Protease inhibitors prevent the protein kinase A-dependent loss of Rap1 GTPase from the particulate fraction of COS1 cells.

Immunoblotting with a monoclonal Rap1 antibody, we found that elevation of cyclic AMP, with forskolin and IBMX or CPT-cAMP, led to a rapid reduction in the levels of Rap1 protein associated with particulate, nuclear/perinuclear fractions from PC12 and COS1 cells. In contrast, cytoplasmic levels of Rap1 remained constant following cyclic AMP stimulation. To gain independent confirmation that cyclic AMP promoted loss of Rap1 in nuclear/perinuclear fractions we used a polyclonal Rap1 antibody, which gave similar results to the monoclonal antibody. This demonstrated that the loss in Rap1 immunoreactivity was not due to phosphorylation-dependent changes that alter immunorecognition. The reduction in Rap1 levels was blocked by PKA inhibitors and by a Rap1 serine to alanine PKA-phosphorylation site mutant (S180A). Peptide inhibitors of the proteasome, cathespin, and calpain II also inhibited the decrease in Rap1 levels, indicating that proteolytic degradation may contribute to maintaining Rap1 levels in the nuclear/perinuclear fraction of cells.