Fig. 6

PPFIA4 activates MTHFD2 via Src-mediated phosphorylation. A. Cell lysates from LNCaP cells with or without androgen deprivation were subjected to immunoprecipitation using anti-phospho-serine/threonine/tyrosine specific antibodies, followed by immunoblotting with indicated antibodies. p-Ser, phospho-serine. p-Thr, phospho-threonine. p-Tyr, phospho-tyrosine. IgG serves as negative control. GAPDH was used as a loading control. B. Cell lysates from LNCaP cells (left) transfected with empty vector or PPFIA4 overexpression plasmid and C4-2B (right) cells transfected with siNC or siPPFIA4 were subjected to immunoprecipitation with anti-phospho-tyrosine antibody, followed by immunoblotting with indicated antibodies. IgG serves as negative control. GAPDH was used as a loading control. C. Cell lysates from HEK293T cells transfected with flag-MTHFD2/WT, flag- MTHFD2/Y84A, flag-MTHFD2/Y170A, flag-MTHFD2/Y234A, or flag- MTHFD2/Y304A plasmids were subjected to immunoprecipitation using anti-phospho-tyrosine antibody, followed by immunoblotting with indicated antibodies. D-F. Measurement of NADPH/NADP⁺ (D), ROS levels (E) and cell proliferation (F) in C4-2B cells transfected with siNC or siPPFIA4 for 24 hours and subsequent flag-MTHFD2/WT or flag-MTHFD2/Y170A for another 48 hours. **p < 0.01, *** p < 0.001. d, days. NS, no significance. G. Tyrosine phosphorylation levels of MTHFD2 were measured by immunoprecipitation and immunoblotting in C4-2B cells treated with Src inhibitor PP2 (10 μM) for 2 hours (top) or transfected with Src siRNA (siSrc) for 48 hours (bottom). IgG serves as negative control. GAPDH was used as a loading control. H. The binding potential between MTHFD2 and Src were performed in the total, cytosolic and mitochondrial lysates by Co-IP assays in C4-2B cells. IgG serves as negative control. COXIV and tubulin were used as mitochondrial and cytosolic markers. I. The binding potential between MTHFD2 and Src were performed by Co-IP assays in C4-2B cells transfected with siNC or siPPFIA4. IgG serves as negative control. GAPDH was used as a loading control. J. Tyrosine phosphorylation levels of MTHFD2 were measured by immunoprecipitation and immunoblotting in C4-2B cells transfected with empty vector or PPFIA4 overexpression plasmid and then treated with PP2 (top) or transfected with siNC or siSrc (bottom). IgG serves as negative control. GAPDH was used as a loading control. K. A putative schematic diagram illustrating the role of PPFIA4 in contributing to CRPC. PPFIA4 proteins translocate mitochondria in response to androgen deprivation, activates MTHFD2 via Src-mediated phosphorylation, and consequently promotes the production of NADPH and reduces the excessive ROS generation, which could enhance mitochondrial activity and promote CRPC progression