Green fluorescence protein (GFP)-based fluorescence resonance energy transfer (FRET) is increasingly used in investigation of inter- and intramolecular interactions in living cells. In this report, we present a modified method for FRET quantification in cultured cells using conventional fluorescence microscopy. To reliably measure FRET, three positive control constructs in which a cyan fluorescence protein and a yellow fluorescence protein were linked by peptides of 15, 24, or 37 amino acid residues were prepared. FRET was detected using a spectrofluorometer, a laser scanning confocal microscope, and an inverted fluorescence microscope. Three calculation methods for FRET quantification using fluorescence microscopes were compared. By normalization against expression levels of GFP fusion proteins, the modified method gave consistent FRET values that could be compared among different cells with varying protein expression levels. Whole-cell global analysis using this method allowed FRET measurement with high spatial resolutions. Using such a procedure, the interaction of synaptic proteins syntaxin and the synaptosomal associated protein of 25 kDa (SNAP-25) was examined in PC12 cells, which showed strong FRET on plasma membranes. These results demonstrate the effectiveness of the modified method for FRET measurement in live cell systems.

Background: Receptor interacting protein 3 (RIP3)-mixed lineage kinase domain-like (MLKL) interaction is essential for necroptosis. Results: Murine RIP3 does not interact with human MLKL and vice versa due to sequence differences in and around the RIP3 phosphorylation sites. Conclusion: Different sequences in human and mouse RIP3 control the functionally conserved RIP3-MLKL interaction. Significance: This study provided new insights into the function of RIP3-MLKL interaction in necroptosis.

SUMMARY Signaling via the Akt serine/threonine protein kinase plays critical roles in the self-renewal of embryonic stem cells and their malignant counterpart, embryonal carcinoma cells (ECCs). Here we show that in ECCs, Akt phosphorylated the master pluripotency factor Oct4 at threonine 235, and that the levels of phosphorylated Oct4 in ECCs correlated with resistance to apoptosis and tumorigenic potential. Phosphorylation of Oct4 increased its stability, and facilitated its nuclear localization and its interaction with Sox2, which promoted the transcription of the core stemness genes POU5F1 and NANOG. Furthermore, in ECCs, unphosphorylated Oct4 bound to the AKT1 promoter and repressed its transcription. Phosphorylation of Oct4 by Akt resulted in dissociation of Oct4 from the AKT1 promoter, which activated AKT1 transcription and promoted cell survival. Therefore, a site-specific, post-translational modification of the Oct4 protein orchestrates the regulation of its stability, subcellular localization and transcriptional activities, which collectively promotes the survival and tumorigenicity of ECCs.

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The Hippo pathway regulates cell proliferation, apoptosis, and stem cell self-renewal, and its inactivation in animal models causes organ enlargement followed by tumorigenesis. Hippo pathway deregulation occurs in many human cancers, but the underlying mechanisms are not fully understood. Here, we report tyrosine phosphorylation of the Hippo pathway tumor suppressor LATS1 as a mechanism underlying its regulation by cell adhesion. A tyrosine kinase library screen identified Src as the kinase to directly phosphorylate LATS1 on multiple residues, causing attenuated Mob kinase activator binding and structural alteration of the substrate-binding pocket in the kinase domain. Cell matrix adhesion activated the Hippo pathway effector transcription coactivator YAP partially through Src-mediated phosphorylation and inhibition of LATS1. Aberrant Src activation abolished the tumor suppressor activity of LATS1 and induced tumorigenesis in a YAP-dependent manner. Protein levels of Src in human breast cancer tissues correlated with accumulation of active YAP dephosphorylated on the LATS1 target site. These findings reveal tyrosine phosphorylation of LATS1 by Src as a novel mechanism of Hippo pathway regulation by cell adhesion and suggest Src activation as an underlying reason for YAP deregulation in tumorigenesis. .