Adenylyl cyclases (ACs) generate cAMP, a second messenger of utmost importance that regulates a vast array of biological processes in all kingdoms of life. However, almost nothing is known about how AC activity is regulated through protein degradation mediated by ubiquitination or other mechanisms. Here, we show that transcriptional regulator interacting with the PHD-bromodomain 1(TRIP-Br1, Sertad1), a newly identified protein with poorly characterized functions, acts as an adaptor that bridges the interaction of multiple AC isoforms with X-linked inhibitor of apoptosis protein (XIAP), a RING-domain E3 ubiquitin ligase. XIAP ubiquitinates a highly conserved Lys residue in AC isoforms and thereby accelerates the endocytosis and degradation of multiple AC isoforms in human cell lines. And XIAP/TRIP-Br1-mediated degradation of ACs forms part of a negative-feedback loop that controls the homeostasis of cAMP signaling in mice. Our findings reveal a previously unrecognized mechanism for degrading multiple AC isoforms and modulating the homeostasis of cAMP signaling.
- Pingbo Huang
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Animal experimentation: All animal procedures were approved by the University Committee on Research Practices at the Hong Kong University of Science and Technology (the ethics protocol number 2014028).
- Volker Dötsch, J.W. Goethe-University, Germany
© 2017, Hu et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
TMEM16F, a Ca2+-activated phospholipid scramblase (CaPLSase), is critical for placental trophoblast syncytialization, HIV infection, and SARS-CoV2-mediated syncytialization, however, how TMEM16F is activated during cell fusion is unclear. Here, using trophoblasts as a model for cell fusion, we demonstrate that Ca2+ influx through the Ca2+ permeable transient receptor potential vanilloid channel TRPV4 is critical for TMEM16F activation and plays a role in subsequent human trophoblast fusion. GSK1016790A, a TRPV4 specific agonist, robustly activates TMEM16F in trophoblasts. We also show that TRPV4 and TMEM16F are functionally coupled within Ca2+ microdomains in a human trophoblast cell line using patch-clamp electrophysiology. Pharmacological inhibition or gene silencing of TRPV4 hinders TMEM16F activation and subsequent trophoblast syncytialization. Our study uncovers the functional expression of TRPV4 and one of the physiological activation mechanisms of TMEM16F in human trophoblasts, thus providing us with novel strategies to regulate CaPLSase activity as a critical checkpoint of physiologically and disease-relevant cell fusion events.
Centrosomes act as the main microtubule organizing center (MTOC) in metazoans. Centrosome number is tightly regulated by limiting centriole duplication to a single round per cell cycle. This control is achieved by multiple mechanisms, including the regulation of the protein kinase PLK4, the most upstream facilitator of centriole duplication. Altered centrosome numbers in mouse and human cells cause p53-dependent growth arrest through poorly defined mechanisms. Recent work has shown that the E3 ligase TRIM37 is required for cell cycle arrest in acentrosomal cells. To gain additional insights into this process, we undertook a series of genome-wide CRISPR/Cas9 screens to identify factors important for growth arrest triggered by treatment with centrinone B, a selective PLK4 inhibitor. We found that TRIM37 is a key mediator of growth arrest after partial or full PLK4 inhibition. Interestingly, PLK4 cellular mobility decreased in a dose-dependent manner after centrinone B treatment. In contrast to recent work, we found that growth arrest after PLK4 inhibition correlated better with PLK4 activity than with mitotic length or centrosome number. These data provide insights into the global response to changes in centrosome number and PLK4 activity and extend the role for TRIM37 in regulating the abundance, localization, and function of centrosome proteins.