A hotspot phosphorylation site on SHP2 drives oncoprotein activation and drug resistance

SHP2, a protein phosphatase encoded by PTPN11 , is a critical mediator of receptor tyrosine kinase (RTK)-driven RAS/mitogen-activated protein kinase (MAPK) signaling and is targeted by allosteric inhibitors that stabilize its autoinhibited conformation. Despite promising preclinical data, SHP2 inhibitors have shown minimal clinical efficacy (monotherapy response rate ~0%), with no defined mechanisms of primary resistance in patients. Here, we elucidate phosphorylation of SHP2 at tyrosine 62 (pY62) as a hotspot phosphorylation site enriched across normal cells and tissues, cancer cells, and varied RTK-driven tumor types in patients. We demonstrate that SRC family kinases (SFKs) directly phosphorylate SHP2 at Y62, downstream but not directly phosphorylated by RTKs. Using biochemical and biophysical analyses, we show that SHP2 pY62 enforces an open, active conformation, resulting in constitutive phosphatase activation that is sufficient to activate MAPK signaling and confer resistance to allosteric SHP2 inhibitors. These findings establish SHP2 pY62 as a phosphorylation hotspot phenocopying mutational activation, as a mechanism of primary resistance to SHP2 inhibitors, and as a cancer drug target distinct from wildtype SHP2.