HUWE1 controls tristetraprolin proteasomal degradation by regulating its phosphorylation

Abstract

Tristetraprolin (TTP) is a critical negative immune regulator. It binds AU-rich elements in the untranslated-regions of many mRNAs encoding pro-inflammatory mediators, thereby accelerating their decay. A key but poorly understood mechanism of TTP regulation is its timely proteolytic removal: TTP is degraded by the proteasome through yet unidentified phosphorylation-controlled drivers. In this study, we set out to identify factors controlling TTP stability. Cellular assays showed that TTP is strongly lysine-ubiquitinated, which is required for its turnover. A genetic screen identified the ubiquitin E3 ligase HUWE1 as a strong regulator of TTP proteasomal degradation, which we found to control TTP stability indirectly by regulating its phosphorylation. Pharmacological assessment of multiple kinases revealed that HUWE1-regulated TTP phosphorylation and stability was independent of the previously characterized effects of MAPK-mediated S52/S178 phosphorylation. HUWE1 function was dependent on phosphatase and E3 ligase binding sites identified in the TTP C-terminus. Our findings indicate that while phosphorylation of S52/S178 is critical for TTP stabilization at earlier times after pro-inflammatory stimulation, phosphorylation of the TTP C-terminus controls its stability at later stages.

Modulation of stress granule dynamics by phosphorylation and ubiquitination in plants

“…A recent genetic analysis revealed that HUWE1 promotes the interaction between TTP C-terminal domain (aa 234-259) and protein phosphatases PP1 and PP2 and inhibits p38 MAPK -MK2/JNK/ERK activities, therefore resulting in dephosphorylation of TTP (within aa 259-279) and leading to an activation of an unknow E3 ligase to deposit K48 ubiquitin chains onto the TZF motif to destabilize TTP protein. This pathway represents the late phase (3-16 h) of the pro-inflammatory stimulus-induced response (65).…”

Guardian ubiquitin E3 ligases target cancer-associated APOBEC3 deaminases for degradation to promote human genome integrity

“…sgRNA CDSs were cloned in pLentiv2-U6-PGK- iRFP670-P2A-Neo 43 to perform knock-outs in RKO cell lines. The Dual-A3H-reporter (pLX303- SFFV-MYC-mCherry-A3H-II-P2A-OLLAS-EGFP-A3H-I) was constructed by cloning the ORF of human A3H-I or A3H-II into a modified pLX303 vector 45,118 . cDNAs encoding A3H-I, A3H-II, A3H-I-G105R, A3H-II-R105-G, A3H-II-W115A, A3H-II-R175/176E, A3H-II-E56A-W115A- R175/176E, A3H-I-K-mutants, A3A, A3B, A3B-E255A, A3C, A3D, A3F, A3G, A3G-RNA-binding mutants were synthesized by Twist Bioscience, or generated by fusion PCR and cloned into a modified pLX303 vector for mammalian expression, or into a modified pET47 containing a decahistidine (10×His) tag followed by a Maltose binding protein (MBP) tag and a 3C site vector for bacterial expression.…”

“…THP-1-DOX-Cas9-P2A-GFP cells were generated by transducing THP-1 cells with the pRRL- TRE3G-Cas9-P2A-GFP-PGK-IRES-rtTA3 lentiviral vector 45 . Cas9 expression was induced with 500 ng/ml of Docycycline hyclate (DOX, Sigma-Aldrich) and single cells were sorted by FACS into 96-well plates using a FACSAria III cell sorter (BD Biosciences) to obtain single- cell-derived clones.…”

“…Primers for qPCR are listed in the Key resource table. TurboID A3H-I and A3H-II, as well as an EGFP control were cloned into lentiviral plasmid pCW-MYC-TurboID-MCS-PGK-mCherry-P2A-rtTA 45 , for the expression of fusion proteins N-terminally tagged with TurboID. For the TurboID experiment, polyclonal cell lines stably expressing DOXinducible TurboID-A3H-I, TurboID-A3H-II and TurboID-EGFP were stimulated with DOX for 48 h. to induce the expression of the TurboID fusion constructs.…”

“…To identify specific protein stability regulators of A3H-I, but not A3H-II, we set up a CRISPR-based genetic screening platform [43][44][45] . First, an RKO cell line was established harboring a doxycycline (DOX)-inducible Cas9-P2A-BFP construct, and an mCherry-A3H-II-P2A-GFP-A3H-I dual reporter (Dual-A3H-reporter), driven from an exogenous promoter (Fig.…”