Sproutys have been shown to negatively regulate growth factor-induced extracellular signal-regulated kinase (ERK) activation, and suggested to be an anti-oncogene. However, molecular mechanism of the suppression has not yet been clarified completely. Sprouty4 inhibits vascular endothelial growth factor (VEGF)-A-induced ERK activation, but not VEGF-C-induced ERK activation. It has been shown that VEGF-A-mediated ERK activation is strongly dependent on protein kinase C (PKC), whereas that by VEGF-C is dependent on Ras. This suggests that Sprouty4 inhibits the PKC pathway more specifically than the Ras pathway. In this study, we confirmed that Sprouty4 suppressed various signals downstream of PKC, such as phosphorylation of MARCKS and protein kinase D (PKD), as well as PKC-dependent nuclear factor (NF)-kappaB activation. Furthermore, Sprouty4 suppressed upstream signals of PKC, such as Ca(2+) mobilization, phosphatidylinositol 4,5-biphosphate (PIP(2)) breakdown and inositol 1,4,5-triphosphate (IP(3)) production in response to VEGF-A. Those effects were dependent on the C-terminal cysteine-rich region, but not on the N-terminal region of Sprouty4, which is critical for the suppression of fibroblast growth factor (FGF)-mediated ERK activation. Sprouty4 overexpression or deletion of the Sprouty4 gene did not affect phospholipase C (PLC) gamma-1 activation, which is an enzyme that catalyzes PIP(2) hydrolysis. Moreover, Sprouty4 inhibited not only VEGF-A-mediated PIP(2) hydrolysis but also inhibited the lysophosphatidic acid (LPA)-induced PIP(2) breakdown that is catalyzed by PLC beta/epsilon activated by G-protein coupled receptor (GPCR). Taken together, Sprouty4 has broader suppression activity for various stimuli than previously thought; it may function as an inhibitor for various types of PLC-dependent signaling as well as for ERK activation.