Impaired cutinase secretion in Saccharomyces cerevisiae induces irregular endoplasmic reticulum (ER) membrane proliferation, oxidative stress, and ER-associated degradation

Impaired secretion of the hydrophobic CY028 cutinase invokes an unfolded protein response (UPR) in Saccharomyces cerevisiae cells. Here we show that the UPR in CY028-expressing S. cerevisiae cells is manifested as an aberrant morphology of the endoplasmic reticulum (ER) and as extensive membrane proliferation compared to the ER morphology and membrane proliferation of wild-type CY000-producing S. cerevisiae cells. In addition, we observed oxidative stress, which resulted in a 21-fold increase in carbonylated proteins in the CY028-producing S. cerevisiae cells. Moreover, CY028-producing S. cerevisiae cells use proteasomal degradation to reduce the amount of accumulated CY028 cutinase, thereby attenuating the stress invoked by CY028 cutinase expression. This proteasomal degradation occurs within minutes and is characteristic of ER-associated degradation (ERAD). Our results clearly show that impaired secretion of the heterologous, hydrophobic CY028 cutinase in S. cerevisiae cells leads to protein aggregation in the ER, aberrant ER morphology and proliferation, and oxidative stress, as well as a UPR and ERAD.     

Mechanisms of cell-surface rerouting of an endoplasmic reticulum-retained mutant of the vasopressin V1b/V3 receptor by a pharmacological chaperone

Cell-surface expression and biological functions of several intracellular-retained G protein-coupled receptors are restored by membrane-permeable ligands called pharmacological chaperones. We have previously demonstrated that a mutation of the hydrophobic motif 341FNX2LLX3L350 in the C terminus of the human pituitary vasopressin V3 receptor (MUT V3R) led to it being retained in the endoplasmic reticulum (ER). Here, we establish the precise role of this motif and investigate whether SSR149415, a non-peptide V3R antagonist, behaves as a pharmacological chaperone for the ER-retained MUT V3R. The absence of the mutated receptor in the plasma membrane is linked to its prolonged association with the molecular chaperone calnexin in the ER and to its intensive degradation by the ubiquitin-proteasomal machinery. However, this is not because of a lack of oligomerization, as demonstrated by the presence of MUT V3R homodimers in the ER. Treatment with SSR149415 restores expression of the mutated receptor on the cell surface and its correct maturation, resulting into the functional recovery of its signaling properties. SSR149415 acts by stabilizing a native-like conformation of the V3R, reducing its association with calnexin and, thus, favoring a secretory pathway rather than the proteasomal degradation pathway. In conclusion, the FN(X)2LL(X)3L sequence is an important motif for the V3R conformation, and the misfolding resulting from its mutation alters the receptor export but can be reverted by SSR149415.     

PDI reductase acts on Akita mutant proinsulin to initiate retrotranslocation along the Hrd1/Sel1L-p97 axis

In mutant INS gene–induced diabetes of youth (MIDY), characterized by insulin deficiency, MIDY proinsulin mutants misfold and fail to exit the endoplasmic reticulum (ER). Moreover, these mutants bind and block ER exit of wild-type (WT) proinsulin, inhibiting insulin production. The ultimate fate of ER-entrapped MIDY mutants is unclear, but previous studies implicated ER-associated degradation (ERAD), a pathway that retrotranslocates misfolded ER proteins to the cytosol for proteasomal degradation. Here we establish key ERAD machinery components used to triage the Akita proinsulin mutant, including the Hrd1-Sel1L membrane complex, which conducts Akita proinsulin from the ER lumen to the cytosol, and the p97 ATPase, which couples the cytosolic arrival of proinsulin with its proteasomal degradation. Surprisingly, we find that protein disulfide isomerase (PDI), the major protein oxidase of the ER lumen, engages Akita proinsulin in a novel way, reducing proinsulin disulfide bonds and priming the Akita protein for ERAD. Efficient PDI engagement of Akita proinsulin appears linked to the availability of Hrd1, suggesting that retrotranslocation is coordinated on the lumenal side of the ER membrane. We believe that, in principle, this form of diabetes could be alleviated by enhancing the targeting of MIDY mutants for ERAD to restore WT insulin production.     

