Protein Phosphatase 2A (PP2A) Regulatory Subunits ParA and PabA Orchestrate Septation and Conidiation and Are Essential for PP2A Activity in Aspergillus nidulans

Abstract

In recent decades, tremendous progress has been made in understanding serine/threonine protein phosphatase-dependent pathways, which are involved in the regulation of numerous processes, including secretion, cell motility, cell cycle, gene transcription, and cell metabolism (1-7). Serine/threonine phosphorylation, which is regulated by many kinases, can be reversed by a few phosphatases that are targeted to substrates via dozens of regulatory subunits (7,8). Currently, serine/threonine protein phosphatases (PPs) are grouped into two structurally distinct families: the PPP family (PP1, PP2A, and PP2B) and the PPM family (PP2C and pyruvate dehydrogenase phosphatase) (9). The PP2A heterotrimeric protein complex, which consists of a cascade of three subunits, namely, a catalytic subunit (C) and a structural subunit (A) associated with a third, hypothetically competitive and variable regulatory subunit (B), represents a highly conserved eukaryotic signal transduction system that is present in many organisms, from yeasts to humans. The activity of PP2A, along with its subcellular localization, is determined by B-family subunits (10-14). In mammals, PP2A is a major intracellular protein phosphatase that regulates multiple aspects of cell growth and metabolism (15). Therefore, PP2A is one of a few serine/threonine-specific phosphatases in the cell, and its complex structure and regulation system guarantee its various functions. The ability of this widely distributed heterotrimeric enzyme to function on a diverse array of substrates is largely controlled by the nature of its regulatory Bfamily subunits. Thus, multiple isoforms of the B-family regulatory subunit have been isolated from different organisms and grouped into three classes (B, B=, and BЉ) based on their structural similarities (16,17). The structural variations between these families support the hypothesis that the B-family regulatory subunit controls enzyme activity and specificity, such that different activities of PP2A and subcellular localization are determined by Bfamily regulatory subunits. However, one challenge we face is the potential for redundancy in the regulatory subunits' function of assigning PP2A function in mammals (18)(19)(20). Therefore, it is difficult to approach the function of PP2A globally by knocking out one of the regulatory subunits. In contrast, simple eukaryotic organisms, such as Saccharomyces cerevisiae (budding yeast) and Schizosaccharomyces pombe (fission yeast), which contain only a few isoforms of B-family regulatory subunits, are excellent model systems for studying how B-family regulatory subunits function in regulating PP2A activity. In fission yeast, there is only one B subunit of PP2A-Pab1 and two of the B=-encoding genes (par1 and par2) (21-24). Moreover, mutational studies in fission yeast have demonstrated that B regulatory subunits play crucial roles during the processes of cytokinesis, cell morphogenesis, and cell wall synthesis (25,26). Similarly, in budding yeast, studies have shown that the B class has only one ...

The Neurospora crassa PP2A Regulatory Subunits RGB1 and B56 Are Required for Proper Growth and Development and Interact with the NDR Kinase COT1

“…Our results demonstrate that RGB1 and B56 are predominantly found in different cellular locations in N. crassa . In F. verticillioides , the RGB1 and B56 homologs were proposed to localize to the cytoplasm and septa, respectively ( Shin et al, 2013 ), while in A. nidulans , both subunit homologs we detected in the septum, where they have been suggested to counteract each other’s function ( Zhong et al, 2014 ). While RGB1 and COT1 share, at least in part, punctate localization at the hyphal tip, B56 was found to concentrate mainly at the septal pores.…”

“…The significance of the PP2A subunits in fungal development has also been reported in other fungal species: deletion mutants of PPR1 and PPR2, the F. verticillioides homologs of RGB1 and B56, respectively, grew at rates of approximately 10–14 and 50% of those observed in the wild type, respectively ( Shin et al, 2013 ). Close to complete abolishment of conidiation, accompanied by defective conidiophore morphology, were observed in the A. nidulans RGB1 and B56 homologs, Δ pabA and Δ parA , respectively ( Zhong et al, 2014 ).…”

“…The PP2A regulatory subunits can dictate the localization of the holoenzyme in the cell, and this localization depends on substrate specificity ( Zolnierowicz, 2000 ; Zhong et al, 2014 ). Thus, various PP2A-AC dimer combinations with various regulatory subunits can be found throughout the cell.…”

“…When crossing these mutants with the wild type strain, all progeny were able to produce a normal fruiting body, with viable ascospores. PP2A regulatory subunits are involved in self-fertilization but not in heterothallic sexual development in A. nidulans ( Zhong et al, 2014 ). When RNAi-based gene silencing was used to reduce rgb1 expression in the plant pathogen S. sclerotiorum , cells were viable, but exhibited slow growth.…”

Molecular Characterization of Gβ-Like Protein CpcB Involved in Antifungal Drug Susceptibility and Virulence in A. fumigatus

“…As previously described ( Zhong et al, 2014 ; Cai et al, 2015 ) for protein extraction from A. fumigatus mycelia, conidia were inoculated into liquid MM and shaken at 220 rpm on a rotary shaker at 37°C for 20 h. The tissue was ground in liquid nitrogen and rapidly suspended in ice-cold extraction buffer [50 mM HEPES (pH 7.4), 137 mM KCl, 10% glycerol, 1 μg/ml pepstatin A, 1 μg/ml leupeptin, 1 mM PMSF]. Equal amounts of protein (40 μg) per lane were subjected to 10% SDS-PAGE and then transferred to a polyvinylidene difluoride (PVDF) membrane (Immobilon-P; Millipore) in 384 mM glycine, 50 mM Tris (pH 8.4), and 20% methanol at 250 mA for 1.5 h. The membrane was blocked with phosphate-buffered saline (PBS) containing 5% milk and 0.1% Tween 20.…”