The role of Aspergillus nidulans polo-like kinase PlkA in microtubule-organizing center control.

Centrosomes are important microtubule-organizing centers (MTOC) in animal cells. In addition, non-centrosomal MTOCs (ncMTOCs) have been described in many cell types. The functional analogs of centrosomes in fungi are the spindle pole bodies (SPBs). In Aspergillus nidulans, additional MTOCs have been discovered at septa (sMTOC). Although the core components are conserved in both MTOCs, their composition and organization are different and dynamic. Here, we show that the polo-like kinase PlkA binds the γ-tubulin ring complex (γ-TuRC) receptor protein ApsB and contributes to targeting ApsB to both MTOCs. PlkA coordinates the activities of the SPB outer plaque and the sMTOC. PlkA kinase activity was required for astral MT formation involving ApsB recruitment. PlkA also interacted with the γ-TuRC inner plaque receptor protein PcpA. Mitosis was delayed without PlkA, and the PlkA protein was required for proper mitotic spindle morphology, although this function was independent of its catalytic activity. Our results suggest that the polo-like kinase is a regulator of MTOC activities and acts as a scaffolding unit through interaction with γ-TuRC receptors.

Genetic evidence for a microtubule-capture mechanism during polarised growth of Aspergillus nidulans.

The cellular switch from symmetry to polarity in eukaryotes depends on the microtubule (MT) and actin cytoskeletons. In fungi such as Schizosaccharomyces pombe or Aspergillus nidulans, the MT cytoskeleton determines the sites of actin polymerization through cortical cell-end marker proteins. Here we describe A. nidulans MT guidance protein A (MigA) as the first ortholog of the karyogamy protein Kar9 from Saccharomyces cerevisiae in filamentous fungi. A. nidulans MigA interacts with the cortical ApsA protein and is involved in spindle positioning during mitosis. MigA is also associated with septal and nuclear MT organizing centers (MTOCs). Super-resolution photoactivated localization microscopy (PALM) analyses revealed that MigA is recruited to assembling and retracting MT plus ends in an EbA-dependent manner. MigA is required for MT convergence in hyphal tips and plays a role in correct localization of the cell-end markers TeaA and TeaR. In addition, MigA interacts with a class-V myosin, suggesting that an active mechanism exists to capture MTs and to pull the ends along actin filaments. Hence, the organization of MTs and actin depend on each other, and positive feedback loops ensure robust polar growth.

F-box protein RcyA controls turnover of the kinesin-7 motor KipA in Aspergillus nidulans.

Fungal filamentous growth depends on continuous membrane insertion at the tip, the delivery of membrane-bound positional markers, and the secretion of enzymes for cell wall biosynthesis. This is achieved through exocytosis. At the same time, polarized growth requires membrane and protein recycling through endocytosis. Endocytic vesicles are thought to enter the protein degradation pathway or recycle their content to the cell surface. In Saccharomyces cerevisiae, the Rcy1 F-box protein is involved in the recycling process of a v-SNARE protein. We identified a Rcy1 orthologue, RcyA, in the filamentous fungus Aspergillus nidulans as a protein interacting with the KipA kinesin-7 motor protein in a yeast two-hybrid screen. The interaction was confirmed through bimolecular fluorescence complementation. RcyA possesses an F-box domain at the N terminus and a prenylation (CaaX) motif at the C terminus. RcyA shows also similarity to Sec10, a component of the exocyst complex. The RcyA protein localized to the hyphal tip and forming septa, likely through transportation on secretory vesicles and partially on early endosomes, but independently of KipA. Deletion of rcyA did not cause severe morphological changes but caused partial defects in the recycling of the SynA v-SNARE protein and the positioning of the cell end markers TeaA and TeaR. In addition, deletion of rcyA led to increased concentrations of the KipA protein, whereas the transcript concentration was unaffected. These results suggest that RcyA probably labels KipA for degradation and thereby controls the protein amount of KipA.

The cell-end marker TeaA and the microtubule polymerase AlpA contribute to microtubule guidance at the hyphal tip cortex of Aspergillus nidulans to provide polarity maintenance.

In the absence of landmark proteins, hyphae of Aspergillus nidulans lose their direction of growth and show a zigzag growth pattern. Here, we show that the cell-end marker protein TeaA is important for localizing the growth machinery at hyphal tips. The central position of TeaA at the tip correlated with the convergence of the microtubule (MT) ends to a single point. Conversely, in the absence of TeaA, the MTs often failed to converge to a single point at the cortex. Further analysis suggested a functional connection between TeaA and AlpA (an ortholog of the MT polymerase Dis1/CKAP5/XMAP215) for proper regulation of MT growth at hyphal tips. AlpA localized at MT plus-ends, and bimolecular fluorescence complementation assays suggested that it interacted with TeaA after MT plus-ends reached the tip cortex. In vitro MT polymerization assays showed that AlpA promoted MT growth up to sevenfold. Addition of the C-terminal region of TeaA increased the catastrophe frequency of the MTs. Thus, the control of the AlpA activity through TeaA might be a novel principle for MT growth regulation after reaching the cortex. In addition, we present evidence that the curvature of hyphal tips also could be involved in the control of MT growth at hyphal tips.

