Protein Kinase A Regulatory Subunit Isoforms Regulate Growth and Differentiation in Mucor circinelloides: Essential Role of PKAR4

Abstract

bThe protein kinase A (PKA) signaling pathway plays a role in regulating growth and differentiation in the dimorphic fungus Mucor circinelloides. PKA holoenzyme is comprised of two catalytic (C) and two regulatory (R) subunits. In M. circinelloides, four genes encode the PKAR1, PKAR2, PKAR3, and PKAR4 isoforms of R subunits. We have constructed null mutants and demonstrate that each isoform has a different role in growth and differentiation. The most striking finding is that pkaR4 is an essential gene, because only heterokaryons were obtained in knockout experiments. Heterokaryons with low levels of wild-type nuclei showed an impediment in the emission of the germ tube, suggesting a pivotal role of this gene in germ tube emergence. The remaining null strains showed different alterations in germ tube emergence, sporulation, and volume of the mother cell. The pkaR2 null mutant showed an accelerated germ tube emission and was the only mutant that germinated under anaerobic conditions when glycine was used as a nitrogen source, suggesting that pkaR2 participates in germ tube emergence by repressing it. From the measurement of the mRNA and protein levels of each isoform in the wild-type and knockout strains, it can be concluded that the expression of each subunit has its own mechanism of differential regulation. The PKAR1 and PKAR2 isoforms are posttranslationally modified by ubiquitylation, suggesting another regulation point in the specificity of the signal transduction. The results indicate that each R isoform has a different role in M. circinelloides physiology, controlling the dimorphism and contributing to the specificity of cyclic AMP (cAMP)-PKA pathway. Mucor circinelloides is a dimorphic fungus from the new subphylum Mucormycotina (formerly classified as Zygomycetes) that displays yeast or filamentous morphology as a consequence of different environmental conditions, including gas atmosphere and level of nutrients (33). Fungi from this subphylum are organized in multinucleated cells. M. circinelloides yeasts display multipolar budding and each cell harbors more than one nucleus, while mycelium is aseptate and has distributed nuclei along the hyphae.Fungal dimorphism is particularly important since genetic evidence indicates that in a number of pathogenic fungi in animals and plants, the morphogenetic transition is directly associated with virulence and pathogenesis (4,21,29). M. circinelloides is a causal agent of the lethal fungal infectious disease mucormycosis (6, 10), which has been reported in patients with impaired immunity (22,27,38,40,49). Recently, it has been reported that the spore size dimorphism is linked to virulence in M. circinelloides (23). The functional analysis of genes involved in the control of dimorphism in M. circinelloides is a contribution to advance the understanding of pathogenic zygomycetes.One of the key regulators of polarity in fungi, as well as of other processes such as development, mating, and virulence, is the cyclic AMP (cAMP)-dependent protein kinase A (PKA) (5, 16). The...

Calcineurin Plays Key Roles in the Dimorphic Transition and Virulence of the Human Pathogenic Zygomycete Mucor circinelloides

“…cAMP then binds to the regulatory subunit of protein kinase A (PKAR) and the protein kinase A (PKA) catalytic subunits are released to result in activation. Previous studies suggested that PKA may play a major role during yeast growth, given that the PKA genes are over-expressed during yeast growth, and the regulatory subunit gene ( pkaR ) that inhibits PKA activity is expressed at a higher level during the yeast to hyphal transition [25], [27], [28]. We further confirmed that Mucor grows as yeast in the presence of high concentrations of bicarbonate in the media.…”

“…We adapted a PKA activity assay used in other studies of Mucor morphogenesis to test how PKA activity is differentially regulated during yeast and hyphal growth [27], [28], [68]. Due to the nature of this assay performed in the presence of cAMP, the maximum possible PKA activity was measured.…”

Transformation of Mucor circinelloides f. lusitanicus Protoplasts

“…In both cases, they contain a selectable marker gene fl anked by DNA sequences (about 1 kb each) up-and downstream the target locus. A total of more than 20 genes have recently been disrupted using both methods described here, although only four disruptions have already been published (Ocampo et al 2012 ;Lee et al 2013 ). The advantageous locus used so far for targeted integration of genes in M. circinelloides is the carRP -carB locus (Velayos et al 2000a , b ) that includes two genes and three enzymatic activities which drive the production of β-carotene from geranyl-geranyl-pyrophosphate (GGPP).…”

“…The result was a comprehensive number of potential recipient strains and molecular markers with which the analysis of different biological problems could be achieved. These include different aspects of basic and applied biology such as carotenoid biosynthesis and regulation (Iturriaga et al 2001 ;Csernetics et al 2011 ), blue-light regulation (Silva et al 2006(Silva et al , 2008, lipid accumulation and metabolism (Xia et al 2011 ;Rodríguez-Frómeta et al 2013 ), dimorphism and differentiation (Wolff et al 2002 ;Ocampo et al 2009Ocampo et al , 2012, or siRNA silencing (Nicolás et al 2009 ;Calo et al 2012 ), among others. The next step was trying to increase the transformation effi ciency and reliability.…”

Phylogenetic analysis of fungal heterotrimeric G protein-encoding genes and their expression during dimorphism in Mucor circinelloides

“…Some molecular components involved in M. circinelloides differentiation have previously been reported. These include protein kinase A (PKA) isoforms, which are relevant during dimorphism, for example pkaR4 is essential during hyphal germination (Wolff et al 2002;Ocampo et al 2012). The cgrA product, which encodes an E3 ubiquitin ligase involved in regulation of carotenogenesis and decrement of sporulation (Nicol as et al 2008).…”