scholarly journals Loss of Septation Initiation Network (SIN) kinases blocks tissue invasion and unlocks echinocandin cidal activity against Aspergillus fumigatus

2021 ◽  
Vol 17 (8) ◽  
pp. e1009806
Author(s):  
Ana Camila Oliveira Souza ◽  
Adela Martin-Vicente ◽  
Ashley V. Nywening ◽  
Wenbo Ge ◽  
David J. Lowes ◽  
...  
Keyword(s):  
Cell Wall ◽  
Protein Kinase ◽  
Protein Kinases ◽  
Wall Stress ◽  
Invasive Growth ◽  
Kinase Gene ◽  
Yeast Fungi ◽  
Kinase Cascade ◽  
Septation Initiation Network ◽  
Residual Tissue

Although considered effective treatment for many yeast fungi, the therapeutic efficacy of the echinocandin class of antifungals for invasive aspergillosis (IA) is limited. Recent studies suggest intense kinase- and phosphatase-mediated echinocandin adaptation in A. fumigatus. To identify A. fumigatus protein kinases required for survival under echinocandin stress, we employed CRISPR/Cas9-mediated gene targeting to generate a protein kinase disruption mutant library in a wild type genetic background. Cell wall and echinocandin stress screening of the 118 disruption mutants comprising the library identified only five protein kinase disruption mutants displaying greater than 4-fold decreased echinocandin minimum effective concentrations (MEC) compared to the parental strain. Two of these mutated genes, the previously uncharacterized A. fumigatus sepL and sidB genes, were predicted to encode protein kinases functioning as core components of the Septation Initiation Network (SIN), a tripartite kinase cascade that is necessary for septation in fungi. As the A. fumigatus SIN is completely uncharacterized, we sought to explore these network components as effectors of echinocandin stress survival. Our data show that mutation of any single SIN kinase gene caused complete loss of hyphal septation and increased susceptibility to cell wall stress, as well as widespread hyphal damage and loss of viability in response to echinocandin stress. Strikingly, mutation of each SIN kinase gene also resulted in a profound loss of virulence characterized by lack of tissue invasive growth. Through the deletion of multiple novel regulators of hyphal septation, we show that the non-invasive growth phenotype is not SIN-kinase dependent, but likely due to hyphal septation deficiency. Finally, we also find that echinocandin therapy is highly effective at eliminating residual tissue burden in mice infected with an aseptate strain of A. fumigatus. Together, our findings suggest that inhibitors of septation could enhance echinocandin-mediated killing while simultaneously limiting the invasive potential of A. fumigatus hyphae.

A yeast mitogen-activated protein kinase homolog (Mpk1p) mediates signalling by protein kinase C

1993 ◽  
Vol 13 (5) ◽  
pp. 3067-3075 ◽  
Author(s):  
K S Lee ◽  
K Irie ◽  
Y Gotoh ◽  
Y Watanabe ◽  
H Araki ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Map Kinase ◽  
Protein Kinases ◽  
Map Kinases ◽  
Pheromone Response ◽  
Pheromone Response Pathway ◽  
Kinase C ◽  
Mitogen Activated Protein ◽  
Kinase Cascade ◽  
Protein Kinase Cascade

Mitogen-activated protein (MAP) kinases are activated in response to a variety of stimuli through a protein kinase cascade that results in their phosphorylation on tyrosine and threonine residues. The molecular nature of this cascade is just beginning to emerge. Here we report the isolation of a Saccharomyces cerevisiae gene encoding a functional analog of mammalian MAP kinases, designated MPK1 (for MAP kinase). The MPK1 gene was isolated as a dosage-dependent suppressor of the cell lysis defect associated with deletion of the BCK1 gene. The BCK1 gene is also predicted to encode a protein kinase which has been proposed to function downstream of the protein kinase C isozyme encoded by PKC1. The MPK1 gene possesses a 1.5-kb uninterrupted open reading frame predicted to encode a 53-kDa protein. The predicted Mpk1 protein (Mpk1p) shares 48 to 50% sequence identity with Xenopus MAP kinase and with the yeast mating pheromone response pathway components, Fus3p and Kss1p. Deletion of MPK1 resulted in a temperature-dependent cell lysis defect that was virtually indistinguishable from that resulting from deletion of BCK1, suggesting that the protein kinases encoded by these genes function in a common pathway. Expression of Xenopus MAP kinase suppressed the defect associated with loss of MPK1 but not the mating-related defects associated with loss of FUS3 or KSS1, indicating functional conservation between the former two protein kinases. Mutation of the presumptive phosphorylated tyrosine and threonine residues of Mpk1p individually to phenylalanine and alanine, respectively, severely impaired Mpk1p function. Additional epistasis experiments, and the overall architectural similarity between the PKC1-mediated pathway and the pheromone response pathway, suggest that Pkc1p regulates a protein kinase cascade in which Bck1p activates a pair of protein kinases, designated Mkk1p and Mkk2p (for MAP kinase-kinase), which in turn activate Mpk1p.

