Protein phosphatase 2A is involved in hyphal growth of Neurospora crassa

Yatzkan, Einat; Szöőr, Balázs; Fehér, Zsigmond; Dombrádi, Viktor; Yarden, Oded

doi:10.1007/s004380050844

Cited by 41 publications

(34 citation statements)

References 41 publications

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“…Because the catalytic subunits of PP1 and PP2A (encoded for by the ppp-1 and pph-1 genes, respectively) are essential for cell survival in Neurospora and other eukaryotes (Yatzkan and Yarden 1995;Zeke et al 2003), we used repeat-induced point mutation (RIP; Cambareri et al 1989) to obtain partially functional To examine whether these mutations led to the decrease of the phosphatase activity of PP1, phosphatase assay (see supplemental materials) was performed using extracts prepared from cultures grown in LL, a condition that the clock is not running. The 32 P-labeled phosphorylase a is used as the substrate of the assay in the presence or absence of inhibitor-2, a specific protein inhibitor for PP1 (Krebs and Fischer 1962;Yatzkan et al 1998;Cohen 2002). As shown in Figure 1A, the inhibitor-2-sensitive phosphatase acitivity was significantly reduced in the ppp-1 RIP strain, indicating that the mutations resulted in a partial functional PPP-1 protein.…”

Section: Resultsmentioning

confidence: 99%

Distinct roles for PP1 and PP2A in the Neurospora circadian clock

Yang¹,

He²,

Cheng³

et al. 2004

Genes Dev.

111

117

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Phosphorylation of the Neurospora circadian clock protein FREQUENCY by several kinases promotes its degradation and is important for the function of the circadian feedback loop. Here, we show that FRQ is less stable in a ppp-1 (catalytic subunit of PP1) mutant, resulting in its advanced phase and short period. In contrast, FRQ stability is not altered in a rgb-1 (a regulatory subunit of PP2A) mutant, but levels of frq protein and mRNA are low, resulting in a low-amplitude and longperiod oscillation of the clock. Furthermore, PP1 and PP2A expressed in Neurospora can dephosphorylate the endogenous FRQ in vitro, suggesting that these two phosphatases may differentially regulate FRQ and, consequently, the behavior of the circadian clock.Supplemental material is available at http://www.genesdev.org.Received September 15, 2003; revised version accepted December 15, 2003. Reversible phosphorylation is an important regulatory mechanism for many biological processes in eukaryotic organisms. The phosphorylation state of a protein is controlled dynamically by protein kinases and phosphatases. Previous studies have demonstrated that phosphorylation of circadian clock proteins is an essential posttranscriptional mechanism in the regulation of circadian clocks (Dunlap 1999;Young and Kay 2001). Several kinases, including casein kinase I (CKI) and CKII, have been shown to phosphorylate and regulate the key clock components in eukaryotic systems Sugano et al. 1999;Lowrey et al. 2000;Gorl et al. 2001;Yang et al. 2002).FREQUENCY (FRQ), WHITE COLLAR-1 (WC-1), and WC-2 proteins are three key components in the Neurospora frq-wc-based circadian oscillator . In addition to being the circadian blue light photoreceptor (Froehlich et al. 2002;He et al. 2002), in the dark, WC-1 forms a heterodimeric complex with WC-2 through their PAS domains and activate frq transcription by binding its promoter (Crosthwaite et al. 1997;Talora et al. 1999;Cheng et al. 2001bCheng et al. , 2002Cheng et al. , 2003Froehlich et al. 2003). FRQ, the negative element of the feedback loop, inhibits its own transcription through interactions with the WC complex (Aronson et al. 1994;Cheng et al. 2001a;Denault et al. 2001;Froehlich et al. 2003). FRQ protein is immediately phosphorylated after its synthesis and becomes extensively phosphorylated prior to its degradation by the ubiquitin/proteasome pathway Liu et al. 2000;He et al. 2003). In constant darkness, FRQ is not only robustly rhythmic in its cellular concentration, but also in its phosphorylation states, so that the level and the phosphorylation status of FRQ define the time of the clock during a circadian cycle .Phoshorylation of FRQ is mediated by CKI, CKII, and a calcium/calmodulin-dependent kinase (Gorl et al. 2001;Yang et al. 2001Yang et al. , 2002Yang et al. , 2003. Molecular, genetic, and biochemical experiments have shown that phosphorylation of FRQ has several functions. Mutations of the FRQ phosphorylation sites lead to the stabilization of the FRQ protein and long period rhythms of the clock (Li...

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Section: Resultsmentioning

confidence: 99%

Distinct roles for PP1 and PP2A in the Neurospora circadian clock

Yang¹,

He²,

Cheng³

et al. 2004

Genes Dev.

111

117

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“…The antifungal spectrum and potency of cantharidin against Dfks mutants of S. cerevisiae differed from those of PLME. The appearance of balloon-like hyphae induced by PP1A and PP2A inhibitors was reported to be limited at the hyphal tips in N. crassa [15]. With PLME, expansion in diameter was observed throughout the hyphae of A. fumigatus (Fig.…”

Section: Fig 2 Effects Of Plme On Hyphal Morphology In Aspergillus Fmentioning

confidence: 90%

“…However, there have been few reports on the physiological role of PP2A in filamentous fungi [15,16]. Cantharidin, an inhibitor of PP1A and PP2A, inhibits the hyphal growth of Neurospora crassa accompanied by swollen hyphal tips and an increase in hyphal branching [15].…”

