Podcasts about ste20

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.21.550060v1?rss=1 Authors: Roberto, G. M., Boutet, A., Keil, S., Emery, G. Abstract: Collective cell migration occurs in various biological processes such as development, wound healing and metastasis. During Drosophila oogenesis, border cells (BC) form a cluster that migrates collectively inside the egg chamber. The Ste20-like kinase Misshapen (Msn) is a key regulator of BC migration coordinating the restriction of protrusion formation and contractile forces within the cluster. Here, we demonstrate that the kinase Tao acts as an upstream activator of Msn in BCs. Depletion of Tao significantly impedes BC migration and produces a phenotype similar to Msn loss-of-function. Furthermore, we show that the localization of Msn relies on its CNH domain, which interacts with the small GTPase Rap2l. Our findings indicate that Rap2l promotes the trafficking of Msn to the endolysosomal pathway. When Rap2l is depleted, the levels of Msn increase in the cytoplasm and at cell-cell junctions between BCs. Overall, our data suggest that Rap2l ensures that the levels of Msn are higher at the periphery of the cluster through the targeting of Msn to the degradative pathway. Together, we identified two distinct regulatory mechanisms that ensure the appropriate distribution and activation of Msn in BCs. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

The cytoskeleton in most eukaryotes consists of actin filaments, intermediate filaments, microtubules and specific associated proteins. It determines the shape and the polarity of a cell and is inevitable for the coordination of cell movement. The regulation of this complex structure requires a highly organised and specialised signalling network. Ste20-like kinases and the regulator protein Mo25 (Morula protein 25) are part of this signalling network. The main objective of this work was the functional characterisation of the regulator protein Mo25 and the Ste20-like kinases Fray1, Fray2 (Frayed kinase 1/2) and DstC (Dictyostelium serine threonine kinase C) in the amoeba Dictyostelium discoideum (D. discoideum). An additional project was to map and characterise the actin and actin related genes in the genome of the fresh water foraminifer Reticulomyxa filosa (R. filosa). Mo25 is a highly conserved 40 kDa scaffolding protein with a 60% identity from amoeba to man. The disruption of the mo25 gene in D. discoideum results in very large, multinucleated cells which are unable to complete cytokinesis. Growth as well as development is severely delayed in the Mo25-minus strain. Furthermore, in phototaxis assays performed with multicellular aggregates (slugs), the Mo25-minus slugs were unable to migrate towards the light source. These findings imply that Mo25 plays an important role in cytokinesis, growth and cell polarity. We could link the Ste 20-like kinase SvkA (severin kinase), a homolog of the human Mst3, Mst4 (Mammalian Ste20-like kinase 3/4) and Ysk1/Sok1 (Yeast Sps1/Ste20-related kinase 1, Suppressor of Kinase 1) kinases to Mo25 as a binding partner. To further elucidate the interaction of Mo25 with SvkA as well as their role in cytokinesis or polarity signalling, we generated a series of GFP–Mo25 rescue constructs with distinct point mutations in protein-protein interaction surfaces and transformed these into the Mo25-minus background. The kinase domains of the Ste20-like kinases, Fray1 and Fray2 in D. discoideum are highly homologous to the catalytic domains of OSR1 (Oxidative stress response kinase 1) and SPAK (Ste20/SPS1-related proline-alanine-rich protein kinase) in humans and Frayed in fruit fly. Here, we generated the knockout clones Fray1-minus, Fray2-minus, and the double knockout Fray2Fray1-minus in D. discoideum. In developmental studies, Fray2-minus did not show an altered phenotype, whereas Fray1-minus and Fray2Fray1-minus developed slightly slower into fruiting bodies. When grown in shaking culture, Fray1-minus and Fray2Fray1-minus showed a reduced growth rate compared to Fray2-minus and the wild type. In addition, by using a GFP-Trap resin we identified a binding partner of Fray1, a yet unknown protein that we named FRIP (Fray Interacting Protein). FRIP mainly consists of a CBS (Cystathionine beta synthase) domain pair and is 30% identical to the gamma subunit of the AMPK (5‘ adenosine mono phosphate-activated protein kinase) complex in D. discoideum. The Ste20-like kinase DstC has been described to be a regulator of the actin driven process of phagocytosis. The catalytic domain of DstC is most similar to the mammalian kinase Mst2 (Mammalian Ste20-like kinase 2) and Hippo (“Hippopotamus”-like phenotype) of D. melanogaster. We could map the sorting signal that localises DstC to phagocytic cups and acidic vesicles to about 90 amino acids. Here we present an array of distinct point mutations for the identification of the exact localisation signal. R. filosa is a fresh water protist which belongs to the the group of Foraminifera within the Rhizaria. The R. filosa genome is the first foraminiferal and only the second rhizarian genome to be deciphered. In this bioinformatics project, we could identify, map and characterise four new actin genes in addition to the already known actin and about 40 genes that code for potential actin related proteins of seven different protein classes.

