Inactivation of the PtdIns(4)P phosphatase Sac1 at the Golgi by H2O2 produced via Ca2+-dependent Duox in EGF-stimulated cells

Abstract

Binding of epidermal growth factor (EGF) to its cell surface receptor induces production of H₂O₂, which serves as an intracellular messenger. We have shown that exogenous H₂O₂ reversibly inactivates the phosphatidylinositol 4-phosphate [PtdIns(4)P] phosphatase Sac1 (suppressor of actin 1) at the Golgi complex of mammalian cells by oxidizing its catalytic cysteine residue and thereby increases both the amount of Golgi PtdIns(4)P and the rate of protein secretion. Here we investigated the effects of EGF on Sac1 oxidation and PtdIns(4)P abundance at the Golgi in A431 cells. EGF induced a transient increase in Golgi PtdIns(4)P as well as a transient oxidation of Sac1 in a manner dependent on elevation of the intracellular Ca²⁺ concentration and on H₂O₂. Oxidation of Sac1 occurred at the Golgi, as revealed with the use of the Golgi-confined Sac1-K2A mutant. Knockdown of Duox enzymes implicated these Ca²⁺-dependent members of the NADPH oxidase family as the major source of H₂O₂ for Sac1 oxidation. Expression of a Golgi-targeted H₂O₂ probe revealed transient EGF-induced H₂O₂ production at this organelle. Our findings have thus uncovered a previously unrecognized EGF signaling pathway that links intracellular Ca²⁺ mobilization to events at the Golgi including Duox activation, H₂O₂ production, Sac1 oxidation, and PtdIns(4)P accumulation.

Keywords: Cysteine oxidation; Epidermal growth factor (EGF); Golgi complex; Hydrogen peroxide; NADPH oxidase; Phosphatidylinositol 4-phosphate; Protein tyrosine phosphatase; Sac1.