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).…”

“…The N-terminal domain of TTP also interacts with PKM2 hence triggering p38 MAPK -MK2 mediated phosphorylation, ubiquitination of TTP, and reduction of target mRNA turnover ability (64). In the late phase (3-16 h) of the pro-inflammatory stimulus-induction, HECT, UBA, and HUWE1 promotes the interaction between TTP C-terminal domain (aa 234-259) and protein phosphatase PP1 and PP2 and inhibits p38 MAPK -MK2/JNK/ERK activities, therefore resulting in dephosphorylation of TTP (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 (65). (B) MAMPs such as flg22 activates MAPK signaling cascade to induce plant defense response.…”

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-RNAbinding 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 singlecell-derived clones.…”

“…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.…”