Fri, 22 Jan 2010 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/11082/ https://edoc.ub.uni-muenchen.de/11082/1/Batsios_Petros.pdf Batsios, Petros

Mitosis is the process by which sister chromatids are equally segregated into two daughter cells. Tight control in various events during mitotic progression is essential for maintaining chromosome stability. Mitotic kinases including Cyclin dependent kinase 1 (Cdk1) and Aurora family are required for regulating proper mitotic progression by phosphorylating mitotic substrates thereby, controlling their activities, localization or abundance. On the other hand, these mitotic kinases are modulated by de-novo synthesis, activators, phosphorylation and ubiquitin-dependent proteolysis. A thorough understanding of the function and regulation of mitotic kinases could further our knowledge on mitotic progression. In the first part of the thesis, we investigated the expression, localization and regulation of human Lats1 kinase, which is a close homologue of the yeast Dbf2 kinase family involved in the mitotic exit network (MEN). Despite the fact that Lats1 has been suggested to be a spindle protein that binds and inactivates Cdk1, we found that Lats1 is mainly cytoplasmic throughout the cell cycle by immunofluorescence microscopy. Both yeast two-hybrid and coimmunoprecipitation showed no significant interaction between Lats1 and Cdk1. Although Lats1 was highly phosphorylated during mitosis, no detectable kinase activity was observed. However, we identified Ste20 like kinase MST2 as the upstream regulator of human Lats1. Phosphorylation of Lats1 by Mst2 resulted in the activation of Lats1 kinase activity both in vivo and in vitro. This kinase-substrate relation was proven to be specific, as another distant Mst2 homolog, Mst4, did not possess this ability. Subsequent mass-spectrometry-based phosphosites analysis revealed that Mst2 phosphorylates Lats1 on more than five residues. Alanine mutations on Lats1T1079 and S909 impaired Lats1 kinase activity. Thus, we could not confirm the suggested role of Lat1 in mitosis. Instead, we show that similar to its Drosophila ortholog, Lats1 is involved in the Mst2 signaling pathway and might control developmentally regulated cell proliferation and apoptosis in mammals. In the second part of this thesis, we characterized hBora, a novel Aurora A interactor originally found in Drosophila. We show that hBora is upregulated and phosphorylated during mitosis. siRNA-mediated knockdown of hBora led to spindle formation defects and aneuploidy. hBora overexpression caused monoastral spindle formation and mislocalization not only of Aurora A but also Plk1. Further investigations showed that Cdk1 phosphorylation on hBoraSer252 leads to Plk1 binding and this may promote the SCF-mediated proteolysis of hBora. Indeed, Plk1 depletion led to an increase in hBora levels. Interestingly, the co-depletion of both hBora and Plk1 (to lower hBora levels in Plk1 depleted cells) rescued the localization of Aurora A to the centrosomes and bipolar spindle formation. Thus, we propose that hBora is a functional link between Plk1 and Aurora A and that by modulating the proteolysis of hBora, Plk1 could regulate Aurora A localization and activity. At the end, we also investigated the function of Aurora A and could show that Aurora A is required for centriole cohesion and centrosome separation.

