Isoforms of the R subunit of PKA: functional and biochemical characterization

OCAMPO, , J; PEREYRA, E; MORENO, S; GARRE MULA, V.; ROSSI S

Tucuman Argentina

Congreso; SAIB - 45 rd Annual Meeting XLV Reunión Anual Sociedad Argentina de Investigación en Bioquímica y Biología Molecula; 2009

SAIB

Isoforms of the R subunit of PKA: functional and biochemical characterization Ocampo, J; Pereyra, E;  Moreno, S; Garre Mula, V.; Rossi, S. Dpto. Qca. Biológica,FCEN, UBA y Dto Genética y Microbiología, Fac.Biología, Universidad de Murcia  E-mail: jocampo@qb.fcen.uba.ar   In M. circinelloides PKA is a tetramer composed of two Regulatory subunits (R) and two Catalytic subunits (C). We demonstrated the existence of 4 genes for R subunit and 10 genes for C subunits. We disrupted pkaR1 and pkaR2 genes generating two KO strains to study its role in the regulation of morphological and cellular development. Analysis of the morphological features associated with these mutations suggests that the genes are involved in several developmental processes in this fungus. The mutant pkaR1D showed a reduction in growth and alterations in germination rates, cell volume, germ tube length, and asexual sporulation. The mutant pkaR2D showed only different germination kinetics, the tube germ emission was earlier than the wild-type strain.  The expression of all genes was detected at the mRNA level by semiquantitative reverse transcription-PCR, and they showed a differential expression at different developmental stages. The expression of the genes in each ko mutan were also analyzed pkar1D, showed no variations in the expression of the other genes but pkaR2D showed that pkaR3 was increased and pkaR4 did not express. Analysis of the nucleotide sequences of pkaR genes revealed that pkaR1, pkaR2, and pkaR4 gene suggest that the M. circinelloides pkaR genes arose by means of gene duplication events, pkaR3gene being less closely related to the other three .Our results suggest that the duplication events, leading to the four extant copies of pkaR genes, occurred very early in the zygomycete evolutionary lineage. The results, taken as a whole, confirm that PKA is involved in multiple processes in M. circinelloides physiology and due to the existence of four R and ten C subunits, there would be a multiplicity of possible PKA holoenzymes which could have a different functional significance.