A combination of SILAC and nucleotide acyl phosphate labelling reveals unexpected targets of the Rsk inhibitor BI-D1870.

Alexander J Edgar,Rossana Zaru,Matthias Trost,Colin Watts

doi:10.1042/bsr20130094

Abstract

Protein kinase inhibitors frequently have interesting effects that cannot be fully ascribed to the intended target kinase(s) but identifying additional targets that might explain the effects is not straightforward. By comparing two different inhibitors of the Rsk (p90 ribosomal S6 kinase) kinases, we found that the increasingly used compound BI-D1870 had biological effects in murine DCs (dendritic cells) that could not be solely ascribed to Rsk or other documented targets. We assessed the ability of BI-D1870 and a second Rsk inhibitor, BIX 02565 to protect enzyme active sites from reaction with biotinylated nucleotide acyl phosphates. Using SILAC (stable isotope labelling by amino acids in cell culture)-labelled DC lysates as a source of enzyme targets, we identify several kinases that interact with BI-D1870 but not with BIX 02565. We confirmed that these kinases, including Slk, Lok and Mst1, are inhibited by BI-D1870 but to a much lesser extent by BIX 02565 and that phosphorylation of some of their substrates is blocked by BI-D1870 in living cells. Our results suggest that the BI-D1870 inhibitor should be used with caution. The SILAC-based methodology we used should be useful for further comparative unbiased profiling of the target spectrum of kinase inhibitors with interesting biological effects under conditions that closely mimic those found in cells.

Highlights

The Rsk (90 kDa ribosomal S6 kinases) are Ser/Thr kinases that have been implicated in the regulation of a wide range of cellular functions such as cell proliferation and growth, apoptosis, metabolism and cell motility
Rsk inhibitors D1870 and BIX differentially affect cytokine production Several studies have shown that activation of Erk1/2 and p38 is crucial for the production of several pro- and anti-inflammatory cytokines in dendritic cell (DC) upon Toll-like receptor (TLR) stimulation [24,25]
In bone marrow-derived dendritic cells (BMDC) stimulated with LPS in the presence of both the MEK1/2 inhibitor PD184352 and the p38 inhibitor BIRB0796, the production of TNFα, interleukin 6 (IL6) and IL10 was blocked (Figure 1A)

Summary

Introduction

The Rsk (90 kDa ribosomal S6 kinases) are Ser/Thr kinases that have been implicated in the regulation of a wide range of cellular functions such as cell proliferation and growth, apoptosis, metabolism and cell motility (reviewed in [1]). Four isoforms have been identified: Rsk and 4, Rsk and 3 being the most abundantly expressed They are composed of two functional kinase domains [3] that are sequentially activated by multiple phosphorylation steps. Mutations in the Rsk gene are thought to be responsible for the Coffin–Lowry syndrome that is characterized by skeletal malformations and severe psychomotor retardation [11]. These studies indicate that it is increasingly important to gain a better understanding of the specific roles of Rsk in different cell systems

Methods

Results

Conclusion