The challenge of rapid macromolecular synthesis enforces the energy-hungry cancer cell mitochondria to switch their metabolic phenotypes, accomplished by activation of oncogenic tyrosine kinases. Precisely how kinase activity is directly exploited by cancer cell mitochondria to meet high-energy demand, remains to be deciphered. Here we show that a non-receptor tyrosine kinase, TNK2/ACK1 (tyrosine kinase non receptor 2), phosphorylated ATP5F1A (ATP synthase F1 subunit alpha) at Tyr243 and Tyr246 (Tyr200 and 203 in the mature protein, respectively) that not only increased the stability of complex V, but also increased mitochondrial energy output in cancer cells. Further, phospho-ATP5F1A (p-Y-ATP5F1A) prevented its binding to its physiological inhibitor, ATP5IF1 (ATP synthase inhibitory factor subunit 1), causing sustained mitochondrial activity to promote cancer cell growth. TNK2 inhibitor, (R)-9b reversed this process and induced mitophagy-based autophagy to mitigate prostate tumor growth while sparing normal prostate cells. Further, depletion of p-Y-ATP5F1A was needed for (R)-9b-mediated mitophagic response and tumor growth. Moreover, Tnk2 transgenic mice displayed increased p-Y-ATP5F1A and loss of mitophagy and exhibited formation of prostatic intraepithelial neoplasia (PINs). Consistent with these data, a marked increase in p-Y-ATP5F1A was seen as prostate cancer progressed to the malignant stage. Overall, this study uncovered the molecular intricacy of tyrosine kinase-mediated mitochondrial energy regulation as a distinct cancer cell mitochondrial vulnerability and provided evidence that TNK2 inhibitors can act as "mitocans" to induce cancer-specific mitophagy.Abbreviations: ATP5F1A: ATP synthase F1 subunit alpha; ATP5IF1: ATP synthase inhibitory factor subunit 1; CRPC: castration-resistant prostate cancer; DNM1L: dynamin 1 like; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; Mdivi-1: mitochondrial division inhibitor 1; Mut-ATP5F1A: Y243,246A mutant of ATP5F1A; OXPHOS: oxidative phosphorylation; PC: prostate cancer; PINK1: PTEN induced kinase 1; p-Y-ATP5F1A: phosphorylated tyrosine 243 and 246 on ATP5F1A; TNK2/ACK1: tyrosine kinase non receptor 2; Ub: ubiquitin; WT: wild type.

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