The Aspergillus nidulans CENP-E kinesin KipA is able to dimerize and to move processively along microtubules.

Kinesin molecular motors serve a variety of cellular functions usually in dynamic processes. One characteristic feature of many kinesins is their ATP-dependent processive movement along polymerized microtubules. However, many kinesins work as stationary polymerases or depolymerases. Therefore, it needs to be determined for each motor, whether it moves processively along microtubules or not. The Schizosaccharomyces pombe kinesin-7, Tea2, has been shown to be involved in cell end marker transportation towards the cortex to organize the actin cytoskeleton. In human, kinesin 7 promotes microtubule polymerization. In Aspergillus nidulans, the machinery for determining growth directionality is conserved, but there is no evidence yet that kinesin 7, KipA is potentially involved in the transportation of the cell end marker proteins, TeaA or TeaR or newly identified proteins such as KatA. We expressed KipA in Escherichia coli and determined the catalytic properties of this kinesin. Here we show that KipA is an active ATPase, which is able to dimerize and move processively along microtubules with a speed of 9.48 µm/min.

The Aspergillus nidulans CENP-E kinesin motor KipA interacts with the fungal homologue of the centromere-associated protein CENP-H at the kinetochore.

Chromosome segregation is an essential process for nuclear and cell division. The microtubule cytoskeleton, molecular motors and protein complexes at the microtubule plus ends and at kinetochores play crucial roles in the segregation process. Here we identified KatA (KipAtarget protein, homologue of CENP-H) as a kinesin-7 (KipA, homologue of human CENP-E) interacting protein in Aspergillus nidulans. KatA located at the kinetochore during the whole cell cycle and colocalized with KipA and partially with the putative microtubule polymerase AlpA (XMAP215) during mitosis. Deletion of katA was lethal at 37°C and caused severe growth and morphology defects at room temperature. KipA was shown before to play an important role in growth directionality determination and our new results suggest a second function of KipA in the interaction between the microtubule plus ends and the kinetochores during mitosis.

Role of mitogen-activated protein kinase Sty1 in regulation of eukaryotic initiation factor 2alpha kinases in response to environmental stress in Schizosaccharomyces pombe.

The mitogen-activated protein kinase (MAPK) Sty1 is essential for the regulation of transcriptional responses that promote cell survival in response to different types of environmental stimuli in Schizosaccharomyces pombe. In fission yeast, three distinct eukaryotic initiation factor 2alpha (eIF2alpha) kinases, two mammalian HRI-related protein kinases (Hri1 and Hri2) and the Gcn2 ortholog, regulate protein synthesis in response to cellular stress conditions. In this study, we demonstrate that both Hri1 and Hri2 exhibited an autokinase activity, specifically phosphorylated eIF2alpha, and functionally replaced the endogenous Saccharomyces cerevisiae Gcn2. We further show that Gcn2, but not Hri1 or Hri2, is activated early after exposure to hydrogen peroxide and methyl methanesulfonate (MMS). Cells lacking Gcn2 exhibit a later activation of Hri2. The activated MAPK Sty1 negatively regulates Gcn2 and Hri2 activities under oxidative stress but not in response to MMS. In contrast, Hri2 is the primary activated eIF2alpha kinase in response to heat shock. In this case, the activation of Sty1 appears to be transitory and does not contribute to the modulation of the eIF2alpha kinase stress pathway. In strains lacking Hri2, a type 2A protein phosphatase is activated soon after heat shock to reduce eIF2alpha phosphorylation. Finally, the MAPK Sty1, but not the eIF2alpha kinases, is essential for survival upon oxidative stress or heat shock, but not upon MMS treatment. These findings point to a regulatory coordination between the Sty1 MAPK and eIF2alpha kinase pathways for a particular range of stress responses.

The cell end marker protein TeaC is involved in growth directionality and septation in Aspergillus nidulans.

Polarized growth in filamentous fungi depends on the correct spatial organization of the microtubule (MT) and actin cytoskeleton. In Schizosaccharomyces pombe it was shown that the MT cytoskeleton is required for the delivery of so-called cell end marker proteins, e.g., Tea1 and Tea4, to the cell poles. Subsequently, these markers recruit several proteins required for polarized growth, e.g., a formin, which catalyzes actin cable formation. The latest results suggest that this machinery is conserved from fission yeast to Aspergillus nidulans. Here, we have characterized TeaC, a putative homologue of Tea4. Sequence identity between TeaC and Tea4 is only 12.5%, but they both share an SH3 domain in the N-terminal region. Deletion of teaC affected polarized growth and hyphal directionality. Whereas wild-type hyphae grow straight, hyphae of the mutant grow in a zig-zag way, similar to the hyphae of teaA deletion (tea1) strains. Some small, anucleate compartments were observed. Overexpression of teaC repressed septation and caused abnormal swelling of germinating conidia. In agreement with the two roles in polarized growth and in septation, TeaC localized to hyphal tips and to septa. TeaC interacted with the cell end marker protein TeaA at hyphal tips and with the formin SepA at hyphal tips and at septa.

