Resorcylic acid lactones (RALs) constitute a family of polyketide mycotoxins with a variety of biological activities.1) Representative examples are the heat shock protein 90 inhibitor radicicol and the estrogen agonist zearalenone. Hypothemycin ( Fig. 1), a RAL containing a cis-enone moiety that initially did not reveal any particularly interesting activity, was later shown to inhibit ras-transformation and T cell activation. [2][3][4] Hypothemycin and other closely related cis-enone RALs gained attention as compounds that irreversibly inhibit certain protein kinases such as mitogen-activated protein kinase kinase (MEK), extracellular signalf-regulated kinase (ERK) and TAK1, but not RAF, protein kinase C (PKC) or protein kinase A (PKA). [5][6][7][8] From their structures and irreversible mode of action, cis-enone RALs were predicted to form stable Michael addition products with cysteine residues of protein kinases. Bioinformatics studies revealed that a cysteine residue that immediately precedes the highly conserved Asp-Phe-Gly (DFG) motif is the cis-enone RAL binding site.9) This cysteine is conserved in 9% (46/ 510) of the protein kinases in the human kinome.Although different kinases contain the RAL target cysteine, cis-enone RALs have been isolated as selective inhibitors of MEK. 7,8) Furthermore, cis-enone RALs share various biological properties with MEK inhibitors. Sonoda et al. identified hypothemycin as a ras-signaling inhibitor.3) We reported that the MEK inhibitor U0126 reverses ras-mediated transformation.10) Hypothemycin and MEK inhibitors showed strong activity in cell lines with the activating BRAF V600E mutation.11) Sonoda et al. reported that the suppression of ras-induced transformation was associated with accelerated cyclin D1 degradation, 3) and a MEK inhibitor caused a loss of cyclin D expression in BRAF mutants. 11)Here, we compared the effects of hypothemycin and MEK inhibitors on signaling pathways in cells. Our results show that even though the RAL target cysteine is not confined to the components of the MEK-ERK pathway, in cellular settings hypothemycin preferentially blocks the MEK-ERK axis with sufficient selectivity to impair transformed phenotypes of cancer cells requiring this pathway. The resorcylic acid lactone hypothemycin has been shown to inactivate protein kinases by binding to a cysteine conserved in 46 protein kinases, including mitogen-activated protein kinase kinase (MEK), extracellular signal-regulated kinase (ERK) and platelet-derived growth factor receptor (PDGFR). We assessed the selectivity of hypothemycin in cellular contexts. Hypothemycin normalized the morphology and inhibited anchorage-independent growth of Ki-ras transformed normal rat kidney (NRK) cells with selectivity and potency comparable to or greater than that of the MEK inhibitor U0126. In Ki-ras-transformed and phorbol 12-myristate 13-acetate (PMA)-treated NRK cells, hypothemycin blocked ERK activation but showed a minimal effect on autophosphorylation of protein kinase D1 (PKD1), another kinase contain...

Many bacteria encode tyrosine kinases that are structurally unrelated to their eukaryotic counterparts and are termed BY-kinases. Two BY-kinases, CapB1 and CapB2, have been identified in the Staphylococcus aureus genome. Although CapB1 and CapB2 share more than 70% homology, earlier studies with purified enzymes did not find any evident kinase activity in CapB1, whereas CapB2 was autophosphorylated on a C-terminal tyrosine cluster in the presence of the kinase modulator proteins CapA1 or CapA2. For the convenient analysis of BY-kinases, we attempted to express CapB2 in an active form in a mammalian cell line. To this end, the C-terminal activation domain of CapA1 was attached to the N-terminus of CapB2, and the resulting CapA1/CT-CapB2 chimera was further fused with various tags and transfected into HEK293T cells. Immunoblotting analyses showed that when fluorescent protein tags were attached to the N-terminus, CapA1/CT-CapB2 was both expressed and tyrosine phosphorylated in HEK293T cells. Mutation of the ATPbinding lysine abrogated tyrosine phosphorylation, indicating that tyrosine phosphorylation was catalyzed by the transfected bacterial kinase and not by endogenous cellular enzymes. Unexpectedly, mutation of the C-terminal tyrosine cluster did not abolish autophosphorylation. Further analyses revealed that CapA1/CT-CapB2 phosphorylated not only itself but also the attached fluorescent protein tag. Several domains and residues important for tyrosine kinase activity were identified from the production of various mutants. We also present data that CapB1, which was previously thought to be catalytically inert, may possess intrinsic kinase activity.

Many bacterial species express tyrosine kinases termed BY-kinases that share no homology with eukaryotic enzymes. We have previously reported that the Staphylococcus aureus BY-kinase CapB2 when fused with the C-terminal activation domain of its modulator CapA1, can translate into an active tyrosine kinase in HEK293T cells. In the present study, full-length CapA1 and CapB2 tagged with different fluorescent proteins were transfected into HEK293T cells. When expressed individually, the modulator CapA1, a membrane protein in bacteria, also appeared to localize to the cell membrane in HEK293T cells. In contrast, the catalytic subunit, CapB2, was found to be cytosolic. Coexpression of the two proteins resulted in apparent translocation of CapB2 to the membrane with concomitant activation of tyrosine kinase activity. This translocation and activation of CapB2 did not occur when the cytoplasmic C-terminal tail of CapA1 was deleted. Conversely, the CapA1 cytoplasmic C-terminal tail alone, when attached to a membrane localization sequence, was sufficient for CapB2 translocation and kinase activation. Our results indicate that the kinase activity of CapB2 is stimulated by direct interaction with the C-terminal cytoplasmic domain of CapA1 and that the process can be reconstituted and visualized in a human cell line. We created various mutants of CapA and CapB, and present data that demonstrate the correlation between CapA-CapB interaction and kinase activation.