Project Summary Oncogenic or tumor suppressive signaling often causes imbalances in posttranslational modifications (PTMs) in human cancer. The receptor tyrosine kinase HER2 is an oncogene amplified or overexpressed in approximately 25% of breast cancers and is associated with tumor aggressiveness and poor prognosis. A recent study demonstrated that HER2 is translocated to mitochondria in a kinase activity-dependent manner to alter metabolism and promote drug resistance in breast cancer cells. Consistent with these findings, our recent study showed that oncogenic fibroblast growth factor receptor (FGFR) 1, which is also localized in mitochondria, directly tyrosine phosphorylates mitochondrial pyruvate dehydrogenase kinase (PDHK) 1, enhancing its enzyme activity to promote tumor growth in mice. Taken together, these results strongly suggest that mitochondrial localization of oncogenic tyrosine kinases, including HER2, regulate mitochondrial metabolic pathways that contribute to tumor growth. However, which mitochondrial metabolic enzymes are regulated by HER2 in breast cancer is totally unknown. Thus, we performed phospho-proteomic analyses of mitochondria- enriched fractions from HER2+ BT474 breast cancer cells and found that mitochondrial carnitine palmitoyltransferase (CPT) 1A is tyrosine phosphorylated at Y514. Further biochemical analyses showed that CPT1A Y514 phosphorylation by HER2 promotes its carnitine palmitoyltransferase (CPTase) activity. Interestingly, we recently found that CPT1A has an additional enzymatic activity as a lysine succinyltransferase (LSTase) to succinylate lysine residues of substrate proteins in vitro and in vivo .To our knowledge, CPT1A is a first and only LSTase in mammalian cells. Importantly, mutation of CPT1A Gly710 (G710E) selectively inactivated CPTase activity but not LSTase activity. Similar to CPT1A WT, CPT1A G710E increased total lysine succinylation in cells without affecting intracellular succinyl-CoA levels. These findings suggest the unprecedented role of CPT1A as LSTase that can regulate lysine succinylation-dependent signal transduction in cells independent of its canonical CPTase activity that facilitates mitochondrial fatty acid oxidation (FAO) pathway. Notably, however, how the LSTase activity of CPT1A is regulated and whether LSTase activity is important for tumorigenesis is not known. Previous studies reported that multiple types of cancers including melanoma, prostate, breast, and ovarian cancers showed elevated expression of CPT1A and knockdown of CPT1A with si/shRNAs decreased cancer cell proliferation. We also found that high CPT1A mRNA expression in HER2+ breast cancer patients strongly correlates with reduced survival while no correlation between CPT1A mRNA expression and reduced patient survival was observed in other types of breast tumors. In addition, we found that knockdown of CPT1A, as well as inhibition of HER2 by lapatinib, decreased CPT1A-subsrate motif LVxxK succinylation and supp...