Mutational activation of BRAF confers sensitivity to transforming growth factor beta inhibitors in human cancer cells

Recent data implicate elevated transforming growth factor-β (TGFβ) signalling in BRAF inhibitor drug-resistance mechanisms, but the potential for targeting TGFβ signalling in cases of advanced melanoma has not been investigated. We show that mutant BRAFV600E confers an intrinsic dependence on TGFβ/TGFβ receptor 1 (TGFBR1) signalling for clonogenicity of murine melanocytes. Pharmacological inhibition of the TGFBR1 blocked the clonogenicity of human mutant BRAF melanoma cells through SMAD4-independent inhibition of mitosis, and also inhibited metastasis in xenografted zebrafish. When investigating the therapeutic potential of combining inhibitors of mutant BRAF and TGFBR1, we noted that unexpectedly, low-dose PLX-4720 (a vemurafenib analogue) promoted proliferation of drug-naïve melanoma cells. Pharmacological or pharmacogenetic inhibition of TGFBR1 blocked growth promotion and phosphorylation of SRC, which is frequently associated with vemurafenib-resistance mechanisms. Importantly, vemurafenib-resistant patient derived cells retained sensitivity to TGFBR1 inhibition, suggesting that TGFBR1 could be targeted therapeutically to combat the development of vemurafenib drug-resistance

A chaperone-dependent GSK3 beta transitional intermediate mediates activation-loop autophosphorylation

Glycogen synthase kinase 3 (GSK3), a key component of the insulin and wnt signaling pathways, is unusual, as it is constitutively active and is inhibited in response to upstream signals. Kinase activity is thought to be increased by intramolecular phosphorylation of a tyrosine in the activation loop (Y216 in GSK3 beta), whose timing and mechanism is undefined. We show that GSK3 beta autophosphorylates Y216 as a chaperone-dependent transitional intermediate possessing intramolecular tyrosine kinase activity and displaying different sensitivity to small-molecule inhibitors compared to mature GSK3 beta. After autophosphorylation, mature GSK3 beta is then an intermolecular serine/threonine kinase no longer requiring a chaperone. This shows that autoactivating kinases have adopted different molecular mechanisms for autophosphorylation; and for kinases such as GSK3, inhibitors that affect only the transitional intermediate would be missed in conventional drug screens

dDYRK2: a novel dual-specificity tyrosine-phosphorylation-regulated kinase in Drosophila

Dual-specificity tyrosine-phosphorylation-regulated kinases (DYRKs) are an emerging family of protein kinases that have been identified in all eukaryotic organisms examined to date. DYRK family members are involved in regulating key developmental and cellular processes such as neurogenesis, cell proliferation, cytokinesis and cellular differentiation. Two distinct subgroups exist, nuclear and cytosolic. In Drosophila, the founding family member minibrain, whose human orthologue maps to the Down syndrome critical region, belongs to the nuclear subclass and affects post-embryonic neurogenesis. In the present paper, we report the isolation of dDYRK2, a cytosolic DYRK and the putative product of the smell-impaired smi35A gene. This is the second such kinase described in Drosophila, but the first to be characterized at the molecular and biochemical level. dDYRK2 is an 81 kDa dual-specificity kinase that autophosphorylates on tyrosine and serine/threonine residues, but appears to phosphorylate exogenous substrates only on serine/threonine residues. It contains a YXY motif in the activation loop of the kinase domain in the same location as the TXY motif in mitogen-activated protein kinases. dDYRK2 is tyrosine-phosphorylated in vivo, and mutational analysis reveals that the activation loop tyrosines are phosphorylated and are essential for kinase activity. Finally, dDYRK2 is active at all stages of fly development, with elevated levels observed during embryogenesis and pupation