Abstract Purpose: Recent evidence suggests that YAP and its paralog TAZ play key roles in tumoriogenesis, tumor progression, and chemotherapeutic resistance. Although verteporfin is known to inhibit YAP by suppressing its interaction with the transcription factor TEAD, it has low efficacy and adverse effects including photosensitivity. Therefore, identification of other molecular targets is essential for developing anticancer drugs to treat YAP-driven cancers. Method: Image-based whole-genome siRNA library screening was performed to identify genes that regulate the abundance of nuclear YAP protein. LATS1/2-null RPE1 cells were used to exclude the influence of cell density that controls the Hippo pathway. Several kinases were identified as positive regulators of nuclear YAP/TAZ intensity. Among them, a member of the MAP kinase family was selected for further study to investigate its YAP-regulating mechanism and its role in BRAF inhibitor-resistant cutaneous melanoma, which has high YAP/TAZ activity. Direct phosphorylation of YAP by the MAP kinase was detected by in vitro kinase assay and LC-MS/MS. Roles of the kinase in BRAF inhibitor-resistant melanoma cells were evaluated by CCK assay, scratch assay, tumor xenograft, and apoptosis analysis. Results: We found that RNAi-mediated inhibition of the MAP kinase decreases total YAP/TAZ protein levels and their transcriptional activity. The MAP kinase physically interacts with and phosphorylates YAP. This phosphorylation influences ubiquitination and lysosomal degradation of YAP. Knockdown of the MAP kinases decreases proliferation, migration, and tumor formation of BRAF inhibitor-resistant melanoma cells. In addition, inhibition of the kinase reverses BRAF inhibitor resistance. Conclusion: These results suggest that the MAP kinase is a novel positive regulator of YAP acting independently of the Hippo pathway. We propose that inhibition of the kinase is an efficient way of suppressing YAP/TAZ and the kinase can serve as a therapeutic target for YAP/TAZ-driven cancers. Citation Format: Sanghyun Park, Joon Kim. Identification of a MAP kinase that regulates YAP abundance [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr A34.