R subunits isoforms of PKA from Mucor circinelloides: different roles and ubiquitination

OCAMPO J,; MORENO, S; ROSSI, S.

Puerto Madrim

Congreso; XLVI REUNIÓN ANUAL SAIB; 2010

In Mucor circinelloides PKA is a tetramer composed of two regulatory subunits (R) and two catalyticsubunits (C). There are 4 isoforms of R (PKAR1-4) which have differential expression pattern during thefungal growth and differentiation. We found that the apparent molecular weight (MW) of R subunits inSDS-PAGE is higher than the MW estimated from their aminoacidic sequences. By Western blot andInmunoprecipitation assay, a 120 kDa ubiquitinated isoform of R was detected, which is associated withkinase and binding activities demonstrated by biochemical analysis. In cAMP-agarosa purifications, the120 kDa protein was not detected by anti-R antibody suggesting that this modification could be changingits capacity to interact with cAMP, however the ubiquitination does not alter its capacity to interact withC. Another protein of 60 kDa was also detected ubiquitinated and it was identified as the PKAR2isofrom. We concluded that R isoforms are modified post-translationally by ubiquitination, particularlywe defined multiubiquitinated high MW isoforms and the monoubiquitination of PKAR2. Theubiquitinated isoform is more abundant in old cultures, suggesting also a differential role during fungalgrowth. By the construction of knock out strains lacking PKAR1 and PKAR2, we demonstrated that theyhave different roles in fungal growth and differentiation.