CREB‑H, an endoplasmic reticulum-anchored transcription factor, plays a key role in regulating secretion and in metabolic and inflammatory pathways, but how its activity is modulated remains unclear. We examined processing of the nuclear active form and identified a motif around S87-S90 with homology to DSG-type phosphodegrons. We show that this region is subject to multiple phosphorylations, which regulate CREB-H stability by targeting it to the SCF(Fbw1a) E3 ubiquitin ligase. Data from phosphatase treatment, use of phosophospecific antibody, and substitution of serine residues demonstrate phosphorylation of candidate serines in the region, with the core S87/S90 motif representing a critical determinant promoting proteasome-mediated degradation. Candidate kinases CKII and GSK-3b phosphorylate CREB-H in vitro with specificities for different serines. Prior phosphorylation with GSK-3 at one or more of the adjacent serines substantially increases S87/S90-dependent phosphorylation by CKII. In vivo expression of a dominant-negative Cul1 enhances steady-state levels of CREB‑H, an effect augmented by Fbw1a. CREB-H directly interacts with Fbw1a in a phosphorylation-dependent manner. Finally, mutations within the phosphodegron, when incorporated into the full-length protein, result in increased levels of constitutively cleaved nuclear protein and increased transcription and secretion of a key endogenous target gene, apolipoprotein A IV.

CREB‐H and activating transcription factor 6 (ATF6) are transmembrane transcription factors that, in response to endoplasmic reticulum (ER) stress, traffic to the Golgi where they are cleaved by specific proteases, producing the N‐terminal domains that effect appropriate transcriptional responses. We show that unlike in ATF6 whose lumenal tail binds BiP and contains determinants for stress sensing and Golgi transport, in CREB‐H the lumenal tail is not involved in ER retention, not required for Golgi transport and does not bind BiP. The main determinant for CREB‐H ER retention resides in a membrane‐proximal cytoplasmic determinant that is conserved in related members of the CREB‐H family, but lacking in ATF6. We refine requirements within the ER‐retention motif (ERM) and show that ERM‐ve variants exhibited constitutive Golgi localization and constitutive cleavage by the Golgi protease, S1P. The ERM also conferred ER retention on a heterologous protein. Furthermore, deletion of the lumenal tail of CREB‐H had no effect on ER retention of parental CREB‐H or Golgi localization of ERM‐ve variants. Importantly, when the lumenal tail of ATF6 was transferred into an ERM‐ve variant, the chimera was now retained in the ER. Together, these data demonstrate novel and qualitatively distinct mechanisms of trafficking and stress signalling in CREB‐H compared to ATF6.

CREB‐H is an ATF6‐related, transmembrane transcription factor that, in response to endoplasmic reticulum (ER)‐associated stress, is cleaved by Golgi proteases and transported to the nucleus to effect appropriate adaptive responses. We characterize the ER processing and turnover of CREB‐H with results which have important implications for ER stress regulation and signalling. We show that CREB‐H is glycosylated and demonstrate that both the ER and nuclear forms of CREB‐H have short half‐lives. We also show that CREB‐H is subject to cycles of retrotranslocation, deglycosylation and degradation through the ER‐associated degradation (ERAD) pathway. Proteasome inhibition resulted in accumulation of a cytosolic intermediate but additionally, in contrast to inhibition of glycosylation, promoted specific cleavage of CREB‐H and nuclear transport of the N‐terminal‐truncated product. Our data indicate that under normal conditions CREB‐H is transported back from the ER to the cytosol, where it is subject to ERAD, but under conditions that repress proteasome function or promote load CREB‐H is diverted from this pathway instead undergoing cleavage and nuclear transport. Finally, we identify a cytoplasmic determinant involved in CREB‐H ER retention, deletion of which results in constitutive Golgi transport and corresponding cleavage. We present a model where cellular stresses may be sensed at different levels by different members of the basic and leucine zipper domain transmembrane proteins.

Regulated intramembrane proteolysis (RIP) of the transmembrane transcription factor ATF6 represents a key step in effecting adaptive response to the presence of unfolded or malfolded protein in the endoplasmic reticulum. Recent studies have highlighted new ATF6-related transmembrane transcription factors. It is likely that current models for ER stress signaling are incomplete and that the expansion of the bZIP transmembrane family reflects selectivity in many aspects of these responses, including the type and duration of any particular stress, the cell type in which it occurs, and the integration with other aspects of cell-type-specific organization or additional intrinsic pathways, and the integration and communication between these pathways, not only in a cell-type-specific manner, but also between different tissues and organs. This review summarizes current information on the bZIP-transmembrane proteins and discusses outstanding questions on the elucidation of the stress signals, the repertoire of components involved in regulating different aspects of the forward transport, cleavage, nuclear import, transcriptional activity, and turnover of each of these factors, and dissection of the integration of the various outputs into broad coordinated responses.