scholarly journals Molecular cloning, characterization and localization of PfPK4, an eIF-2α kinase-related enzyme from the malarial parasite Plasmodium falciparum

1997 ◽  
Vol 328 (2) ◽  
pp. 677-687 ◽  
Author(s):  
Jörg J. MÖHRLE ◽  
Yi ZHAO ◽  
Barbara WERNLI ◽  
M. Richard FRANKLIN ◽  
Barbara KAPPES
Keyword(s):  
Amino Acids ◽  
Plasmodium Falciparum ◽  
Protein Kinase ◽  
Protein Kinases ◽  
Initiation Factor ◽  
Malarial Parasite ◽  
Kinase Gene ◽  
Western Blots ◽  
Parasite Plasmodium ◽  
Parasite Plasmodium Falciparum

PfPK4, a protein kinase gene from the human malarial parasite Plasmodium falciparum, has been cloned utilizing oligonucleotide probing. The gene encodes a protein of a predicted length of 1123 amino acids, and within this amino acid sequence all the conserved regions characteristic of protein kinases can be identified. The catalytic kinase domain possesses highest identities (34-37%) with eukaryotic initiation factor-2α (eIF-2α) kinases, especially haem-regulated inhibitory (HRI) protein kinases. There are two kinase inserts in PfPK4, located at positions common to eIF-2α kinases. The first insert separates kinase subdomains IV and VI by 559 amino acids, and the second subdomains VII and VIII by 41 amino acids. Both inserts are larger than their homologues in eIF-2α kinases. The sequence of PfPK4 has one putative haemin-binding site. The recombinant protein, expressed in Escherichia coli, phosphorylates a synthetic peptide representing a substrate of eIF-2α kinases. Autophosphorylation and substrate phosphorylation are inhibited by haemin. Thus PfPK4 appears to be the first protozoan protein kinase related to eIF-2α kinases and might be the first non-mammalian HRI kinase. Western blots indicated that the protein is expressed as major forms of 80 and 90 kDa. Whereas the 80 kDa form is present throughout the intraerythrocytic development and in merozoites, the two 90 kDa forms are only found in mature parasites. One of the latter is also present in the membrane fraction of erythrocytes harbouring segmenters. Confocal microscopy detected the protein distributed throughout the trophozoite, whereas it was found in discrete foci (punctate distribution) in segmenters. PfPK4 co-localizes with P. falciparum 83 kDa antigen/apical membrane antigen-1 at the apical complex in segmenters and merozoites, but does not co-localize with rhoptry-associated protein-1.

scholarly journals Cell Wall Stress Depolarizes Cell Growth via Hyperactivation of Rho1

1999 ◽  
Vol 147 (1) ◽  
pp. 163-174 ◽  
Author(s):  
Pierre-Alain Delley ◽  
Michael N. Hall
Keyword(s):  
Cell Wall ◽  
Cell Growth ◽  
Actin Cytoskeleton ◽  
Mammalian Cells ◽  
Wall Stress ◽  
Mitogen Activated Protein Kinase ◽  
Regulatory Subunit ◽  
Cell Wall Stress ◽  
Mitogen Activated Protein ◽  
Kinase Cascade

