Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2007 Jan 1;401(1):29-38.
doi: 10.1042/BJ20061088.

BI-D1870 is a specific inhibitor of the p90 RSK (ribosomal S6 kinase) isoforms in vitro and in vivo

Gopal P Sapkota  1 , Lorna Cummings, Felicity S Newell, Christopher Armstrong, Jennifer Bain, Morten Frodin, Matthias Grauert, Matthias Hoffmann, Gisela Schnapp, Martin Steegmaier, Philip Cohen, Dario R Alessi
Affiliations

BI-D1870 is a specific inhibitor of the p90 RSK (ribosomal S6 kinase) isoforms in vitro and in vivo

Gopal P Sapkota et al. Biochem J. .

Abstract

Hormones and growth factors induce the activation of a number of protein kinases that belong to the AGC subfamily, including isoforms of PKA, protein kinase B (also known as Akt), PKC, S6K p70 (ribosomal S6 kinase), RSK (p90 ribosomal S6 kinase) and MSK (mitogen- and stress-activated protein kinase), which then mediate many of the physiological processes that are regulated by these extracellular agonists. It can be difficult to assess the individual functions of each AGC kinase because their substrate specificities are similar. Here we describe the small molecule BI-D1870, which inhibits RSK1, RSK2, RSK3 and RSK4 in vitro with an IC(50) of 10-30 nM, but does not signi-ficantly inhibit ten other AGC kinase members and over 40 other protein kinases tested at 100-fold higher concentrations. BI-D1870 is cell permeant and prevents the RSK-mediated phorbol ester- and EGF (epidermal growth factor)-induced phosphoryl-ation of glycogen synthase kinase-3beta and LKB1 in human embry-onic kidney 293 cells and Rat-2 cells. In contrast, BI-D1870 does not affect the agonist-triggered phosphorylation of substrates for six other AGC kinases. Moreover, BI-D1870 does not suppress the phorbol ester- or EGF-induced phosphorylation of CREB (cAMP-response-element-binding protein), consistent with the genetic evidence indicating that MSK, and not RSK, isoforms mediate the mitogen-induced phosphorylation of this transcription factor.

PubMed Disclaimer

Figures

Figure 1
Figure 1. Effect of BI-D1870 on RSK activity in vitro
(A) The ATP-competitive kinase inhibitor, BI-D1870, derived from a novel chemical series (WO 03/020722), potently inhibits RSK1/2. Activated His6–RSK1 (B) or His6–RSK2 (C) were assayed in the absence or presence of the indicated concentration of BI-D1870 with 100 μM ATP (●) or 10 μM ATP (○). The results are presented as percentage kinase activity relative to the control measured in the absence of BI-D1870. (D) As in (B) and (C), except that an active fragment of RSK comprising the isolated N-terminal catalytic domain (RSK1–389:S381E) activated by phosphorylation with PDK1, as described in the Materials and methods section, was assayed in the absence or presence of BI-D1870 with 100 μM ATP. Results are presented as the average for two experiments with each determination carried out in triplicate. The error for each data point is less than 5%.
Figure 2
Figure 2. BI-D1870 inhibits RSK activity in vivo
(A) HEK-293 cells that had been deprived of serum for 16 h, were incubated in the absence or presence of 10 μM BI-D1870 for the indicated times, then stimulated with 400 ng/ml PMA (TPA) for 20 min. The cells were lysed and the lysates were immunoblotted with the indicated antibodies as described in the Materials and methods section. (B) As in (A), except that cells were incubated for 30 min with the indicated concentrations of BI-D1870 prior to stimulation with PMA. Similar results were obtained in two separate experiments. Phosphorylation of GSK3 isoforms was quantitated by densitometric analysis of autorads.
Figure 3
Figure 3. Effect of BI-D1870 on activation of RSK and MSK1
(A) HEK-293 cells that had been deprived of serum for 16 h, were incubated in the absence or presence of 10 μM BI-D1870 or 2 μM PD 184352 as indicated. After 30 min the cells were stimulated with PMA (TPA) for 20 min, then the cells were lysed and the RSK isoforms immunoprecipitated with an antibody that recognized both RSK1 and RSK2. The immunoprecipitates were washed to remove BI-D1870 and assayed in the absence of BI-D1870. (B) As in (A) except that the cell lysates were immunoblotted with the indicated antibodies, as described in the Materials and methods section. (C) As in (A), except that MSK1 was immunoprecipitated from the lysates and assayed. In (A) and (C) the results are presented as means±S.E.M. for two separate experiments with each determination carried out in triplicate.
Figure 4
Figure 4. Effect of BI-D1870 on EGF-stimulated Rat-2 cells
Serum-deprived Rat-2 cells were incubated for 30 min in the presence of the indicated concentrations of BI-D1870. The cells were then stimulated with 100 ng/ml EGF for 10 min, lysed and immunoblotted with the indicated antibodies.
Figure 5
Figure 5. Effect of BI-D1870, PD 184352 and LY294002 on signalling pathways in Rat-2 cells
Serum-deprived Rat-2 cells were incubated for 30 min in the absence or presence of 10 μM BI-D1870, 2 μM PD 184352 or 100 μM LY 294002 as indicated. The cells were then stimulated with 100 ng/ml EGF for 10 min, lysed and the cell lysates were immunoblotted with indicated antibodies as described in the Materials and methods section. Similar results were obtained in two separate experiments.
Figure 6
Figure 6. BI-D1870 does not inhibit PKA in vivo
Serum-deprived Rat-2 cells or HEK-293 cells were incubated for 30 min in the absence or presence of 10 μM BI-D1870. The cells were either left unstimulated or stimulated with 20 μM forskolin for 10 min. The cells were lysed and the lysates immunoblotted with the indicated antibodies. Similar results were obtained in two separate experiments.
Figure 7
Figure 7. BI-D1870 activates ERKs in Rat-2 cells
(A) Serum-deprived Rat-2 cells were incubated for the indicated times in the presence of 10 μM BI-D1870 in the absence of EGF. The cells were lysed and the lysates immunoblotted with the indicated antibodies. (B) As in (A), except that cells were incubated with the indicated concentrations of BI-D1870 for 30 min and then either left unstimulated or stimulated with 100 ng/ml EGF for 10 min. For the immunoblotting studies similar results were obtained in two separate experiments.
See this image and copyright information in PMC

Comment in

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Scott J. D. Cyclic nucleotide-dependent protein kinases. Pharmacol. Therapeut. 1991;50:123–145. - PubMed
    1. Avruch J., Belham C., Weng Q., Hara K., Yonezawa K. The p70 S6 kinase integrates nutrient and growth signals to control translational capacity. Prog. Mol. Subcell. Biol. 2001;26:115–154. - PubMed
    1. Parekh D. B., Ziegler W., Parker P. J. Multiple pathways control protein kinase C phosphorylation. EMBO J. 2000;19:496–503. - PMC - PubMed
    1. Vanhaesebroeck B., Alessi D. R. The PI3K-PDK1 connection: more than just a road to PKB. Biochem. J. 2000;346:561–576. - PMC - PubMed
    1. Dalby K. N., Morrice N., Caudwell F. B., Avruch J., Cohen P. Identification of regulatory phosphorylation sites in mitogen-activated protein kinase (MAPK)-activated protein kinase-1a/p90rsk that are inducible by MAPK. J. Biol. Chem. 1998;273:1496–1505. - PubMed

Publication types

MeSH terms

LinkOut - more resources