R subunit isoforms of pka from Mucor circinelloides: different roles and ubiquitination.

OCAMPO, J; MORENO, S; ROSSI, S

Puerto Madryn, Chubut

Congreso; XLVI Reunión Anual de la Sociedad Argentina de Investigación en; 2010

SAIB

In Mucor circinelloides PKA is a tetramer composed of two regulatory subunits (R) and two catalytic subunits (C). There are 4 isoforms of R (PKAR1-4) which have differential expression pattern during the fungal growth and differentiation. We found that the apparent molecular weight (MW) of R subunits in SDS-PAGE is higher than the MW estimated from their aminoacidic sequences. By western blot and inmunoprecipitation assay, a 120 KDa ubiquitinated isoform of R was detected, which is associated with kinase and binding activities demonstrated by biochemical analysis. In cAMP-agarosa purifications, the 120 KDa protein was not detected by anti-R antibody suggesting that this modification could be changing its capacity to interact with cAMP, however the ubiquitination does not alter its capacity to interact with C. Another protein of 60 KDa was also detected ubiquitinated and it was identified as the PKAR2 isofrom. We concluded that R isoforms are modified post-translationally by ubiquitination, particularly we defined multiubiquitinated high MW isoforms and the monoubiquitination of PKAR2. The ubiquitinated isoform is more abundant in old cultures, suggesting also a differential role during fungal growth. By the construction of knock out strains lacking PKAR1 and PKAR2, we demonstrated that they have different roles in fungal growth and differentiation.