To investigate potential interplay between the SUMO1 (small ubiquitin-related modifier-1) and ubiquitin pathways of post-translational protein modification, we examined aspects of their localization and conjugation status during proteasome inhibition. Our results indicate that these pathways converge upon the discrete sub-nuclear domains known as PML (promyelocytic leukaemia protein) NBs (nuclear bodies). Proteasome inhibition generated an increased number of PML bodies, without any obvious increase in size. Using a cell line that constitutively expresses an epitope-tagged version of SUMO1, which was incorporated into high-molecular-mass conjugates, we observed SUMO1 accumulating in clusters around a subset of the NBs. Nuclear ubiquitin was initially observed in numerous speckles and foci, which bore no relationship to PML NBs in the absence of proteasome inhibition. However, during proteasome inhibition, total ubiquitin-conjugated species increased in the cell, as judged by Western blotting. Concomitantly the number of nuclear ubiquitin clusters decreased, and were almost quantitatively associated with the PML NBs, co-localizing with the SUMO-conjugated pool. Proteasome inhibition depleted the pool of free SUMO1 in the cell. Reversal of proteasome inhibition in the presence or absence of protein synthesis demonstrated that free SUMO1 was regenerated from the conjugated pool. The results indicate that a significant fraction of the free SUMO1 pool could be accounted for by recycling from the conjugated pool and indeed it may be that, as for ubiquitin, SUMO1 needs to be removed from conjugated species prior to processing by the proteasome. Taken together with other recent reports on the proteasome and PML NBs, these results suggest that the PML NBs may play an important role in integrating these pathways.

Modification of proteins by small ubiquitin-like modifier (SUMO) plays an important role in the function, compartmentalization, and stability of target proteins, contributing to the regulation of diverse processes. SUMO-1 modification can be regulated not only at the level of conjugation; it may also be reversed by a class of proteases known as the SUMO-specific proteases. However, current understanding of the regulation, specificity, and function of these proteases remains limited. In this study, we characterize aspects of the compartmentalization and proteolytic activity of the mammalian SUMO-specific protease, SENP1, providing insight into its function and regulation. We demonstrate the presence of a single nonconsensus nuclear localization signal within the N terminus of the protein, the mutation of which results in pronounced cytoplasmic accumulation in contrast to the nuclear accumulation of the parental protein. In addition, we observe that the N terminus of the protein may be essential for the correct regulation of the protease, since expression of the core domain alone results in limited expression and loss of SUMO-1, indicative of constitutive catalytic activity. Consistent with the prediction that the protease is a member of the cysteine family of proteases, we mutated a key cysteine residue and observed that expression of this catalytic mutant had a dominant negative phenotype, resulting in the accumulation of high molecular weight SUMO-1 conjugates. Furthermore, we demonstrate that SENP1 may itself be a target for SUMO-1 modification occurring at a nonconsensus site. Finally, we demonstrate that SENP1 localization is influenced by expression and localization of SUMO-1-conjugated target proteins within the cell.

Early during infection, the herpes simplex regulatory protein ICP0 promotes the proteasome-dependent degradation of a number of cellular proteins and the loss of a number of SUMO-1-modified protein isoforms, including PML. Recently, ICP0 has been shown to induce the accumulation of conjugated ubiquitin and function as a ubiquitin E3 ligase. However, certain aspects of the biochemistry, cell biology and the links between SUMO-1 conjugation/deconjugation and protein degradation remain unclear. For example, it is not currently known whether SUMO-1 deconjugation is a prerequisite for ubiquitination or degradation and, if so, by what mechanism this may occur. To help address these questions, a SUMO-specific protease (SENP1) was cloned and its expression and localization in relation to ICP0 examined. A cell line was established which constitutively expresses SUMO-1 to facilitate studies of localization and biochemistry. SENP1 localized to the nucleus mainly in discrete subdomains, a subset of which co-localized with the PML bodies. Both ICP0 and SENP1 protease promoted the loss of SUMO-1 from the nucleus, observed both for the endogenous species and the cell line expressing the epitope-tagged SUMO-1. The tagged SUMO-1 was recruited into high molecular mass conjugates in the cell line, and expression of SENP1 promoted loss of these species, including the modified species of PML. Finally, in co-transfection experiments ICP0 promoted the recruitment of SENP1 to nuclear domains, a result which was also observed early during infection. The significance of these findings is discussed in relation to the function of ICP0.