Section: Fig 2 Effects Of Plme On Hyphal Morphology In Aspergillus Fmentioning

confidence: 99%

Identification of Phoslactomycin E as a Metabolite Inducing Hyphal Morphological Abnormalities in Aspergillus fumigatus IFO 5840

et al. 2007

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In our survey for antifungal compounds, a fermentation broth of Streptomyces sp. HA81-2 was found to inhibit the in vitro growth of Aspergillus fumigatus IFO 5840 accompanied by hyphal morphological abnormalities. One of the isolated antibiotics was identified as phoslactomycin E based on LC-MS and NMR spectral data. In a preliminary assay using the membrane fractions of A. fumigatus, phoslactomycin E was found to inhibit the activity of 1,3-b glucan synthase.Keywords antifungal activity, phoslactomycin E, Aspergillus fumigatus, hyphal morphological abnormalities, 1,3-b glucan synthase Opportunistic fungal infections are a major cause of serious morbidity and mortality in immunocompromised patients. However, there are few drugs for antifungal chemotherapy of deep-seated mycosis. As both human and fungal cells are eukaryotes, it is difficult to develop antifungal drugs without side effects in human hosts. Current targets are limited to ergosterol, cell wall components, biosynthetic pathways of cell wall and cytosine deaminase [1,2]. Therefore, there is a great need for novel antifungal drugs with new mechanisms of action. Morphological deformation of fungal hyphae is often induced by antifungal drugs [3]. As this phenomenon is specific to fungi, it could be used to screen for selective antifungal antibiotics [4]. In addition, this hyphal deformation is expected to reflect the disruption of cell wall biosynthesis, which is a specific target of antifungal agents. Moreover, investigating the mechanism of antifungal-induced hyphal morphological abnormalities is potentially useful for understanding the morphogenesis of fungal hyphae.In the course of our search for antifungal antibiotics, we found that a fermentation broth of Streptomyces sp. HA81-2 exhibited antifungal activity against Aspergillus fumigatus IFO 5840 accompanied by hyphal abnormalities. The isolated active fraction was identified as phoslactomycin E (PLME) [5] based on LC-MS and NMR spectral data. We herein report the isolation procedures for PLME, its molecular characteristics, and its induction of morphological changes in fungal cells.Active principles were isolated as follows; fermentation broth was filtered, the filtrate was applied on Diaion HP-20 column, and then eluted using an increasing gradient of aq MeOH (10ϳ100%). The 80ϳ90% MeOH fractions were concentrated in vacuo to afford crude extract, which was partitioned between water and CHCl 3 . Purification of

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“…Of particular interest is whether CPP1 affects other fumonisin biosynthesis regulatory genes, particularly the genes FUM21, ZFR1 and GBB1 that are positively associated with fumonisin biosynthesis (Brown et al, 2007;Flaherty & Woloshuk, 2004;Sagaram & Shim, 2007). Meanwhile, it is well recognized that type 2A protein phosphatases are associated with cell differentiation and development in eukaryotes, including filamentous fungi (Dickman & Yarden, 1999;Hunter, 1995;Mayer-Jaekel & Hemmings, 1994;Shenolikar, 1994;Yatzkan et al, 1998), and multiple PP179 phenotypes suggested that CPP1 is also involved in multiple downstream signalling pathways in F. verticillioides. Strikingly, CPP1 deletion led to a hyphal-swelling phenotype, thereby suggesting the functional role of CPP1 in morphogenesis.…”

Section: Discussionmentioning

confidence: 99%

Functional characterization of Fusarium verticillioides CPP1, a gene encoding a putative protein phosphatase 2A catalytic subunit

Choi

Shim

2008

Microbiology

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Fusarium verticillioides produces the mycotoxin fumonisin B 1 (FB 1 ) on maize kernels. In this study, we identified a putative protein phosphatase gene CPP1 in F. verticillioides, and investigated its role in FB 1 regulation. Previous work has shown that CPP1 expression is elevated in an FB 1 -suppressing genetic background. Thus, we hypothesized that CPP1 is negatively associated with FB 1 production. To test this hypothesis, we generated a CPP1 knockout mutant, PP179, and studied the effects of gene deletion on FB 1 biosynthesis and fungal development. PP179 showed elevated expression of FUM genes, and in turn produced higher levels of FB 1 than the wild-type progenitor. Other significant mutant phenotypes included reduced radial growth on agar plates, reduced conidia germination rates, significantly increased macroconidia formation, and hyphal swelling. To verify that these phenotypes were directly due to CPP1 deletion, we complemented PP179 with the wild-type CPP1 gene. The complemented strain PPC4 showed FUM1 expression and FB 1 production similar to that of the wild-type, providing evidence that CPP1 is negatively associated with FB 1 biosynthesis. Other PP179 phenotypes, such as macroconidiation and hyphal swelling, were also restored to that of wild-type progenitor. Furthermore, we complemented F. verticillioides PP179 strain with Neurospora crassa wild-type ppe-1 gene, demonstrating that Cpp1 and PPE-1 proteins are functionally conserved. Pleiotropic effects of CPP1 deletion led us to hypothesize that CPP1 is associated with multiple downstream signalling pathways in F. verticillioides. Identification and functional characterization of downstream Cpp1-interacting proteins are necessary to better understand the complex regulatory mechanisms associated with Cpp1.

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Protein phosphatase 2A is involved in hyphal growth of Neurospora crassa

Cited by 41 publications

References 41 publications

Distinct roles for PP1 and PP2A in the Neurospora circadian clock

Distinct roles for PP1 and PP2A in the Neurospora circadian clock

Identification of Phoslactomycin E as a Metabolite Inducing Hyphal Morphological Abnormalities in Aspergillus fumigatus IFO 5840

Functional characterization of Fusarium verticillioides CPP1, a gene encoding a putative protein phosphatase 2A catalytic subunit

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