Structure/Function Analysis of Tristetraprolin (TTP): p38 Stress-Activated Protein Kinase and Lipopolysaccharide Stimulation Do Not Alter TTP Function

Abstract

Tristetraprolin (TTP) is the only trans-acting factor shown to be capable of regulating AU-rich element-dependent mRNA turnover at the level of the intact animal; however, the mechanism by which TTP mediated RNA instability is unknown. Using an established model system, we performed structure/function analysis with TTP as well as examined the current hypothesis that TTP function is regulated by p38-MAPKAP kinase 2 (MK2) activation. Deletion of either the N- or C-terminal domains inhibited TTP function. Extensive mutagenesis, up to 16%, of serines and threonines, some of which were predicted to mediate proteasomal targeting, did not alter human TTP function. Mutation of the conserved MK2 phosphorylation sites enhanced human TTP function in both resting and p38-stress-activated protein kinase-MK2-activated cells. However, p38-stress-activated protein kinase-MK2 activation did not alter the activity of either wild-type or mutant TTP. TTP localized to the stress granules, with arsenite treatment reducing this localization. In contrast, arsenite treatment enhanced stress granule localization of the MK2 mutant, consistent with the involvement of additional pathways regulating this event. Finally, we determined that, in response to LPS stimulation, human TTP moves onto the polysomes, and this movement occurs in the absence of 14-3-3. Taken together, these data indicate that, although p38 activation alters TTP entry into the stress granule, it does not alter TTP function. Moreover, the interaction of TTP with 14-3-3, which may limit entry into the stress granule, is not involved in the downstream message stabilization events.

Cullin 4B Is Recruited to Tristetraprolin-Containing Messenger Ribonucleoproteins and Regulates TNF-α mRNA Polysome Loading

“…LPS stimulation (100 ng/ml, 90 min) did not alter pGL3-Control luciferase expression in either of the cell lines, consistent with previous findings with pGL3-Control luciferase expression (19,30). Luciferase expression from the pGL3-TNF-39 UTR vector is significantly lower than expression from the pGL3-Control vector, due to the presence of the TNF-a 39 UTR, consistent with our previous findings (30,32). Importantly, TNF-39 UTR luciferase expression is only 5% higher in the Cul4B shRNA cells compared with the control shRNA cells (Fig.…”

“…5A) (30-32). Use of the TTP-AA mutant (S52A, S178A) (18), which cannot be inhibited by p38/MK2 phosphorylation, results in a further reduction in TNF-39 UTR luciferase expression (51% decrease) that is significantly different from wild-type TTP (p , 0.01), consistent with previous reports (32). Use of a TTP zinc finger mutant (TTP-M1,2) (34), which cannot bind RNA due to mutations in both zinc finger domains, abrogates the effect of TTP transfection.…”

“…The NT condition is transfected with an equal amount of pcDNA 3.1 HisC, the backbone vector for the TTP expression construct. Consistent with previous results (19,(30)(31)(32), transfection of TTP into control shRNA cells results in an ∼25% decrease in TNF-39 UTR luciferase expression in resting cells (p , 0.01) (Fig. 5A) (30-32).…”

“…These studies employ pGL3-Control and TNF-39 UTR luciferase reporters, which differ only in the presence of the TNF-a 39 UTR, as well as several TTP expression constructs (5,19,(30)(31)(32)(33). Our hypothesis for these experiments was that TNF-a luciferase expression would be lower in the Cul4B shRNA cells compared with the control shRNA cells.…”

“…We have previously established that TTP localizes to polysome and nonpolysome cellular fractions (32). To establish the distribution of Cul4B, we performed sucrose density fractionation (5-45%) of resting and LPS-activated RAW264.7 cell cytoplasm to determine the localization of Cul4B within the gradient and whether localization is altered by LPS stimulation (Fig.…”