Effect of the thermostable protein kinase inhibitor on intracellular localization of the catalytic subunit of cAMP-dependent protein kinase.
cAMP-dependent protein kinase mediates a variety of cellular responses in most eukaryotic cells. Many of these responses are cytoplasmic, whereas others appear to require nuclear localization of the catalytic subunit. In order to understand further the molecular basis for subcellular localization of the catalytic subunit, the effect of the heat stable protein kinase inhibitor (PKI) was investigated. The subcellular localization of the catalytic (C) subunit was determined both in the presence and absence of PKI, by microinjecting fluorescently labeled C subunit into single living cells. When injected alone, a significant fraction of the dissociated C subunit localized to the nucleus. When coin-injected with an excess of PKI, little of the C subunit localized to the nucleus, suggesting that accumulation of catalytic subunit in the nucleus requires either enzymatic activity or a nuclear localization signal. Inactivation of the catalytic subunit in vitro by treatment with N-ethylmaleimide did not prevent localization in the nucleus, indicating that enzymatic activity was not a prerequisite for nuclear localization. In an effort to search for a specific signal that might mediate nuclear localization, a complex of the catalytic subunit with a 20-residue inhibitory peptide derived from PKI (PKI(5-24)) was microinjected. In contrast to intact PKI, the peptide was not sufficient to block nuclear accumulation. In the presence of PKI(5-24), the C subunit localized to the nucleus in a fashion analogous to that of dissociated, active C subunit despite evidence of no catalytic activity in situ. Thus, nuclear localization of the C subunit appears to be independent of enzymatic activity but most likely dependent upon a signal. The signal is apparently masked by both the regulatory subunit and PKI but not by the inhibitory peptide.
Fantozzi
D A
DA
Department of Chemistry, University of California, San Diego, La Jolla 92093.
Taylor
S S
SS
Howard
P W
PW
Maurer
R A
RA
Feramisco
J R
JR
Meinkoth
J L
JL
D000818:Animals; D002352:Carrier Proteins / Q000494:pharmacology*; D002460:Cell Line; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D004355:Drug Stability; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005033:Ethylmaleimide / Q000494:pharmacology; D016650:Fluorescein-5-isothiocyanate; D047908:Intracellular Signaling Peptides and Proteins; D046911:Macromolecular Substances; D051379:Mice; D010446:Peptide Fragments / Q000494:pharmacology; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism
D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D046911:Macromolecular Substances; D010446:Peptide Fragments; D011994:Recombinant Proteins; C015482:protein kinase modulator; D011494:Protein Kinases; D016650:Fluorescein-5-isothiocyanate; D005033:Ethylmaleimide