OBJECTIVE: To determine the molecular mechanism underlying the synergistic response of mitogen-activated protein kinase phosphatase-1 (MKP-1), which is induced by thrombin and epidermal growth factor (EGF). METHODS AND RESULTS: MKP-1 induction by thrombin (approximately 6-fold) was synergistically increased (approximately 18-fold) by cotreatment with EGF in cultured endothelial cells. EGF alone did not induce MKP-1 substantially (<2-fold). The synergistic induction of MKP-1 was not mediated by matrix metalloproteinases. The EGF receptor kinase inhibitor AG1478 blocked approximately 70% of MKP-1 induction by thrombin plus EGF (from 18- to 6-fold) but not the response to thrombin alone. An extracellular signal-regulated kinase (ERK)-dependent protease-activated receptor-1 (PAR-1) signal was required for the thrombin alone effect; an ERK-independent PAR-1 signal was necessary for the approximately 12-fold MKP-1 induction by thrombin plus EGF. VEGF induction of MKP-1 was also approximately 12-fold and c-Jun N-terminal kinase (JNK) dependent. Inhibitors of extracellular signal-regulated kinase and JNK activation blocked thrombin plus EGF-induced MKP-1 completely. Furthermore, VEGF receptor 2 depletion blocked the synergistic response without affecting the induction of MKP-1 by thrombin alone. CONCLUSIONS: We have identified a novel signaling interaction between protease-activated receptor-1 and EGF receptor that is mediated by VEGF receptor 2 and results in synergistic MKP-1 induction.