Integrated Interactome and Phosphoproteome Analysis Reveals Novel MAP4K3 ‐Regulated Pathways and Unexpected Cellular Roles for MAP4K3

ABSTRACT MAP4K3, also known as germinal‐center kinase‐like kinase (GLK), is a member of the Ste20 sub‐family of MAPKs. Numerous studies have shown that MAP4K3 is required for mTORC1 activation in response to amino acids, and MAP4K3 represses autophagy by initiating inhibitory suppression of transcription factor EB. Furthermore, MAP4K3 is ubiquitously expressed; thus, MAP4K3 likely plays a central role in regulating the metabolic disposition of the cell. To define the basis for MAP4K3 regulation of these cellular pathways and to identify novel cellular processes subject to MAP4K3 regulation, we performed mass spectrometry interactome analysis of MAP4K3 and unbiased phosphoproteomics to define the MAP4K3 phosphoproteome landscape. MAP4K3 interactome and phosphoproteome analysis confirmed the existence of numerous MAP4K3 interactors and substrates involved in mTORC1 regulation, while suggesting a potential role for MAP4K3 in controlling the subcellular localization of mTORC1 via phosphorylation of Mios, a component of the GATOR2 complex. In addition to linking MAP4K3 to processes occurring at the lysosome, MAP4K3 interactome and phosphoproteome data revealed an unexpected role for MAP4K3 in the nucleus, implicating MAP4K3 in DNA damage response and repair. When we examined MAP4K3 subcellular localization, we confirmed that MAP4K3 is present in the nucleus, and found that MAP4K3 interacts with the DNA damage response regulator PARP1. Our unbiased interactome and phosphoproteome analysis of MAP4K3 provides a powerful resource for further study of MAP4K3 function in the mTORC1 pathway, but also in the regulation of DNA damage response and repair pathways in the nucleus.