Transient CRE- and kappa B site-binding is cross-regulated by cAMP-dependent protein kinase and a protein phosphatase in mouse splenocytes.
Cyclic AMP regulates a variety of cellular responses through activation of cAMP-dependent protein kinase (PKA). The catalytic subunit of PKA, in turn, activate cAMP responsive element (CRE) and nuclear factor-kappa B (NF-kappa B) binding proteins. In this study, we demonstrated that binding activity to both CRE and kappa B sites in nuclear extracts from spleen cells is modulated by PKA in a time-dependent manner. Electrophoretic mobility shift assays showed that binding by transcription factors to either the CRE or kappa B motif was rapidly up-regulated by cAMP, with maximum binding detected at 30 min in response to forskolin stimulation of splenocytes. This was followed by a steady decline in CRE and kappa B thereafter reaching basal levels by 2 hr. This up-regulation in CRE and kappa B binding was closely associated with an enhancement of PKA activity which was maximum at 30 min following forskolin stimulation. However, unlike the binding of regulatory factors to CRE and kappa B motifs which was very transient, peak PKA activity was sustained for 2 hr. Interestingly, okadaic acid, a protein phosphatase inhibitor, prevented the decline in protein binding to CRE and kappa B motifs 2 hr following forskolin stimulation and actually produced a slight increase at 30 min. These data suggest that binding by transcription factors to CRE and kappa B sites are up-regulated concomitantly with PKA activation but subsequently down-regulated by a protein phosphatase.
Koh
W S
WS
Department of Pharmacology and Toxicology, Michigan State University, East Lansing 48824, USA.
Jeon
Y J
YJ
Herring
A C
AC
Kaminski
N E
NE
D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004789:Enzyme Activation; D005260:Female; D007700:Kinetics; D051379:Mice; D008815:Mice, Inbred Strains; D016328:NF-kappa B / Q000378:metabolism*; D019319:Okadaic Acid / Q000494:pharmacology; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D012045:Regulatory Sequences, Nucleic Acid; D013154:Spleen / Q000166:cytology / Q000378:metabolism*
D017362:Cyclic AMP Response Element-Binding Protein; D016328:NF-kappa B; D009838:Oligodeoxyribonucleotides; D005576:Colforsin; D019319:Okadaic Acid; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D010749:Phosphoprotein Phosphatases