Effective intracellular inhibition of the cAMP-dependent protein kinase by microinjection of a modified form of the specific inhibitor peptide PKi in living fibroblasts.
In order to obtain a peptide retaining its biological activity following microinjection into living cells, we have modified a synthetic peptide [PKi(m)(6-24)], derived from the specific inhibitor protein of the cAMP-dependent protein kinase (A-kinase) in two ways: (1) substitution of the arginine at position 18 for a D-arginine; (2) blockade of the side chain on the C-terminal aspartic acid by a cyclohexyl ester group. In an in vitro assay, PKi(m) has retained a specific inhibitory activity against A-kinase as assessed against six other kinases, with similar efficiency to that of the unmodified PKi(5-24) peptide. Microinjection of PKi(m) into living fibroblasts reveals its capacity to prevent the changes in cell morphology and cytoskeleton induced by drugs which activate endogenous A-kinase, whereas the original PKi peptide failed to do so. This inhibition of A-kinase in vivo by PKi(m) lasts between 4 and 6 h after injection. In light of its effective half-life, this modified peptide opens a route for the use of biologically active peptides in vivo, an approach which has been hampered until now by the exceedingly short half-life of peptides inside living cells. By providing a direct means of inhibiting A-kinase activity for sufficiently long periods to observe effects on cellular functions in living cells, PKi(m) represents a powerful tool in studying the potential role of cAMP-dependent phosphorylation in vivo.
Fernandez
A
A
Cell Biology Unit, CRBM, CNRS-INSERM, Montpellier, France.
Mery
J
J
Vandromme
M
M
Basset
M
M
Cavadore
J C
JC
Lamb
N J
NJ
D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins; D002478:Cells, Cultured; D005347:Fibroblasts / Q000187:drug effects* / Q000378:metabolism; D006207:Half-Life; D047908:Intracellular Signaling Peptides and Proteins; D008845:Microinjections; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000494:pharmacology*; D010455:Peptides / Q000494:pharmacology*; D047428:Protein Kinase Inhibitors; D051381:Rats
D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; D010446:Peptide Fragments; D010455:Peptides; D047428:Protein Kinase Inhibitors; C061479:protein kinase inhibitor peptide; C069890:protein kinase inhibitor M (6-24); C069889:protein kinase inhibitor peptide (6-24)