RESUMEN
Smk1 is a MAPK homolog in the yeast Saccharomyces cerevisiae that controls the postmeiotic program of spore wall assembly. During this program, haploid cells are surrounded by a layer of mannan and then a layer of glucan. These inner layers of the spore wall resemble the vegetative cell wall. Next, the outer layers consisting of chitin/chitosan and then dityrosine are assembled. The outer layers are spore-specific and provide protection against environmental stressors. Smk1 is required for the proper assembly of spore walls. However, the protective properties of the outer layers have limited our understanding of how Smk1 controls this morphogenetic program. Mutants lacking the chitin deacetylases, Cda1 and Cda2, form spores that lack the outer layers of the spore wall. In this study, cda1,2∆ cells were used to demonstrate that Smk1 promotes deposition of the glucan layer of the spore wall through the partially redundant glucan synthases Gsc2 and Fks3. Although Gsc2 is localized to sites of spore wall assembly in the wild type, it is mislocalized in the mother cell cytoplasm in the smk1∆ mutant. These findings suggest that Smk1 controls assembly of the spore wall by regulating the localization of Gsc2 during sporogenesis.
Asunto(s)
Pared Celular , Glucanos , Proteínas Quinasas Activadas por Mitógenos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Esporas Fúngicas , Pared Celular/metabolismo , Pared Celular/genética , Glucanos/metabolismo , Glucosiltransferasas/genética , Glucosiltransferasas/metabolismo , Proteínas de la Membrana , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Esporas Fúngicas/genética , Esporas Fúngicas/crecimiento & desarrollo , Esporas Fúngicas/metabolismoRESUMEN
Many eukaryotic protein kinases are activated by the intramolecular autophosphorylation of activation loop residues. Smk1 is a meiosis-specific mitogen-activated protein kinase (MAPK) in yeast that autophosphorylates its activation loop tyrosine and thereby upregulates catalytic output. This reaction is controlled by an inhibitor, Isc10, that binds the MAPK during meiosis I and an activator, Ssp2, that binds Smk1/Isc10 during meiosis II. Upon completion of the meiotic divisions, Isc10 is degraded, and Smk1 undergoes autophosphorylation to generate the high activity form of the MAPK that controls spore formation. How Isc10 inhibits Smk1 is not clear. Here, we use a bacterial coexpression/reconstitution system to define a domain in the carboxy-terminal half of Isc10 that specifically inhibits Smk1 autophosphorylation. Nevertheless, Smk1 bound by this domain is able to phosphorylate other substrates, and it phosphorylates the amino-terminal half of Isc10 on serine 97. In turn, the phosphorylated motif in Isc10 inhibits the Smk1 active site. These data show that Isc10 inhibits autophosphorylation and the phosphorylation of substrates by separate mechanisms. Furthermore, we demonstrate Isc10 can inhibit the autophosphorylation of the mammalian intestinal cell kinase ICK1 (also known as CILK1), suggesting a conserved mechanism of action. These findings define a novel class of developmentally regulated molecules that prevent the self-activation of MAPKs and MAPK-like enzymes.
Asunto(s)
Proteínas Quinasas Activadas por Mitógenos , Proteínas de Saccharomyces cerevisiae , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fosforilación , Esporas Fúngicas/metabolismo , Saccharomyces cerevisiaeRESUMEN
Smk1 is a mitogen-activated protein kinase (MAPK) family member in the yeast Saccharomyces cerevisiae that controls the postmeiotic program of spore formation. Ssp2 is a meiosis-specific protein that activates Smk1 and triggers the autophosphorylation of its activation loop. A fragment of Ssp2 that is sufficient to activate Smk1 contains two segments that resemble RNA recognition motifs (RRMs). Mutations in either of these motifs eliminated Ssp2's ability to activate Smk1. In contrast, deletions and insertions within the segment linking the RRM-like motifs only partially reduced the activity of Ssp2. Moreover, when the two RRM-like motifs were expressed as separate proteins in bacteria, they activated Smk1. We also find that both motifs can be cross-linked to Smk1 and that at least one of the motifs binds near the ATP-binding pocket of the MAPK. These findings demonstrate that motifs related to RRMs can directly activate protein kinases.
Asunto(s)
Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Motivo de Reconocimiento de ARN/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Sitios de Unión/genética , Activación Enzimática/genética , Meiosis/genética , Proteínas Quinasas Activadas por Mitógenos/química , Modelos Moleculares , Mutación , Conformación Proteica , ARN de Hongos/genética , ARN de Hongos/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Esporas Fúngicas/genética , Esporas Fúngicas/metabolismoRESUMEN
Commitment to cellular differentiation programs can be controlled by self-activating transcription factors that trigger the expression of cell-type-specific genes. A new study shows that, although commitment to meiosis in yeast is controlled in this manner, additional signaling interactions promote the committed state.
Asunto(s)
Meiosis , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
Smk1 is a meiosis-specific mitogen-activated protein kinase (MAPK) in yeast that controls spore differentiation. It is activated by a MAPK binding protein, Ssp2, upon completion of the meiotic divisions. The activation of Smk1 by Ssp2 is positively regulated by a meiosis-specific coactivator of the anaphase promoting complex (APC/C) E3 ubiquitin ligase, Ama1. Here, we identify Isc10 as an inhibitor that links APC/CAma1 to Smk1 activation. Isc10 and Smk1 form an inhibited complex during meiosis I (MI). Ssp2 is produced later in the program, and it forms a ternary complex with Isc10 and Smk1 during MII that is poised for activation. Upon completion of MII, Isc10 is ubiquitylated and degraded in an AMA1-dependent manner, thereby triggering the activation of Smk1 by Ssp2. Mutations that caused Ssp2 to be produced before MII, or isc10Δ mutations, modestly reduced the efficiency of spore differentiation whereas spores were nearly absent in the double mutant. These findings define a pathway that couples spore differentiation to the G0-like phase of the cell cycle.