PEST sequences in cAMP-dependent protein kinase subunits of the aquatic fungus Blastocladiella emersonii are necessary for in vitro degradation by endogenous proteases

During Blastocladiella emersonii germination, the regulatory (R) and the ca talytic (C) subunits of the cAMP-dependent protein kinase (PKA) are rapidly and concurrently degraded, after PKA activation in response to a transient increase in intracellular cAMP levels. The possibility that PEST sequences could be acting as proteolytic recognition signals in this process was inv estigated, and high score PEST sequences were found in both B. emersonii R and C subunits. Deletions in the PEST sequences were obtained by site-direc ted mutagenesis and the different PKA subunits were independently expressed in Escherichia coli. Proteolysis assays of the various R and C recombinant forms, using B. emersonii cell extracts as the source of proteases, showed a strong correlation between the presence of high score PEST sequences and susceptibility to degradation. Furthermore, the amino-terminal sequence of the proteolytic fragments indicated that the cleavage sites in both subuni ts are located at or near the PEST regions. The PEST sequence in B. emerson ii C subunit, which when deleted or disrupted leads to resistance to proteo lysis, is entirely contained in the 72-amino-acid extension located in the N-terminus of the protein. C subunit mutants carrying deletions in this reg ion displayed little difference in their kinetic properties or enzyme therm ostability. These results suggest that the N-terminal extension may only pl ay a role in C subunit degradation.