No abstract

Unlike other members of the MAPK family, ERK5 contains a large C-terminal domain with transcriptional activation capability in addition to an N-terminal canonical kinase domain. Genetic deletion of ERK5 is embryonic lethal, and tissue-restricted deletions have profound effects on erythroid development, cardiac function, and neurogenesis. In addition, depletion of ERK5 is antiinflammatory and antitumorigenic. Small molecule inhibition of ERK5 has been shown to have promising activity in cell and animal models of inflammation and oncology. Here we report the synthesis and biological characterization of potent, selective ERK5 inhibitors. In contrast to both genetic depletion/deletion of ERK5 and inhibition with previously reported compounds, inhibition of the kinase with the most selective of the new inhibitors had no antiinflammatory or antiproliferative activity. The source of efficacy in previously reported ERK5 inhibitors is shown to be off-target activity on bromodomains, conserved protein modules involved in recognition of acetyl-lysine residues during transcriptional processes. It is likely that phenotypes reported from genetic deletion or depletion of ERK5 arise from removal of a noncatalytic function of ERK5. The newly reported inhibitors should be useful in determining which of the many reported phenotypes are due to kinase activity and delineate which can be pharmacologically targeted.) is a member of the mitogen-activated protein kinase (MAPK) family, which includes ERK1/2, JNK1/2/3, and p38α/β/δ/γ (1). However, unlike the other MAPK members, ERK5 contains a unique 400-amino-acid C-terminal domain in addition to the kinase domain. Through the MAPK signaling cascade, mitogenactivated protein kinase kinase 5 (MEK5) activates ERK5 by phosphorylating the TEY motif in the N-terminal activation loop (2). This event unlocks the N-and C-terminal halves, allowing ERK5 to auto-phosphorylate multiple sites in its C-terminal region, which can then regulate nuclear shuttling and gene transcription (3, 4). Noncanonical pathways (including cyclin-dependent kinases during mitosis and ERK1/2 during growth factor stimulation) also exist for phosphorylation of sites in the ERK5 tail (5-7). Although ERK5 has been demonstrated to directly phosphorylate transcription factors (8-10), the noncatalytic C-terminal tail of ERK5 can also interact with transcription factors and influence gene expression (4,11,12).ERK5 can be activated in response to a range of mitogenic stimuli [e.g., growth factors, G protein-coupled receptor (GPCR) agonists, cytokines] and cellular stresses (e.g., hypoxia, shear stress) (13). Like most kinases including MAPK members, ERK5 function is assumed to be driven by its kinase activity. ERK5 deletion is embryonic lethal in mice and a variety of tissue-or development-stage restricted KOs have shown clear phenotypes, suggesting that the catalytic function and/or an aspect of the nonkinase domain(s) have key roles in development and mature organ function (14-18). The availability of the first ERK5 inhibitor ...

In those proteins, an essential metal ion is bound by a metal ion-dependent adhesion site (MIDAS). The MIDAS is presented at the apex of a larger protein module called an I domain. The metal ligands in the MIDAS can be separated into three distantly spaced clusters of oxygenated residues. These three coordination sites also appear to exist in the integrin ␤3 and ␤5 subunits. Here, we examined the putative metal binding site within ␤3 and ␤5 using site-directed mutagenesis and ligand binding studies. We also investigated the fold of the domain containing the putative metal binding site using the PHD structural algorithm. The results of the study point to the similarity between the integrin ␤ subunits and the MIDAS motif at two of three key coordination points. Importantly though, the study failed to identify a residue in either ␤ subunit that corresponds to the second metal coordination group in the MIDAS. Moreover, structural algorithms indicate that the fold of the ␤ subunits is considerably different than the I domains. Thus, the integrin ␤ subunits appear to present a MIDAS-like motif in the context of a protein module that is structurally distinct from known I domains.Integrins are ␣␤ heterodimers that mediate cell adhesion (1, 2). Integrins participate in development and tissue remodeling and are linked to several diseases. The integrins bind to many adhesive and extracellular matrix proteins. The focal points of this study are the ␣v␤3 and ␣v␤5 integrins, both of which recognize the Arg-Gly-Asp (RGD) 1 tripeptide motif. The ␣v␤3 integrin binds to at least nine adhesive proteins and has two important biological functions. First, ␣v␤3 mediates the adhesion of osteoclasts to the bone surface (3), an event often considered to be the first step in bone resorption (4). Second, the ␣v␤3 integrin is expressed on the surface of angiogenic endothelial cells, where it is required for cell survival and further vessel development (5-7). It has been suggested that inhibitors of the ␣v␤3 integrin could be applied as antagonists of osteoporosis and tumor angiogenesis. The biological function of the ␣v␤5 integrin is less clear. This integrin can mediate cell adhesion to vitronectin. The ␣v␤5 integrin is also required for the internalization of adenovirus (8, 9), and it may be associated with angiogenesis (7).All integrins require divalent cations to bind their ligands. An important clue to the structural basis for ion binding was revealed by the crystal structures of the I domains from the integrin ␣ L and ␣ M subunits (10, 11). Each I domain spans approximately 200 residues and is homologous to an "inserted" domain in a number of other proteins including von Willebrand factor (12). In ␣ L and ␣ M , the I domain is necessary and sufficient for ligand contact. These I domains contain a metal binding site called a MIDAS (metal ion-dependent adhesion site). This ion binding site consists of five liganding residues that can be separated into three groups. Each group of coordinating residues is located at separate positions wit...