Heat-stable inhibitor protein derived peptide substrate analogs: phosphorylation by cAMP-dependent and cGMP-dependent protein kinases.
The phosphorylation of substrate peptides derived from PKI, the heat-stable inhibitor protein of the cAMP-dependent protein kinase (PKA), has been studied with both PKA and the cGMP-dependent protein kinase (PKG) using a variety of substitution and deletion analogs. On the basis of Km, kcat and kcat/Km values, (Ser21)PKI alpha(14-22) amide (numbering based upon native PKI alpha) is the most effective peptide substrate yet discovered for either kinase, although other peptides, while phosphorylated considerably less efficiently by PKG, are more specific. Although the inhibitory peptide corresponding to this sequence (i.e., with an Ala at position 21) is a much more potent inhibitor of PKA than of PKG (approximately 250-fold), PKG actually exhibits a 60% higher kcat than does PKA with the (Ser21)PKI alpha(14-22) amide substrate peptide, with only a 20-fold higher Km value. The two key PKI residues within this peptide which were found to be essential for substrate activity with both kinases were Arg18 (P-3) and Ile22 (P+1). The Arg19 (P-2) residue, which contributes significantly to both PKI-based peptide inhibitors and substrates of PKA, was only a more minor contributor to PKG substrate efficacy. Of particular note, the Phe10 (P-11) residue, which contributes very substantially to high affinity binding of both PKI and longer PKI peptide inhibitors, neither positively nor negatively affects the kinetics of either PKA or PKG with PKI-based substrates.(ABSTRACT TRUNCATED AT 250 WORDS)
Mitchell
R D
RD
Department of Biological Chemistry, School of Medicine, University of California, Davis 95616.
Glass
D B
DB
Wong
C W
CW
Angelos
K L
KL
Walsh
D A
DA
D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002417:Cattle; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D017869:Cyclic GMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006358:Hot Temperature; D047908:Intracellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D010766:Phosphorylation; D013379:Substrate Specificity
D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; D010446:Peptide Fragments; C015482:protein kinase modulator; D017868:Cyclic AMP-Dependent Protein Kinases; D017869:Cyclic GMP-Dependent Protein Kinases