Proteasome inhibition compromises direct retention of cytochrome P450 2C2 in the endoplasmic reticulum

To determine whether protein degradation plays a role in the endoplasmic reticulum (ER) retention of cytochromes P450, the effects of proteasomal inhibitors on the expression and distribution of green fluorescent protein chimeras of CYP2C2 and related proteins was examined. In transfected cells, expression levels of chimeras of full-length CYP2C2 and its cytosolic domain, but not its N-terminal transmembrane sequence, were increased by proteasomal inhibition. Redistribution of all three chimeras from the reticular ER into a perinuclear compartment and, in a subset of cells, also to the cell surface was observed after proteasomal inhibition. Redistribution was blocked by the microtubular inhibitor, nocodazole, suggesting that redistribution to the cell surface followed the conventional vesicular transport pathway. Similar redistributions were detected for BAP31, a CYP2C2 binding chaperone; CYP2E1 and CYP3A4, which are also degraded by the proteasomal pathway; and for cytochrome P450 reductase, which does not undergo proteasomal degradation; but not for the ER membrane proteins, sec61 and calnexin. Redistribution does not result from saturation of an ER retention “receptor” since in some cases protein levels were unaffected. Proteasomal inhibition may, therefore, alter ER retention by affecting a protein critical for ER retention, either directly, or indirectly by affecting the composition of the ER membranes.     

Impaired Cutinase Secretion in Saccharomyces cerevisiae Induces Irregular Endoplasmic Reticulum (ER) Membrane Proliferation, Oxidative Stress, and ER-Associated Degradation

Impaired secretion of the hydrophobic CY028 cutinase invokes an unfolded protein response (UPR) in Saccharomyces cerevisiae cells. Here we show that the UPR in CY028-expressing S. cerevisiae cells is manifested as an aberrant morphology of the endoplasmic reticulum (ER) and as extensive membrane proliferation compared to the ER morphology and membrane proliferation of wild-type CY000-producing S. cerevisiae cells. In addition, we observed oxidative stress, which resulted in a 21-fold increase in carbonylated proteins in the CY028-producing S. cerevisiae cells. Moreover, CY028-producing S. cerevisiae cells use proteasomal degradation to reduce the amount of accumulated CY028 cutinase, thereby attenuating the stress invoked by CY028 cutinase expression. This proteasomal degradation occurs within minutes and is characteristic of ER-associated degradation (ERAD). Our results clearly show that impaired secretion of the heterologous, hydrophobic CY028 cutinase in S. cerevisiae cells leads to protein aggregation in the ER, aberrant ER morphology and proliferation, and oxidative stress, as well as a UPR and ERAD.     

Suppression of viral replication by stress-inducible GADD34 protein via the mammalian serine/threonine protein kinase mTOR pathway

GADD34 is a protein that is induced by a variety of stressors, including DNA damage, heat shock, nutrient deprivation, energy depletion, and endoplasmic reticulum stress. Here, we demonstrated that GADD34 induced by vesicular stomatitis virus (VSV) infection suppressed viral replication in wild-type (WT) mouse embryo fibroblasts (MEFs), whereas replication was enhanced in GADD34-deficient (GADD34-KO) MEFs. Enhanced viral replication in GADD34-KO MEFs was reduced by retroviral gene rescue of GADD34. The level of VSV protein expression in GADD34-KO MEFs was significantly higher than that in WT MEFs. Neither phosphorylation of eIF2alpha nor cellular protein synthesis was correlated with viral replication in GADD34-KO MEFs. On the other hand, phosphorylation of S6 and 4EBP1, proteins downstream of mTOR, was suppressed by VSV infection in WT MEFs but not in GADD34-KO MEFs. GADD34 was able to associate with TSC1/2 and dephosphorylate TSC2 at Thr1462. VSV replication was higher in TSC2-null cells than in TSC2-expressing cells, and constitutively active Akt enhanced VSV replication. On the other hand, rapamycin, an mTOR inhibitor, significantly suppressed VSV replication in GADD34-KO MEFs. These findings demonstrate that GADD34 induced by VSV infection suppresses viral replication via mTOR pathway inhibition, indicating that cross talk between stress-inducible GADD34 and the mTOR signaling pathway plays a critical role in antiviral defense.     

ER‐to‐lysosome‐associated degradation of proteasome‐resistant ATZ polymers occurs via receptor‐mediated vesicular transport

Maintenance of cellular proteostasis relies on efficient clearance of defective gene products. For misfolded secretory proteins, this involves dislocation from the endoplasmic reticulum (ER) into the cytosol followed by proteasomal degradation. However, polypeptide aggregation prevents cytosolic dislocation and instead activates ill‐defined lysosomal catabolic pathways. Here, we describe an ER‐to‐lysosome‐associated degradation pathway (ERLAD) for proteasome‐resistant polymers of alpha1‐antitrypsin Z (ATZ). ERLAD involves the ER‐chaperone calnexin (CNX) and the engagement of the LC3 lipidation machinery by the ER‐resident ER‐phagy receptor FAM134B, echoing the initiation of starvation‐induced, receptor‐mediated ER‐phagy. However, in striking contrast to ER‐phagy, ATZ polymer delivery from the ER lumen to LAMP1/RAB7‐positive endolysosomes for clearance does not require ER capture within autophagosomes. Rather, it relies on vesicular transport where single‐membrane, ER‐derived, ATZ‐containing vesicles release their luminal content within endolysosomes upon membrane:membrane fusion events mediated by the ER‐resident SNARE STX17 and the endolysosomal SNARE VAMP8. These results may help explain the lack of benefits of pharmacologic macroautophagy enhancement that has been reported for some luminal aggregopathies.     

