhibits a polarized localization pattern in the cell and is required for differential cell fates resulting from asymmetric cell division [3]. The polarization of BASL is made possible by a positive feedback loop with a canonical Mitogen-activated protein kinase (MAPK) pathway that recruits the MAPKK Kinase YODA (YDA) and MAPK 6 (MPK6) to the cortical polarity site [4]. Here we study BASL intracellular dynamics and show that the membrane-associated BASL is slowly replenished at the cortical polarity site and the mobility is tightly linked to its phosphorylation status. Because BASL polarity is only exhibited by one daughter cell after an asymmetric cell division, we study how BASL differentially functions in the two daughter cells. The YDA MAPK cascade transduces upstream ligand-receptor signaling [5–13] to the transcription factor SPEECHLESS (SPCH) that controls stomatal initiation and is directly suppressed by MPK3/6-mediated phosphorylation [14, 15]. We show that BASL polarization leads to elevated nuclear MPK6 signaling and lowered SPCH abundance in one of the two daughter cells. Therefore, two daughter cells are differentiated by BASL polarity-mediated differential suppression of SPCH, which may provide developmental plasticity in plant stem cell ACD.

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Section: Discussionmentioning

confidence: 99%

Phosphorylation of the Polarity Protein BASL Differentiates Asymmetric Cell Fate through MAPKs and SPCH

2016

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“…Moreover, MAPK activation in cumulus cells requires one or more oocyte paracrine factors to induce GVBD and cumulus expansion. As a MAP kinase kinase kinase, c‐mos is a proto‐oncogene activates extracellular signal‐regulated protein kinases (ERK) 1 and 2, and cyclinb1 and cdc2, which are essential regulators of MPF (Kubiak ). We selected c‐mos, cyclinb1 and cdc2 as maternal transcripts for a detailed study of gene expression patterns and polyadenylation status.…”

Section: Discussionmentioning

confidence: 99%

Effects of Growth Differentiation Factor 9 and Bone Morphogenetic Protein 15 on the in vitro Maturation of Porcine Oocytes

et al. 2013

Reprod Domestic Animals

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Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are members of the transforming growth factor-β (TGF-β) family, and their roles in oocyte maturation and cumulus expansion are well known in the mouse and human, but not in the pig. We investigated GDF9 and BMP15 expressions in porcine oocytes during in vitro maturation. A significant increase in the mRNA levels of GDF9 and BMP15 was observed at germinal vesicle breakdown, with expression levels peaking at metaphase I (MI), but decreasing at metaphase II (MII). GDF9 and BMP15 protein localized to the oocyte cytoplasm. While treatment with GDF9 and BMP15 increased the expression of genes involved in both oocyte maturation (c-mos, cyclinb1 and cdc2) and cumulus expansion (has2, ptgs2, ptx3 and tnfaip6), SB431542 (a TGFβ-GDF9 inhibitor) decreased meiotic maturation at MII. Following parthenogenetic activation, the percentage of blastocysts in SB431542 treatment was lower than in the control (41.3% and 74.4%, respectively). Treatment with GDF9 and BMP15 also increased the mRNA levels of maternal genes such as c-mos [a regulatory subunit of mitogen-activated protein kinase (MAPK)], and cyclinb1 and cdc2 [regulatory subunits of maturation/M-phase-promoting factor (MPF)]; however, SB431542 significantly decreased their mRNA levels. These data were supported by poly (A)-test PCR and protein activity analyses. Our results show that GDF9 and BMP15 participate in cumulus expansion and that they stimulate MPF and MAPK activities in porcine oocytes during in vitro maturation.

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“…Kinase assays performed with casein and MPK10 or MPK10-K51A at pH 5.5, 6.5, 7.5 and 8.5 revealed different pH optima for auto- and substrate-specific phosphorylation at pH 6.5 and 7.5, respectively (Figure S2, lower panel). In conclusion, recombinant MPK10 shows some minor auto-phophorylation activity, but largely fails to phosphorylate the canonical MAPK substrate MBP [37]–[39], which may either depend on activation through an upstream M2K, parasite-specific kinase substrate interactions, or auto-inhibition.…”

Section: Resultsmentioning

confidence: 92%

“…We used the bacterially expressed and purified proteins in an in vitro kinase assay at 30°C or 37°C, monitoring the transfer of radiolabeled phosphate from γ- 33 P-ATP to MPK10 (auto-phosphorylation) or to the canonical MAPK substrate myelin basic protein (MBP) [37]–[39]. The kinase reactions were subjected to SDS-PAGE, proteins were visualized by Coomassie staining for normalization (Figure 1A, upper panel), and phosphotransferase activity was revealed by auto-radiography (Figure 1A, lower panel).…”

Section: Resultsmentioning

confidence: 99%

Transgenic Analysis of the Leishmania MAP Kinase MPK10 Reveals an Auto-inhibitory Mechanism Crucial for Stage-Regulated Activity and Parasite Viability

et al. 2014

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Protozoan pathogens of the genus Leishmania have evolved unique signaling mechanisms that can sense changes in the host environment and trigger adaptive stage differentiation essential for host cell infection. The signaling mechanisms underlying parasite development remain largely elusive even though Leishmania mitogen-activated protein kinases (MAPKs) have been linked previously to environmentally induced differentiation and virulence. Here, we unravel highly unusual regulatory mechanisms for Leishmania MAP kinase 10 (MPK10). Using a transgenic approach, we demonstrate that MPK10 is stage-specifically regulated, as its kinase activity increases during the promastigote to amastigote conversion. However, unlike canonical MAPKs that are activated by dual phosphorylation of the regulatory TxY motif in the activation loop, MPK10 activation is independent from the phosphorylation of the tyrosine residue, which is largely constitutive. Removal of the last 46 amino acids resulted in significantly enhanced MPK10 activity both for the recombinant and transgenic protein, revealing that MPK10 is regulated by an auto-inhibitory mechanism. Over-expression of this hyperactive mutant in transgenic parasites led to a dominant negative effect causing massive cell death during amastigote differentiation, demonstrating the essential nature of MPK10 auto-inhibition for parasite viability. Moreover, phosphoproteomics analyses identified a novel regulatory phospho-serine residue in the C-terminal auto-inhibitory domain at position 395 that could be implicated in kinase regulation. Finally, we uncovered a feedback loop that limits MPK10 activity through dephosphorylation of the tyrosine residue of the TxY motif. Together our data reveal novel aspects of protein kinase regulation in Leishmania, and propose MPK10 as a potential signal sensor of the mammalian host environment, whose intrinsic pre-activated conformation is regulated by auto-inhibition.

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Protein Kinase Assays for Measuring MPF and MAPK Activities in Mouse and Rat Oocytes and Early Embryos

Cited by 9 publications

References 21 publications

Phosphorylation of the Polarity Protein BASL Differentiates Asymmetric Cell Fate through MAPKs and SPCH

Phosphorylation of the Polarity Protein BASL Differentiates Asymmetric Cell Fate through MAPKs and SPCH

Effects of Growth Differentiation Factor 9 and Bone Morphogenetic Protein 15 on the in vitro Maturation of Porcine Oocytes

Transgenic Analysis of the Leishmania MAP Kinase MPK10 Reveals an Auto-inhibitory Mechanism Crucial for Stage-Regulated Activity and Parasite Viability

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