Moreover, CCG-1423, an inhibitor of MICAL2, was shown to inhibit HNSCC cell proliferation, invasion, and migration. Consistently, ectopic expression of non-phosphorylatable MICAL2 mutants (MICAL2Y445F and MICAL2Y463F, not MICAL2Y488F) failed to ameliorate HNSCC cell growth, whereas expression of wild-type MICAL2 or MICAL2Y488F rescued the impaired proliferation induced by MICAL2 knockdown. Substitution of the Tyr445 or Tyr463 residue of purified recombinant MICAL2-redox with phenylalanine (generating a non-phosphorylatable mutant) abolishes the enhanced MICAL2-mediated F-actin disassembly induced by ARG. Direct phosphorylation assay and mass spectrometry confirmed that ARG phosphorylates MICAL2 at Tyr445, Tyr463, and Tyr488. The kinase ARG interacts with MICAL2 and augments MICAL2-mediated actin disassembly. MICAL2 serves as a flavoprotein monooxygenase and directly induces actin filament depolymerization by specifically oxidizing the methionine 44 and 47 residues of F-actin. Here, we show that MICAL2, which is increased in head and neck squamous cell carcinoma (HNSCC) and inversely associated with patient survival, promotes HNSCC growth, invasion, and migration. However, the underlying mechanisms remain largely unknown. In cancer cells, changes in the actin cytoskeleton may serve as prerequisites for proliferation, invasion, and metastatic dissemination. The actin cytoskeletal architecture provides the structural underpinnings for crucial cellular behaviors. A recently developed analog, CCG-257081, which co-crystallizes with pirin, is also effective in the prevention of bleomycin-induced dermal fibrosis. Finally, using both siRNA and a previously validated pirin inhibitor, we show a role for pirin in TGF-β induced gene expression in primary dermal fibroblasts. We also show with genetic approaches that pirin modulates MRTF-dependent SRE.L Luciferase activation. Using biophysical techniques including isothermal titration calorimetry and X-ray crystallography, we verify that pirin binds these compounds in vitro. Here, we describe affinity isolation-based target identification efforts which yielded pirin, an iron-dependent co-transcription factor, as a target of this series of compounds. Although these compounds are efficacious, the molecular target is unknown. These data identify redox modification of nuclear actin as a regulatory switch that mediates SRF/MRTF-A-dependent gene transcription.Ī series of compounds (including CCG-1423 and CCG-203971) discovered through an MRTF/SRF dependent luciferase screen has shown remarkable efficacy in a variety of in vitro and in vivo models, including melanoma metastasis and bleomycin-induced fibrosis. Furthermore, we show that MICAL-2 is a target of CCG-1423, a small molecule inhibitor of SRF/MRTF-A-dependent transcription that exhibits efficacy in various preclinical disease models. MICAL-2 induces redox-dependent depolymerization of nuclear actin, which decreases nuclear G-actin and increases MRTF-A in the nucleus. Here, we show that MICAL-2, an atypical actin-regulatory protein, mediates SRF/MRTF-A-dependent gene transcription elicited by nerve growth factor and serum. However, pathways that regulate nuclear actin levels are poorly understood. The level of nuclear MRTF-A is regulated by nuclear G-actin, which binds to MRTF-A and promotes its nuclear export. SRF/MRTF-A-dependent gene transcription is activated when nuclear MRTF-A levels increase, enabling the formation of transcriptionally active SRF/MRTF-A complexes. The serum response factor (SRF) binds to coactivators, such as myocardin-related transcription factor-A (MRTF-A), and mediates gene transcription elicited by diverse signaling pathways.
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