Cells sense and physiologically respond to environmental stress via signaling pathways. Saccharomyces cerevisiae cells respond to cell wall stress by transiently depolarizing the actin cytoskeleton. We report that cell wall stress also induces a transient depolarized distribution of the cell wall biosynthetic enzyme glucan synthase FKS1 and its regulatory subunit RHO1, possibly as a mechanism to repair general cell wall damage. The redistribution of FKS1 is dependent on the actin cytoskeleton. Depolarization of the actin cytoskeleton and FKS1 is mediated by the plasma membrane protein WSC1, the RHO1 GTPase switch, PKC1, and a yet-to-be defined PKC1 effector branch. WSC1 behaves like a signal transducer or a stress-specific actin landmark that both controls and responds to the actin cytoskeleton, similar to the bidirectional signaling between integrin receptors and the actin cytoskeleton in mammalian cells. The PKC1-activated mitogen-activated protein kinase cascade is not required for depolarization, but rather for repolarization of the actin cytoskeleton and FKS1. Thus, activated RHO1 can mediate both polarized and depolarized cell growth via the same effector, PKC1, suggesting that RHO1 may function as a rheostat rather than as a simple on-off switch.

scholarly journals Yeast Mpk1 Cell Wall Integrity Mitogen-activated Protein Kinase Regulates Nucleocytoplasmic Shuttling of the Swi6 Transcriptional Regulator

2010 ◽  
Vol 21 (9) ◽  
pp. 1609-1619 ◽  
Author(s):  
Ki-Young Kim ◽  
Andrew W. Truman ◽  
Stefanie Caesar ◽  
Gabriel Schlenstedt ◽  
David E. Levin
Keyword(s):  
Cell Wall ◽  
Protein Kinase ◽  
Transcriptional Activation ◽  
Transcriptional Response ◽  
Wall Stress ◽  
Cell Wall Integrity ◽  
Mitogen Activated Protein Kinase ◽  
Nucleocytoplasmic Shuttling ◽  
Nuclear Entry ◽  
Mitogen Activated Protein

The yeast SBF transcription factor is a heterodimer comprised of Swi4 and Swi6 that has a well defined role in cell cycle-specific transcription. SBF serves a second function in the transcriptional response to cell wall stress in which activated Mpk1 mitogen-activated protein kinase of the cell wall integrity signaling pathway forms a complex with Swi4, the DNA binding subunit of SBF, conferring upon Swi4 the ability to bind DNA and activate transcription of FKS2. Although Mpk1–Swi4 complex formation and transcriptional activation of FKS2 does not require Mpk1 catalytic activity, Swi6 is phosphorylated by Mpk1 and must be present in the Mpk1-Swi4 complex for transcriptional activation of FKS2. Here, we find that Mpk1 regulates Swi6 nucleocytoplasmic shuttling in a biphasic manner. First, formation of the Mpk1-Swi4 complex recruits Swi6 to the nucleus for transcriptional activation. Second, Mpk1 negatively regulates Swi6 by phosphorylation on Ser238, which inhibits nuclear entry. Ser238 neighbors a nuclear localization signal (NLS) whose function is blocked by phosphorylation at Ser238 in a manner similar to the regulation by Cdc28 of another Swi6 NLS, revealing a mechanism for the integration of multiple signals to a single endpoint. Finally, the Kap120 β-importin binds the Mpk1-regulated Swi6 NLS but not the Cdc28-regulated NLS.

scholarly journals The fission yeast septation initiation network (SIN) kinase, Sid2, is required for SIN asymmetry and regulates the SIN scaffold, Cdc11

2012 ◽  
Vol 23 (9) ◽  
pp. 1636-1645 ◽  
Author(s):  
Anna Feoktistova ◽  
Jennifer Morrell-Falvey ◽  
Jun-Song Chen ◽  
N. Sadananda Singh ◽  
Mohan K. Balasubramanian ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Asymmetric Distribution ◽  
Kinase Cascade ◽  
Septation Initiation Network ◽  
Ring Constriction ◽  
Signaling Components ◽  
Protein Kinase Cascade

The Schizosaccharomyces pombe septation initiation network (SIN) is an Spg1-GTPase–mediated protein kinase cascade that triggers actomyosin ring constriction, septation, and cell division. The SIN is assembled at the spindle pole body (SPB) on the scaffold proteins Cdc11 and Sid4, with Cdc11 binding directly to SIN signaling components. Proficient SIN activity requires the asymmetric distribution of its signaling components to one of the two SPBs during anaphase, and Cdc11 hyperphosphorylation correlates with proficient SIN activity. In this paper, we show that the last protein kinase in the signaling cascade, Sid2, feeds back to phosphorylate Cdc11 during mitosis. The characterization of Cdc11 phosphomutants provides evidence that Sid2-mediated Cdc11 phosphorylation promotes the association of the SIN kinase, Cdc7, with the SPB and maximum SIN signaling during anaphase. We also show that Sid2 is crucial for the establishment of SIN asymmetry, indicating a positive-feedback loop is an important element of the SIN.

