Project Summary Many important biological processes are regulated by protein-tyrosyl phosphorylation. Tyrosyl phosphorylation, in turn, is controlled by protein-tyrosine kinases (PTKs) and protein-tyrosine phosphatases (PTPs). Abnormal regulation of these pathways can lead to developmental defects and diseases such as cancer. A complete understanding of cellular regulation by tyrosyl phosphorylation requires defining the PTKs and PTPs involved and determining how they interact. Such understanding may lead to the development of new drugs that selectively target elements of these signaling pathways, agents that may be useful for the treatment of human disease. The goal of our research program has been to delineate the mechanism of action of non-transmembrane PTPs. During this MERIT award, we made major progress in understanding the normal function and role in disease of SHP2, SHP1, and PTP1B. Over the past year, we discovered a new pathway involving PTP1B, and its putative substrate, the Moyamoya disease-associated AAA+ ATPase/E3 ligase RNF213, in the control of α-ketoglutarate-dependent dioxygenase (aKGDD) activity, non-mitochondrial O2 consumption (NMOC) and survival of HER2+ BC cells and at least other BC subtypes under hypoxic conditions. Preliminary data suggest a model in which tyrosyl phosphorylation activates RNF213, which has K6 E3-ubiquitin ligase activity, and may regulate cystine uptake, intracellular redox tone, ascorbate levels and global αKGDD activity. We propose to elucidate the molecular details of this pathway, and its relevance to HER2 tumorigenesis and to other types of BC. We will ask how PTP1B regulates RNF213, how RNF213 controls αKGDDs/NMOC, and how generally this pathway regulates hypoxia and tumorigenesis. Our results will provide new insights into the hypoxia response, regulation of ubiquitylation by K6 ligases, control of the αKGDDs, the possible therapeutic effects of PTP1B inhibitors and ascorbate, and Moyamoya disease pathogenesis.