Novel (Rp)-cAMPS Analogs as Tools for Inhibition of cAMP-kinase in Cell Culture

doi:10.1074/jbc.270.35.20599

Novel (Rp)-cAMPS Analogs as Tools for Inhibition of cAMP-kinase in Cell Culture

BASAL cAMP-KINASE ACTIVITY MODULATES INTERLEUKIN-1β ACTION (*)

From the (1) Department of Anatomy and Cell Biology, University of Bergen, 19, N-5009 Bergen, Norway, the
(2) Department of Pharmacology, School of Medicine, SJ-30, University of Washington, Seattle, Washington 98195, the
(3) BIOLOG Life Science Institute, Bremen, Flughafendamm 9, P. O. Box 107125 D-28071 Bremen, Germany, and the
(4) Universität Bremen, Institut für Organische Chemie, NW 2. Leobener Strasse, 28359 Bremen, Germany

^¶ To whom correspondence should be addressed. Tel.: +47-55206376; Fax: +47-55206360.

Abstract

Novel (Rp)-cAMPS analogs differed widely in ability to antagonize cAMP activation of pure cAMP-dependent protein kinase I and II and to antagonize actions of cAMP on gene expression, shape change, apoptosis, DNA replication, and protein phosphorylation in intact cells. These differences were related to different abilities of the analogs to stabilize the holoenzyme form relative to the dissociated form of cAMP kinase type I and II.

(Rp)-8-Br-cAMPS and (Rp)-8-Cl-cAMPS were the most potent cAMP antagonists for isolated type I kinase and for cells expressing mostly type I kinase, like IPC-81 leukemia cells, fibroblasts transfected with type I regulatory subunit (RI), and primary hepatocytes. It is proposed that (Rp)-8-Br-cAMPS or (Rp)-8-Cl-cAMPS should replace (Rp)-cAMPS as the first line cAMP antagonist, particularly for studies in cells expressing predominantly type I kinase.

The phosphorylation of endogenous hepatocyte proteins was affected oppositely by (Rp)-8-Br-cAMPS and increased cAMP, indicating that (Rp)-8-Br-cAMPS inhibited basal cAMP-kinase activity. The inhibition of basal kinase activity was accompanied by enhanced DNA replication, an effect which could be reproduced by microinjected mutant cAMP-subresponsive RI. It is concluded that the basal cAMP-kinase activity exerts a tonic inhibition of hepatocyte replication. (Rp)-8-Br-cAMPS and microinjected RI also desensitized hepatocytes toward inhibition of DNA synthesis by interleukin-1β. This indicates that basal cAMP-kinase activity can have a permissive role for the action of another (interleukin-1β) signaling pathway.

Footnotes

↵^§ The first two authors are equal.
↵* This work was supported by grants from the Medical Research Council of Norway, the Novo Nordisk Foundation, National Institutes of Health Grants ML 44948 and GM 32875, Grant Ja 246/6 from the Deutsche Forschungsgemeinschaft, Der Fonds der Chemischen Industrie, and the EU Human Capital and Mobility program. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵1 The abbreviations used are:

cAKI and cAKII

isozyme type I and II of cAMP-dependent protein kinase

R and C

the regulatory and catalytic subunits, respectively, of cAMP-dependent protein kinase

RI and RII

the regulatory subunits of cAMP-dependent protein kinase isozymes I and II, respectively

AI, BI, AII and BII

the cAMP-binding sites in RI and RII, respectively

Sp-cAMPS and Rp-cAMPS

the diastereoisomers (Sp)-cAMPS and (Rp)-cAMPS of adenosine 3′,5′(cyclic)phosphoro[thioate]

Rp-8-Br/Cl-cAMPS

Rp-8-Br-cAMPS as well as Rp-8-Cl-cAMPS

IL-1β

interleukin-1β

PGE¹

prostaglandin E¹

CHAPS

3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.
↵² The RI/RII ratio was higher in the RI-transfected (RN1.25) fibroblasts than in IPC cells (Table I). Nevertheless, isozyme II-directed cAMP-analog pairs synergized significantly in activating cAMP-responsive gene activity and cell rounding in RN1.25 cells (Fig. 6B), but not in inducing IPC cell apoptosis (16). This demonstrates that endogenous holoenzyme-associated RII was still present in RN1.25 cells overexpressing RIα, as supported by the finding of a substantial amount of type II holoenzyme by DEAE chromatography of RN1.25 cell extracts (data not shown). The RN1.25 cells thus cannot be considered a pure type I system, but they have much more type I kinase than the RIIα expressing H1.9 cell line, which showed very little type I directed analog synergism (Fig. 7A).
- Received May 11, 1995.