scholarly journals Novel Protein Kinase Induced during Sporangial Cleavage in the Oomycete Phytophthora infestans

2002 ◽  
Vol 1 (5) ◽  
pp. 687-695 ◽  
Author(s):  
Howard S. Judelson ◽  
Samuel Roberts
Keyword(s):  
Protein Kinase ◽  
Phytophthora Infestans ◽  
Protein Kinases ◽  
Kinase Inhibitors ◽  
De Novo ◽  
Protein Kinase Inhibitors ◽  
Kinase Gene ◽  
Mrna Accumulation ◽  
Calmodulin Antagonist ◽  
Zoospore Release

ABSTRACT A study of the effect of inhibitors on zoospore development in Phytophthora infestans demonstrated the involvement of protein kinases and calcium and led to the discovery of a gene induced during zoosporogenesis that encoded a protein resembling Ca+2- and calmodulin-regulated serine/threonine protein kinases. The calcium channel blocker verapamil and the calmodulin antagonist trifluoroperazine inhibited zoosporogenesis and encystment. The protein kinase inhibitors K-252a and KN-93 inhibited zoospore release, encystment, and cyst germination, and K-252a reduced zoospore viability. In contrast, the inhibitors had minor or no effects on sporangia directly germinating in media. Spurred by these findings, a survey of putative protein kinase genes was performed to identify any that were up-regulated during zoosporogenesis. A kinase-encoding gene was identified for which mRNA accumulation was first detected soon after chilling sporangia in water, conditions that induce sporangial cytoplasm to cleave and release zoospores. The transcript persisted in motile zoospores and in germinated cysts but was not detected in other tissues, including hyphae, hyphae placed in water, or directly germinating sporangia. The structure of the predicted protein was novel, as its C-terminal region, which binds calmodulin in related proteins, was unusually short. Concentrations of actinomycin D previously used in experiments that suggested that de novo transcription was not needed for zoosporogenesis or encystment only partially inhibited transcription of the kinase gene, probably due to poor uptake into sporangia.

scholarly journals Hypermorphic SERK1 mutations function via a SOBIR1 pathway to activate floral abscission signaling

2018 ◽  
Author(s):  
Isaiah Taylor ◽  
John Baer ◽  
Ryan Calcutt ◽  
John C. Walker
Keyword(s):  
Protein Kinase ◽  
Protein Kinases ◽  
Negative Regulator ◽  
Peptide Ligand ◽  
Protein Kinase Activity ◽  
Loss Of Function ◽  
Allelic Series ◽  
Kinase Gene ◽  
Floral Organs ◽  
Suppression Effect

AbstractIn Arabidopsis, the abscission of floral organs is regulated by two related receptor-like protein kinases (RLKs), HAESA and HAESA-like 2 (HAE/HSL2). HAE/HSL2, in complex with members of the SERK family of coreceptor protein kinases, are activated by the binding of the proteolytically processed peptide ligand IDA. This leads to expression of genes encoding secreted cell wall remodeling and hydrolase enzymes. hae hsl2 mutants fail to induce expression of these genes and retain floral organs indefinitely. In this paper we report identification of an allelic series of hae hsl2 suppressor mutations in the SERK1 coreceptor protein kinase gene. Genetic and transcriptomic evidence indicates these alleles represent a novel class of gain of function mutations that activate signaling independent of HAE/HSL2. We show that the suppression effect surprisingly does not rely on protein kinase activity of SERK1, and that activation of signaling relies on the RLK gene SOBIR1. The effect of these mutations can be mimicked by loss of function of BIR1, a known negative regulator of SERK-SOBIR1 signaling. These results suggest BIR1 functions to negatively regulate SERK-SOBIR1 signaling during abscission, and that the identified SERK1 mutations likely interfere with this negative regulation.

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