Heme responsiveness in vitro is a common feature shared by the eukaryotic initiation factor 2á kinase family

Four distinct eukaryotic initiation factor 2á (eIF2á) kinases phosphorylate eIF2á at Ser-51 and regulate protein synthesis in response to cellular stress conditions. This kinase family includes the hemin-regulated inhibitor (HRI); the doublestranded RNA-dependent kinase (PKR); the GCN2 protein kinase; and the endoplasmic reticulum-resident kinase (PERK). HRI mediates protein synthesis inhibition in heme-deficient reticulocyte lysates. Although HRI contains two putative heme regulatory motifs (HRMs) that are not present in other eIF2á kinases, the significance of these motifs in heme regulation is not clear. In fact, it had been characterized two novel eIF2á kinases from Schizosaccharomyces pombe that lacked any of the HRMs, but were sensitive to heme regulation in vitro. To investigate the importance of different regions in the regulation of HRI by heme, specific HRI mutants were generated, and kinase activities and heme responsiveness were analyzed in vitro. Mutational analysis indicated that the heme regulatory motifs were spread around some regions in the HRI catalytic domain, outside of the HRMs. In accordance with these results, both the autokinase and the eIF2á kinase activities of three distinct eIF2á kinases, including the human PKR, the mouse GCN2 and the Drosophila PERK were inhibited in vitro by hemin. Although the known regulatory mechanisms of these eIF2á kinases are very different, the data reported here indicate that all known eIF2á kinases are regulated in vitro by hemin. This finding provides evidence that hemin represents a regulatory mechanism unique to eIF2á kinases and underscores the role of hemin in the translational regulation of eukaryotic cells

Las cuatro eIF2á quinasas eucarióticas fosforilan el residuo Ser-51 de la subunidad alfa del factor de iniciación 2 y regulan la síntesis de proteínas en respuesta a situaciones de estrés celular. Esta familia de proteínas quinasas está formada por el inhibidor regulado por hemina (HRI); la quinasa dependiente de RNA de doble cadena (PKR); la proteína quinasa GCN2 y la quinasa residente en el retículo endoplásmico (PERK). El HRI inhibe la síntesis de proteínas en lisados de reticulocitos de conejo deficientes de hemina. Aunque el HRI contiene dos supuestos motivos reguladores de hemina (HRMs), que no están presentes en las otras eIF2á quinasas, no está claro aún el papel de estos motivos en la regulación por hemina. De hecho, se han caracterizado dos nuevas eIF2á quinasas de Schizosaccharomyces pombe que carecen de dichos HRMs, pero son sensibles a la regulación por hemina in vitro. Un análisis mutacional indicó que los motivos reguladores de hemina estaban dispersos a lo largo del dominio catalítico, fuera de los HRMs. De acuerdo con estos resultados, las actividades autoquinasa y eIF2á quinasa de tres eIF2á quinasas distintas, la PKR humana, la GCN2 de ratón y la PERK de Drosophila, se inhibían por hemina in vitro. Aunque los mecanismos de regulación de todas estas eIF2á quinasas son muy diferentes, nuestros resultados indican que todas las eIF2á quinasas se regulan por hemina in vitro. Este descubrimiento soporta la evidencia de que la hemina representa un mecanismo de regulación específico de las eIF2á quinasas, y subraya su papel en la regulación de la traducción de células eucarióticas

Structural analysis of protein kinase A mutants with Rho-kinase inhibitor specificity.

Controlling aberrant kinase-mediated cellular signaling is a major strategy in cancer therapy; successful protein kinase inhibitors such as Tarceva and Gleevec verify this approach. Specificity of inhibitors for the targeted kinase(s), however, is a crucial factor for therapeutic success. Based on homology modeling, we previously identified four amino acids in the active site of Rho-kinase that likely determine inhibitor specificities observed for Rho-kinase relative to protein kinase A (PKA) (in PKA numbering: T183A, L49I, V123M, and E127D), and a fifth (Q181K) that played a surprising role in PKA-PKB hybrid proteins. We have systematically mutated these residues in PKA to their counterparts in Rho-kinase, individually and in combination. Using four Rho-kinase-specific, one PKA-specific, and one pan-kinase-specific inhibitor, we measured the inhibitor-binding properties of the mutated proteins and identify the roles of individual residues as specificity determinants. Two combined mutant proteins, containing the combination of mutations T183A and L49I, closely mimic Rho-kinase. Kinetic results corroborate the hypothesis that side-chain identities form the major determinants of selectivity. An unexpected result of the analysis is the consistent contribution of the individual mutations by simple factors. Crystal structures of the surrogate kinase inhibitor complexes provide a detailed basis for an understanding of these selectivity determinant residues. The ability to obtain kinetic and structural data from these PKA mutants, combined with their Rho-kinase-like selectivity profiles, make them valuable for use as surrogate kinases for structure-based inhibitor design.