p300 Modulates ATF4 Stability and Transcriptional Activity Independently of Its Acetyltransferase Domain

Abstract

ATF4 plays a crucial role in the cellular response to stress and multiple stress responses pathways converge to the translational up-regulation of ATF4. ATF4 is a substrate of the SCF ␤TrCP ubiquitin ligase that binds to ␤TrCP through phosphorylation on a DSGXXXS motif. We show here that ATF4 stability is also modulated by the histone acetyltransferase p300, which induces ATF4 stabilization by inhibiting its ubiquitination. Despite p300 acetylates ATF4, we found that p300-mediated ATF4 stabilization is independent of p300 catalytic activity, using either the inactive form of p300 or the acetylation mutant ATF4-K311R. ATF4 deleted of its p300 binding domain is no more stabilized by p300 nor recruited into nuclear speckles. In consequence of ATF4 stabilization, both p300 and the catalytically inactive enzyme increase ATF4 transcriptional activity.ATF4, a member of the ATF/CREB bZIP transcription factor family, plays a crucial role in response to stress, because multiple intracellular stress pathways (endoplasmic reticulum stress, amino acid deprivation, and exposure to oxidant or reactive metals) converge on a single event, phosphorylation of eIF2␣, which leads both to a general inhibition of protein synthesis but also to the translational up-regulation of the mRNA encoding ATF4 (1-4). Thus the targets of ATF4 are of paramount importance in a generalized stress response. In fact, higher eucaryotes have conserved through activation of ATF4 the same fundamental mechanism used by yeast through up-regulation of GCN4 in response to amino acid starvation (5). In addition, ATF4 is important for cell proliferation and differentiation, because ATF4 knock-out mice display abnormal lens formation (6) and defects in cell proliferation in fetal liver, embryonic lens and hair follicles, as well as an overall reduction in size of the animals (7). ATF4 is a critical regulator of osteoblast differentiation and function (8) and bone resorption (9). ATF4 is also involved in long term memory induction (10). Hence ATF4 is a master transcription factor for which temporal expression and activity are under tight cellular control. ATF4 interacts with several general transcription factors such as TBP, TFIIB, and RAP30 (11). The transcriptional selectivity of ATF4 is modulated by the formation of heterodimers with multiple C/EBP bZIP or AP-1 family members (12-14). CBP and p300 acetylate ATF4 in its bZIP domain (15) and enhance its transcriptional activity (11,15).Acetylation of histone and non-histone proteins is emerging as a central process in transcriptional activation. Nuclear histone acetyltransferases (HATs) 5 act as transcriptional co-activators that have been shown to acetylate different transcription factors, including p53, ␤cate-nin, MyoD, E2F-1, ATF4, and SREBP1 (15-19). The consequences of acetylation on protein function vary from one protein to another depending on where within the protein the acetylation takes place. Acetylation has also been reported to modulate protein-protein interactions, to inhibit nuclear export (20), a...

ATF4, A Novel Mediator of the Anabolic Actions of PTH on Bone

“…As shown in Fig. 8A, in the absence of TFIIA␥ overexpression, ATF4 protein was rapidly degraded and almost undetectable on Western blot by 3 h after CHX addition, which is consistent with a previous study (51). However, overexpression of TFIIA␥ greatly delayed the degradation process with the levels of ATF4 protein only slightly reduced by 3 h after CHX addition.…”

“…Taken together, these results indicate that PKA is the major pathway mediating PTH activation of ATF4 in osteoblasts with PKC and MAPK/ERK pathways playing lesser roles in the PTH response. The concentrations of the inhibitors or activators used in this study are in the range reported to selectively affect the relevant pathways (33,(51)(52)(53). We found no evidence of toxicity; compounds did not reduce cell DNA or protein under the current condition (data not shown).…”

“…to completely block de novo protein synthesis) and harvested at different time points of CHX addition (0, 0.5, 1, and 3 h) followed by Western blot analysis for ATF4 and Runx2. This technique has been widely used to study protein stability (51). As shown in Fig.…”

“…TFIIA␥ Increases ATF4 Protein Stability-Lassot et al (51) recently showed that acetylase p300 markedly increased the levels of ATF4 protein and ATF4-dependent transcriptional activity by inhibiting ATF4 protein degradation via a proteasomal ubiquitin pathway. As an initial step to determine whether TFIIA␥ alters ATF4 protein stability, C3H10T1/2 cells were transiently transfected with ATF4 expression vector in the presence of ␤-galactosidase, TFIIA␥, or Runx2 expression vectors.…”

Role of ATF4 in regulation of autophagy and resistance to drugs and hypoxia

“…Phosphorylationdependent degradation of ATF4 is tightly modulated by the interactions with an E3 ubiquitin ligase-the SCFβTrCP (Skp1/ Cullin/F-box protein) complexes; however, the kinase(s) regulating stability of ATF4 has not been identified. 36,37 Further evidence that ATF4 is degraded by the UPS is provided by studies using inhibitors of the proteasome.…”