Journal: Molecular Biology of the Cell

Article Title: Phosphorylation of the RNA-binding protein Dazl by MAPKAP kinase 2 regulates spermatogenesis

doi: 10.1091/mbc.E15-11-0773

Figure Lengend Snippet: Dazl is a novel substrate of MK2. (A) Reduction of MK2 expression by shRNA. Cell lysates from SUM149 cells stably expressing either control or MK2 shRNA were resolved by SDS–PAGE (on two separate gels) and immunoblotted with the indicated antibodies. (B) Schematic of the screen used to identify novel MK2 substrates. Lysates from control and MK2 shRNA cells were incubated with human protein microarrays and probed with anti-phosphoserine and anti-phosphothreonine antibodies, followed by detection with Alexa 488–conjugated secondary antibodies. Arrays were scanned and quantified. See Materials and Methods for additional details. (C) Bar graph of the top 20 putative MK2 substrates identified from the screen. Data are plotted as the average fold signal reduction obtained from the MK2- knockdown shRNA array compared with the control shRNA knockdown array. Error bars indicate SD. (D) In vitro kinase assay using GST-Dazl and MK2. GST-Dazl (or no substrate) was incubated in the presence or absence of active recombinant MK2 and ATPγS, followed by treatment with PNBM. Kinase reactions were resolved by SDS–PAGE (on three separate gels), followed by immunoblotting with the indicated antibodies.

Article Snippet: Human MK2 was PCR amplified as an Eco RI– Not I fragment from a cDNA clone (OriGene) and cloned into the corresponding sites of pCMV-myc (Clontech, Mountain View, CA).

Techniques: Expressing, shRNA, Stable Transfection, SDS Page, Incubation, In Vitro, Kinase Assay, Recombinant, Western Blot

Journal: Molecular Biology of the Cell

Article Title: Phosphorylation of the RNA-binding protein Dazl by MAPKAP kinase 2 regulates spermatogenesis

doi: 10.1091/mbc.E15-11-0773

Figure Lengend Snippet: MK2 phosphorylates Dazl at S65. (A) Domain architecture of human Dazl. DAZ, deleted in azoospermia domain; RRM, RNA recognition motif. The putative MK2 phosphorylation site (S65) is indicated in red. (B) MK2-mediated phosphorylation of Dazl-S65 in vitro. Wild-type or S65A GST-Dazl-RRM (residues 32–117) fusion proteins were incubated with active recombinant MK2 and ATPγS, followed by incubation with PNBM. Kinase reactions were resolved by SDS–PAGE (on two separate gels), followed by immunoblotting with the indicated antibodies. (C) Detection of Dazl-S65 phosphorylation using a phosphospecific antibody. GST-Dazl RRM (wild-type or S65A) fusion proteins were incubated with active recombinant MK2 and ATP. Kinase reactions were resolved by SDS–PAGE (on two separate gels), followed by immunoblotting with the indicated antibodies. (D) Alteration of Dazl-S65 phosphorylation status via genetic manipulation of the p38-MK2 signaling pathway. HEK293 cells were transfected with the indicated constructs and either stimulated or not with sodium arsenite (Ars) and lysed. Cell lysates were split, resolved by SDS–PAGE (on eight separate gels), and immunoblotted with the indicated antibodies. Western blots shown are representative of three independent experiments. (E) Reduced Dazl-S65 phosphorylation by chemical inhibition of the p38-MK2 signaling pathway. HEK293 cells were transfected with the indicated constructs and pretreated for 30 min with DMSO (vehicle) only, the p38 inhibitor SB20350 (10 μM), or MK2 inhibitor III (20 μM). Cells were then stimulated with sodium arsenite and lysed. Cell lysates were split, resolved by SDS–PAGE (on seven separate gels), and immunoblotted with the indicated antibodies. Western blots shown are representative of three independent experiments.

Article Snippet: Human MK2 was PCR amplified as an Eco RI– Not I fragment from a cDNA clone (OriGene) and cloned into the corresponding sites of pCMV-myc (Clontech, Mountain View, CA).

Techniques: In Vitro, Incubation, Recombinant, SDS Page, Western Blot, Transfection, Construct, Inhibition

Journal: Molecular Biology of the Cell

Article Title: Phosphorylation of the RNA-binding protein Dazl by MAPKAP kinase 2 regulates spermatogenesis

doi: 10.1091/mbc.E15-11-0773

Figure Lengend Snippet: Expression of Dazl, but not Dazl-S65D, rescues meiotic defects and infertility in boule mutant male flies. (A) Expression of human myc-tagged Dazl or Dazl-S65D in the Drosophila male germline. Testes from flies of the indicated genotypes were dissected, and protein extracts were resolved by SDS–PAGE and immunoblotted with the indicated antibodies. (B) Transgenic expression of wild-type human Dazl in the Drosophila male germline rescues the sterility of boule mutants. Hatch-rate analysis from crosses of male flies of the indicated genotype to w 1118 virgin females. Mean values from three independent experiments. Error bars denote SD. * p < 0.05 and ** p < 0.005 as calculated by a Student’s t test. n.s., not significant. (C) Phase contrast microscopy of live testes from flies of the indicated genotypes. Yellow arrows denote bundles of elongating spermatids that are absent in testes of bol 1 flies and flies expressing the human DAZL-S65D transgene in the bol 1 genetic background. Green boxes denote areas displayed in magnified insets. Green arrowheads indicate examples of round (“onion-stage”) spermatids characterized by a phase-light nucleus juxtaposed to a phase-dark mitochondrial aggregate. Pink arrows indicate cysts of degenerating primary spermatocytes. Red asterisks denote the apical end of the testis. Scale bar, 400 μm. (D) Expression of twine in testes from flies of the indicated genotypes. Testes were dissected, and protein extracts were resolved by SDS–PAGE, followed by immunoblotting with the indicated antibodies. Western blots shown are representative of three independent experiments. (E) Model. Cellular stress induces phosphorylation of Dazl S65 via the p38-MK2 kinase pathway in male germ cells. This posttranslational modification decreases the binding of Dazl to PABP, leading to reduced translation of the proteins encoded by those mRNAs.

Article Snippet: Human MK2 was PCR amplified as an Eco RI– Not I fragment from a cDNA clone (OriGene) and cloned into the corresponding sites of pCMV-myc (Clontech, Mountain View, CA).

Techniques: Expressing, Mutagenesis, SDS Page, Transgenic Assay, Sterility, Microscopy, Western Blot, Modification, Binding Assay