Abstract MAP kinase kinase kinase kinases (MAP4Ks) belong to the mammalian Ste20-like family of serine/threonine kinases and recently have been reported to regulate Hippo pathway via LATS1/2 activation. However, the diverse roles of MAP4Ks in biologic process are still unclear. To identify the MAP4K family kinase interaction network, we employed a tandem affinity purification-mass spectrometry (TAP-MS) approach using SFB tag-conjugated MAP4K1-7. Through a proteomic analysis, we could guess that MAP4K family kinases, especially MAP4K2, may regulate autophagy or interact with striatin-interacting phosphatase and kinase (STRIPAK) components. These results offer a framework for further studies on Hippo pathway and biologic processes involving MAP4Ks. Note: This abstract was not presented at the conference. Citation Format: Gayoung Seo, Han Han, Bing Yang, Rebecca Vargas, Yuxuan Chen, Wenqi Wang. Proteomic profiling of tandem affinity purified MAP4K family kinases [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 B21.