Abstract Background: Tumor growth, especially in the late stage, requires adequate nutrients and rich vasculature, in which PKM2 plays a convergent role. It has been reported that PKM2, together with FOXM1D, is upregulated in late stage colorectal cancer and associated with metastasis; however, their underlying mechanism for promoting tumor progression remains elusive. Methods: Western blot, coimmunoprecipitation (Co-IP), glutathione S-transferase (GST)-pull down and Immunofluorescence (IF) were performed to investigate the interaction between FOXM1D with PKM2 and other proteins. Extracellular acidification (ECAR) and oxygen consumption rate (OCR) assays, Protein Cross-linking, Gel chromatography, Pyruvate Kinase (PK) activity assay were used to examine the effect of FOXM1D on glycolysis. The angiogenesis effect was verified by Human umbilical vein endothelial cell (HUVEC) tube formation assay, tumor tissue microangiography, VEGFA Elisa assay and ChIP assay. Results: FOXM1D potentiates PKM2-mediated glycolysis and angiogenesis through multiple protein-protein interactions. In the presence of FBP, FOXM1D binds to tetrameric PKM2 and assembles a heterooctamer, restraining PKM2 metabolic activity by about a half and thereby promoting aerobic glycolysis. Furthermore, FOXM1D interacts with PKM2 and NF-κB and induces their nuclear translocation with the assistance of the nuclear transporter importin 4, which subsequently augments VEGFA transcription that is regulated by the PKM2 and NF-κB complex. The increased VEGFA is secreted extracellularly via exosome, which is potentiated by the interaction of FOXM1 with VPS11, eventually promoting tumor angiogenesis. Conclusion: Our study indicates that FOXM1D plays a crucial role in promoting glycolysis and angiogenesis through its protein-protein interaction. The above findings provide another insight into the role of PKM2 in the regulation of glycolysis and angiogenesis.
Significance Ykt6 is a conserved SNARE that plays critical roles along multiple vesicular pathways. To achieve its function, Ykt6 cycles between the cytosol and membrane-bound compartments through reversible lipidation. The mechanism that regulates these transitions is unknown. Ykt6 function is disrupted by α-synuclein, a protein critically implicated in synucleinopathies such as Parkinson’s Disease. Through a multidisciplinary approach, we report that phosphorylation regulated by Ca 2+ signaling drives a conformational change that allows Ykt6 to switch from a closed cytosolic to an open membrane-bound form. Phosphorylation is also a critical determinant for Ykt6 protein interactions with functional consequences in the secretory and autophagy pathways under normal and α-synuclein conditions. This work provides a mechanistic insight into Ykt6 regulation with therapeutic implications for synucleinopathies.
Using nuclear magnetic resonance spectroscopy, we establish that the N-terminal domain of the yeast vacuolar R-SNARE Nyv1p adopts a longin-like fold similar to those of Sec22b and Ykt6p. Nyv1p is sorted to the limiting membrane of the vacuole via the adaptor protein (AP)3 adaptin pathway, and we show that its longin domain is sufficient to direct transport to this location. In contrast, we found that the longin domains of Sec22p and Ykt6p were not sufficient to direct their localization. A YXXΦ-like adaptin-dependent sorting signal (Y 31 GTI 34 ) unique to the longin domain of Nyv1p mediates interactions with the AP3 complex in vivo and in vitro. We show that amino acid substitutions to Y 31 GTI 34 (Y31Q;I34Q) resulted in mislocalization of Nyv1p as well as reduced binding of the mutant protein to the AP3 complex. Although the sorting of Nyv1p to the limiting membrane of the vacuole is dependent upon the Y 31 GTI 34 motif, and Y31 in particular, our findings with structure-based amino acid substitutions in the mu chain (Apm3p) of yeast AP3 suggest a mechanistically distinct role for this subunit in the recognition of YXXΦ-like sorting signals.
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