Ubiquitination of R subunits isoforms of PKA from Mucor circinelloides

JOSEFINA OCAMPO; SILVIA MORENO; SILVIA ROSSI

¨Puerto Madryn

Congreso; XLVI Reunion Nacional de SAIB; 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, an ubiquitinated protein form of R of 120 KDa was detected, which is associated with kinase and binding activities as demonstrate analysis in sucrose gradients. In cAMP-agarosa purifications, the 120 KDa protein was not detected by anti-R antibody and suggests that this modification could be changing its capacity to interact with cAMP. By DEAE-Celulosa fractioning, we determined that R subunits of lower molecular weight were only responsible for the  cAMP binding activity. This modification does not alter its capacity to interact with C. The ubiquitinated form is also more abundant in old cultures, suggesting also a role differential during fungal growth. We also detected a 60 KDa protein, with anti-R and anti-Ub antibodies, identified as the PKAR2 isofrom. From these results, we concluded that R isoforms are modified post-translationally by ubiquitination, particularly we defined multiubiquitinated high MW isoforms and the monoubiquitination of PKAR2.