Inefficient maturation of the rat luteinizing hormone receptor. A putative way to regulate receptor numbers at the cell surface

Increasing evidence suggests that the folding and maturation of monomeric proteins and assembly of multimeric protein complexes in the endoplasmic reticulum (ER) may be inefficient not only for mutants that carry changes in the primary structure but also for wild type proteins. In the present study, we demonstrate that the rat luteinizing hormone receptor, a G protein-coupled receptor, is one of these proteins that matures inefficiently and appears to be very prone to premature degradation. A substantial portion of the receptors in stably transfected human embryonic kidney 293 cells existed in immature form of M(r) 73,000, containing high mannose-type N-linked glycans. In metabolic pulse-chase studies, only approximately 20% of these receptor precursors were found to gain hormone binding ability and matured to a form of M(r) 90,000, containing bi- and multiantennary sialylated N-linked glycans. The rest had a propensity to form disulfide-bonded complexes with a M(r) 120,000 protein in the ER membrane and were eventually targeted for degradation in proteasomes. The number of membrane-bound receptor precursors increased when proteasomal degradation was inhibited, and no cytosolic receptor forms were detected, suggesting that retrotranslocation of the misfolded/incompletely folded receptors is tightly coupled to proteasomal function. Furthermore, a proteasomal blockade was found to increase the number of receptors that were capable of hormone binding. Thus, these results raise the interesting possibility that luteinizing hormone receptor expression at the cell surface may be controlled at the ER level by regulating the number of newly synthesized proteins that will mature and escape the ER quality control and premature degradation.     

Deubiquitinases USP20/33 promote the biogenesis of tail-anchored membrane proteins

Numerous proteins that have hydrophobic transmembrane domains (TMDs) traverse the cytosol and posttranslationally insert into cellular membranes. It is unclear how these hydrophobic membrane proteins evade recognition by the cytosolic protein quality control (PQC), which typically recognizes exposed hydrophobicity in misfolded proteins and marks them for proteasomal degradation by adding ubiquitin chains. Here, we find that tail-anchored (TA) proteins, a vital class of membrane proteins, are recognized by cytosolic PQC and are ubiquitinated as soon as they are synthesized in cells. Surprisingly, the ubiquitinated TA proteins are not routed for proteasomal degradation but instead are handed over to the targeting factor, TRC40, and delivered to the ER for insertion. The ER-associated deubiquitinases, USP20 and USP33, remove ubiquitin chains from TA proteins after their insertion into the ER. Thus, our data suggest that deubiquitinases rescue posttranslationally targeted membrane proteins that are inappropriately ubiquitinated by PQC in the cytosol.     

TRAM1 is involved in disposal of ER membrane degradation substrates

ER quality control consists of monitoring protein folding and targeting misfolded proteins for proteasomal degradation. ER stress results in an unfolded protein response (UPR) that selectively upregulates proteins involved in protein degradation, ER expansion, and protein folding. Given the efficiency in which misfolded proteins are degraded, there likely exist cellular factors that enhance the export of proteins across the ER membrane. We have reported that translocating chain-associated membrane protein 1 (TRAM1), an ER-resident membrane protein, participates in HCMV US2- and US11-mediated dislocation of MHC class I heavy chains (Oresic, K., Ng, C.L., and Tortorella, D. 2009). Consistent with the hypothesis that TRAM1 is involved in the disposal of misfolded ER proteins, cells lacking TRAM1 experienced a heightened UPR upon acute ER stress, as evidenced by increased activation of unfolded protein response elements (UPRE) and elevated levels of NF-κB activity. We have also extended the involvement of TRAM1 in the selective degradation of misfolded ER membrane proteins Cln6^M241T and US2, but not the soluble degradation substrate α₁-antitrypsin null_HK. These degradation model systems support the paradigm that TRAM1 is a selective factor that can enhance the dislocation of ER membrane proteins.     