The major goal of the project was the investigation of proteins that regulate dynamic rearrangements of the actin cytoskeleton in Dictyostelium discoideum amoebae. Among the proteins studied in detail were (i) D. discoideum vasodilator stimulated phosphoprotein DdVASP and a new profilin isoform as putative regulators of filopodia formation, and (ii) the Ste20-like kinases Krs1 and Severin-Kinase as members of signalling cascades towards the actin cytoskeleton. Filopodia are bundles of actin filaments projecting from the cell surface. They are found on a variety of cell types and are needed among others, for cell adhesion, sensory and exploratory functions. Filopodia are frequently found associated with sheet-like arrays of actin filaments called lamellipodia and membrane ruffles. The function of the VASP homolog from D. discoideum in filopodia formation was studied using molecular, biochemical and cell biological approaches. The protein sequence of DdVASP shares a significant homology to the members from other species. The protein harbours two Ena/VASP homology domains EVH1 and EVH2 separated by a polyproline stretch. The EVH2 domain is characterised by a G-actin binding site (GAB), an F-actin binding site (FAB) and a tetramerisation domain. As a tetramer the DdVASP protein can nucleate actin polymerization and bundle actin filaments. The in vitro nucleating activity of DdVASP is salt dependent and its nucleating activity is completely abolished at 100 mM salt. However, the F-actin bundling activity as determined by the low speed sedimentation assay was not disturbed. The ability of DdVASP to influence the binding of capping protein to the growing ends of the actin filaments was tested through elongation of capped F-actin seeds and by depolymerization of capped filaments upon dilution below the critical concentration of the barbed ends. Results from both sets of experiments showed that DdVASP cannot remove capping protein from the barbed ends. The D. discoideum formin dDia2, which was previously reported to be essential for filopodia formation could elongate the capped F-actin seeds. In vitro biochemical data led to the conclusion that the bundling activity of DdVASP is the essential in vivo function to stabilise actin filaments in emerging filopodia. To test this hypothesis, a DdVASP null mutant was isolated. As expected the mutant failed to produce any filopodia. Expression of wild type DdVASP, but not DdVASPFAB, rescued the phenotype suggesting the importance of the bundling activity of DdVASP in filopodia formation. To confirm that the data obtained with DdVASP were not species specific, key biochemical functions of HsVASP were also tested. The results indicated that VASPs are functionally well conserved throughout evolution. During this study, a third profilin isoform, profilin III, was further characterised. Specific interaction between profilin III and DdVASP was discovered. Profilin III shares a limited homology at the amino acid level with the other two and well studied profilins. Polyclonal antibodies that recognise only the profilin III isoform showed that in wild type cells profilin III represents less than 1% of all profilins. This suggests a distinct role for profilin III, because a low protein concentration argues against an actin sequestering function. Immunolocalisation studies showed profilin III in filopodia and enriched at their tips. Cells lacking the profilin III protein show defects in cell motility during chemotaxis. The second part of the project dealt with the characterisation of two D. discoideum Germinal Centre Kinases (GCK). The catalytic domain of Krs1 was found to be highly homologous to the catalytic region of human MST1 and MST2 from the GCK-II subfamily. The regulatory region harbours the putative inhibitory domain (aa 330-379) and a possible multimerization (SARAH) domain (aa 412-458) described for GCKs in higher organisms. This SARAH region spans about 50 amino acid residues, is located at the extreme carboxyl terminus and most likely forms an  - helical coiled-coil motif. GFP-Krs1 overexpressing wild type cells showed an enrichment of the kinase in the cell cortex, and motility of these cells during aggregation was reduced. Krs1 knockout strains exhibited only subtle differences to wild type cells. Severin kinase is encoded by the gene svkA, and phylogenetic analysis groups it into subfamily GCK-III, along with human MST3, MST4 and YSK/SOK-1. Immunoblot analysis with polyclonal antibodies showed an uniform expression level throughout development. Gene disruption of svkA resulted in cells that had problems to divide both in submerged or in shaking cultures. Though the motility and chemotaxis of these cells remain unaltered compared to the wild type cells, the movement of the multicellular slugs is disturbed. In addition, development was delayed and the mutant formed aberrant fruiting bodies.

In this study a new Dictyostelium STE20-like protein kinase DST2 (Dictyostelium STE20-like kinase 2) was cloned and characterised. STE20 (Sterile 20) kinase was first identified in yeast as a pheromone-induced serine/threonine protein kinase that acts upstream of a MAP kinase cascade. Based on the domain structure, DST2 belongs to the GCK subfamily of STE20-like protein kinases, which include the mammalian STE20-like kinases (MST1/2/3), oxidant stress response kinase SOK-1, and DST1 in Dictyostelium discoideum which phosphorylates severin, a gelsolin-like F-actin fragmenting protein. DST2 was discovered by screening of the D. discoideum cDNA project database using DST1 as query. To confirm the existence of the DST2 gene and its expression, Southern, Northern and Western analyses of DST2 were carried out. It revealed that DST2 may have two copies in the Dictyostelium genome and that DST2 was expressed during all stages of D. discoideum development. In vitro kinase assays with bacterially expressed fusion protein of full length DST2 (aa461), the catalytic domain (aa287) and the regulatory domain (aa174) showed that autophosphorylation of DST2 occurson the regulatory domain and phosphorylates severin in the presence of a Mn2+ or Mg2+. Purified catalytic domain of PKA phosphorylated the regulatory domain of DST2 and caused an increase in the basal autophosphorylation activity of DST2, suggesting that PKA may be a potential upstream kinase of DST2 through the phosphorylation of its regulatory domain. To understand the function of the non-catalytic domain of DST2, three C-terminal truncation constructs (aa1-421, aa1-368 and aa1-326) were used in comparison to full length DST2 in in vitro kinase assays. Deletion of C-terminal regions revealed an inhibitory region amino acids 326-461 of DST2. Gel filtration chromatography showed that DST2 was eluted in a broad peak ranging from approximately 63 kDa to 400 kDa, suggesting that DST2 may exist in vivo as a monomer as well as a high molecular weight complex. The influence of phosphorylated and unphosphorylated severin on F-actin solutions was investigated using falling-ball viscometry and fluorescence spectroscopy. It turned out that phosphorylation by DST2 inhibits the F-actin fragmenting activity of severin, suggesting that DST2 may be directly involved in actin-cytoskeleton rearrangements.