BIRB796 Inhibits All p38 MAPK Isoforms in Vitro and in Vivo

摘要

The compound BIRB796 inhibits the stress-activated protein kinases p38α and p38β and is undergoing clinical trials for the treatment of inflammatory diseases. Here we report that BIRB796 also inhibits the activity and the activation of SAPK3/p38γ. This occurs at higher concentrations of BIRB796 than those that inhibit p38α and p38β and at lower concentrations than those that inhibit the activation of JNK isoforms. We also show that at these concentrations, BIRB796 blocks the stress-induced phosphorylation of the scaffold protein SAP97, further establishing that this is a physiological substrate of SAPK3/p38γ. Our results demonstrate that BIRB796, in combination with SB203580, a compound that inhibits p38α and p38β, but not the other p38 isoforms, can be used to identify physiological substrates of SAPK3/p38γ as well as those of p38α and p38β. The compound BIRB796 inhibits the stress-activated protein kinases p38α and p38β and is undergoing clinical trials for the treatment of inflammatory diseases. Here we report that BIRB796 also inhibits the activity and the activation of SAPK3/p38γ. This occurs at higher concentrations of BIRB796 than those that inhibit p38α and p38β and at lower concentrations than those that inhibit the activation of JNK isoforms. We also show that at these concentrations, BIRB796 blocks the stress-induced phosphorylation of the scaffold protein SAP97, further establishing that this is a physiological substrate of SAPK3/p38γ. Our results demonstrate that BIRB796, in combination with SB203580, a compound that inhibits p38α and p38β, but not the other p38 isoforms, can be used to identify physiological substrates of SAPK3/p38γ as well as those of p38α and p38β. The stress-activated protein kinase (SAPK) 1The abbreviations used are: SAPK, stress-activated protein kinase; MAPK, mitogen-activated protein kinase; JNK, c-Jun N-terminal kinase; ERK, extracellular signal-regulated kinase; MKK, MAPK kinase; MAPKAP, MAPK-activated protein; MAPKAP-K, MAPKAP-kinase; HEK, human embryonic kidney; EGF, epidermal growth factor. 1The abbreviations used are: SAPK, stress-activated protein kinase; MAPK, mitogen-activated protein kinase; JNK, c-Jun N-terminal kinase; ERK, extracellular signal-regulated kinase; MKK, MAPK kinase; MAPKAP, MAPK-activated protein; MAPKAP-K, MAPKAP-kinase; HEK, human embryonic kidney; EGF, epidermal growth factor. p38 isoforms are mitogen-activated protein kinase (MAPK) family members that are activated by changes in the cellular environment, such as alterations in the concentration of nutrients, cytokines, cell-damaging agents, and changes in osmolarity of the surrounding medium (1Cohen P. Trends Cell Biol. 1997; 7: 353-361Abstract Full Text PDF PubMed Scopus (515) Google Scholar). They comprise p38α, p38β, SAPK3/p38γ (also known as ERK6), and SAPK4/p38δ. Each p38 isoform may have different biological functions and different physiological substrates, but they all phosphorylate substrates containing the minimal consensus sequence Ser/Thr-Pro. A major challenge of current research in this field is to identify the downstream physiological substrates and processes that each p38 MAPK regulates in the cell, as well as determining which “upstream” components regulate their activities. One of the most successful aids to the identification of physiological substrates has been the use of small cell-permeable compounds that are specific inhibitors of particular protein kinases. These compounds enter cells within minutes and act rapidly to suppress the activity of a particular kinase so that indirect effects caused, for example, by changes in gene expression or protein activity, a potential risk when cells deficient in a particular kinase are used, are excluded. Moreover, the use of protein kinase inhibitors avoids the need for transfection-based approaches, which have the potential to give misleading results since the fidelity of signaling can break down when components are overexpressed. Identification of p