SUMMARY Receptor tyrosine kinases (RTKs) and protein phosphatases comprise protein families that play crucial roles in cell signaling. We used two protein-protein interaction (PPI) approaches, the Membrane Yeast Two-Hybrid (MYTH) and the Mammalian Membrane Two-Hybrid (MaMTH), to map the PPIs between human RTKs and phosphatases. The resulting RTK-phosphatase interactome reveals a considerable number of previously unidentified interactions and suggests specific roles for different phosphatase families. Additionally, the differential PPIs of some protein tyrosine phosphatases (PTPs) and their mutants suggest diverse mechanisms of these PTPs in the regulation of RTK signaling. We further found that PTPRH and PTPRB directly dephosphorylate EGFR and repress its downstream signaling. By contrast, PTPRA plays a dual role in EGFR signaling: besides facilitating EGFR dephosphorylation, it enhances downstream ERK signaling by activating SRC. This comprehensive RTK-phosphatase interactome study provides a broad and deep view of RTK signaling.

Deregulation of the ubiquitin ligase E6AP is causally linked to the development of human disease, including cervical cancer. In complex with the E6 oncoprotein of human papillomaviruses, E6AP targets the tumor suppressor p53 for degradation, thereby contributing to carcinogenesis. Moreover, E6 acts as a potent activator of E6AP by a yet unknown mechanism. However, structural information explaining how the E6AP-E6-p53 enzyme-substrate complex is assembled, and how E6 stimulates E6AP, is largely missing. Here, we develop and apply different crosslinking mass spectrometry-based approaches to study the E6AP-E6-p53 interplay. We show that binding of E6 induces conformational rearrangements in E6AP, thereby positioning E6 and p53 in the immediate vicinity of the catalytic center of E6AP. Our data provide structural and functional insights into the dynamics of the full-length E6AP-E6-p53 enzyme-substrate complex, demonstrating how E6 can stimulate the ubiquitin ligase activity of E6AP while facilitating ubiquitin transfer from E6AP onto p53.

Diadenosine polyphosphates (Ap n As) such as diadenosine tri-and tetraphosphates are formed in prokaryotic as well as eukaryotic cells. Since upon stress intracellular Ap n A concentrations increase, it was postulated that Ap n As are alarmones triggering stress-adaptive processes. The major synthesis pathway of Ap n As is assumed to be a side reaction of amino acid activation. How this process is linked to stress adaptation remains enigmatic. The first step of one of the most prominent eukaryotic posttranslational modification systems-the conjugation of ubiquitin (Ub) and ubiquitin-like proteins (Ubl) to target proteins-involves the formation of an adenylate as intermediate. Like Ap n A formation, Ub and Ubl conjugation is significantly enhanced during stress conditions. Here, we demonstrate that diadenosine tri-and tetraphosphates are indeed synthesized during activation of Ub and Ubls. This links one of the most prevalent eukaryotic protein-modification systems to Ap n A formation for the first time.

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Simple andr obust assays to monitore nzymatic ATPc leavage with high efficiency in real-time are scarce.T o address this shortcoming, we developed fluorescently labelled adenosine tri-, tetra-and pentaphosphate analogues of ATP. The novel ATPa naloguesb ear -i nc ontrast to earlier reports -o nly as ingle acridone-based dye at the terminal phosphate group. The dye's fluorescencei sq uenched by the adeninec omponent of the ATPa nalogue and is restored upon cleavage of the phosphate chain and dissociation of the dye from the adenosine moiety.T hereby the activity of ATP-cleaving enzymes can be followed in real-time. We demonstrate this proficiency for ubiquitin activation by the ubiq-uitin-activating enzymesU BA1 and UBA6 which represents the first step in an enzymatic cascadel eading to the covalent attachment of ubiquitin to substrate proteins,aprocess that is highly conserved from yeast to humans. We found that the efficiency to serve as cofactor forU BA1/UBA6 very much dependso nt he length of the phosphate chain of the ATPa nalogue:t riphosphatesa re used poorly while pentaphosphates are most efficiently processed. Notably,t he novel pentaphosphate-harbouring ATPa nalogues upersedes the efficiency of recently reported dual-dye labelleda nalogues and thus, is ap romising candidate for broad applications.