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 catalytic (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...

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Bibliographic Details
Published in:Molecular microbiology 2000-05, Vol.36 (4), p.926-939
Main Authors: Borges, A.C.C, Gomes, S.L
Format: Article
Language:English
Subjects:
Amino Acid Sequence
amino acid sequences
aquatic fungi
Blastocladiella - enzymology
Blastocladiella emersonii
Catalytic Domain
Cyclic AMP-Dependent Protein Kinases - genetics
Cyclic AMP-Dependent Protein Kinases - metabolism
Endopeptidases - metabolism
Gene Expression
glutamic acid
Holoenzymes - metabolism
Molecular Sequence Data
proline
proteolysis
Recombination, Genetic
serine
threonine
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Summary:During Blastocladiella emersonii germination, the regulatory (R) and the catalytic (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 investigated, and high score PEST sequences were found in both B. emersonii R and C subunits. Deletions in the PEST sequences were obtained by site‐directed 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 subunits are located at or near the PEST regions. The PEST sequence in B. emersonii C subunit, which when deleted or disrupted leads to resistance to proteolysis, is entirely contained in the 72‐amino‐acid extension located in the N‐terminus of the protein. C subunit mutants carrying deletions in this region displayed little difference in their kinetic properties or enzyme thermostability. These results suggest that the N‐terminal extension may only play a role in C subunit degradation.
ISSN:0950-382X
1365-2958
DOI:10.1046/j.1365-2958.2000.01912.x