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Threshold level of protein kinase A activity and polarized growth in Mucor rouxii

Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pabellón 2, Piso 4, 1428 Buenos Aires, Argentina¹

Author for correspondence: Silvia Moreno. Tel: +54 11 4576 3342. Fax: +54 11 4576 3342. e-mail: smoreno{at}qb.fcen.uba.ar

A model system to study the involvement of cAMP-mediated regulation of a cellular process such as hyphal morphogenesis was investigated. Impairment of polarized growth was observed when Mucor rouxii sporangiospores were grown in the presence of N⁶-cAMP analogues and of SQ 65,442, a cAMP phosphodiesterase inhibitor. Together with an effect on isodiametric growth, there was increased pigmentation, increased cell fragility and loss of cell adhesiveness. The total effect on morphology was attained even after adding the compounds shortly before germ-tube emergence; when added after this time growth continued in a non-polarized form and rounding of the germ tip was observed. The morphological effect was observed under all the nutritional and environmental conditions studied (aerobic conditions and defined medium with maltose or glucose, Casamino acids medium with glucose, or rich medium; anaerobic conditions with rich medium; and following a shift from anaerobiosis to aerobiosis). The time of germ-tube emergence, and the size of the cell at this time, was dependent on the growth medium. Protein kinase A (PKA) specific activity was followed during the germination process under three growth conditions. It was found that the total activity of PKA correlated with differentiation and not with growth, and that the total specific activity at the time of germination was the same, independent of the culture medium. The time of germ-tube emergence correlated with the time of attainment of a threshold level of PKA total specific activity. The concentration of dibutyryl-cAMP needed to promote isodiametric growth correlated with the total units of activity of PKA to be activated per cell. It was concluded that PKA is involved in the morphogenetic process of the fungus grown under all the nutritional and ambient conditions tested.

Keywords: cAMP, morphogenesis, Mucor rouxii, protein kinase A, polarized growth

Abbreviations: CI-930, 3(2H)-pyridazinone,4,5-dihydro-6-[4-(1H-imidazol-1-yl) phenyl-5-methylmonohydrochloride; dbut-cAMP, dibutyryl-cAMP; IBMX, isobutyl-methylxanthine; PDE, phosphodiesterase; PKA, cAMP-dependent protein kinase; SQ 65,442, 1-ethyl-4-(ethylthio)-1H-pyrazolo[3,4-b]-pyridine-5-carboxylic acid ethyl ester.

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