AAA ATPase p97/valosin-containing protein interacts with gp78, a ubiquitin ligase for endoplasmic reticulum-associated degradation

Endoplasmic reticulum-associated degradation (ERAD) is a protein quality control mechanism that eliminates unwanted proteins from the endoplasmic reticulum (ER) through a ubiquitin-dependent proteasomal degradation pathway. gp78 is a previously described ER membrane-anchored ubiquitin ligase (E3) involved in ubiquitination of ER proteins. AAA ATPase (ATPase associated with various cellular activities) p97/valosin-containing protein (VCP) subsequently dislodges the ubiquitinated proteins from the ER and chaperones them to the cytosol, where they undergo proteasomal degradation. We now report that gp78 physically interacts with p97/VCP and enhances p97/VCP-polyubiquitin association. The enhanced association correlates with decreases in ER stress-induced accumulation of polyubiquitinated proteins. This effect is abolished when the p97/VCP-interacting domain of gp78 is removed. Further, using ERAD substrate CD3delta, gp78 consistently enhances p97/VCP-CD3delta binding and facilitates CD3delta degradation. Moreover, inhibition of endogenous gp78 expression by RNA interference markedly increases the levels of total polyubiquitinated proteins, including CD3delta, and abrogates VCP-CD3delta interactions. The gp78 mutant with deletion of its p97/VCP-interacting domain fails to increase CD3delta degradation and leads to accumulation of polyubiquitinated CD3delta, suggesting a failure in delivering ubiquitinated CD3delta for degradation. These data suggest that gp78-p97/VCP interaction may represent one way of coupling ubiquitination with retrotranslocation and degradation of ERAD substrates.     

Liver cytochrome P450 3A endoplasmic reticulum-associated degradation: a major role for the p97 AAA ATPase in cytochrome P450 3A extraction into the cytosol

The CYP3A subfamily of hepatic cytochromes P450, being engaged in the metabolism and clearance of >50% of clinically relevant drugs, can significantly influence therapeutics and drug-drug interactions. Our characterization of CYP3A degradation has indicated that CYPs 3A incur ubiquitin-dependent proteasomal degradation (UPD) in an endoplasmic reticulum (ER)-associated degradation (ERAD) process. Cytochromes P450 are monotopic hemoproteins N-terminally anchored to the ER membrane with their protein bulk readily accessible to the cytosolic proteasome. Given this topology, it was unclear whether they would require the AAA-ATPase p97 chaperone complex that retrotranslocates/dislocates ubiquitinated ER-integral and luminal proteins into the cytosol for proteasomal delivery. To assess the in vivo relevance of this p97-CYP3A association, we used lentiviral shRNAs to silence p97 (80% mRNA and 90% protein knockdown relative to controls) in sandwich-cultured rat hepatocytes. This extensive hepatic p97 knockdown remarkably had no effect on cellular morphology, ER stress, and/or apoptosis, despite the well recognized strategic p97 roles in multiple important cellular processes. However, such hepatic p97 knockdown almost completely abrogated CYP3A extraction into the cytosol, resulting in a significant accumulation of parent and ubiquitinated CYP3A species that were firmly ER-tethered. Little detectable CYP3A accumulated in the cytosol, even after concomitant inhibition of proteasomal degradation, thereby documenting a major role of p97 in CYP3A extraction and delivery to the 26 S proteasome during its UPD/ERAD. Intriguingly, the accumulated parent CYP3A was functionally active, indicating that p97 can regulate physiological CYP3A content and thus influence its clinically relevant function.     

Salsolinol causing parkinsonism activates endoplasmic reticulum-stress signaling pathways in human dopaminergic SK-N-SH cells

The endoplasmic reticulum (ER) is a small intracellular organelle to which one-third of cellular proteins are translocated after translation and post-translational modification, folding and the formation of a three- or four-dimensional structure. ER also has a role in the transportation of proteins to other intracellular organelles, the cell surface or the outer space of the cell membrane. Thus, ER is an important intermediate which maintains intracellular homeostasis through complex control systems. Once these control systems are disrupted, serious disturbances occur. Many neurodegenerative diseases including Parkinson's disease involve aggregation and deposition of misfolded proteins such as alpha-synuclein. Endogenously occurring neurotoxins such as Salsolinol and 1-benzyl-1,2,3,4-tetrahydroisoquinoline (1BnTIQ) causing Parkinsonism may foster misfolded proteins and bring forth ER stress in dopaminergic neurons. In the present study we examined translational changes fostered by ER stress and mediated by the Parkinsonian endogenous neurotoxins, salsolinol and 1BnTIQ, in dopaminergic cell line. Treatment with salsolinol and 1BnTIQ induced several genes involved in ER stress and unfolded protein response (UPR), such as ER chaperones and GADD153 (CHOP). Immunoblotting confirmed phosphorylation of the key endoplasmic reticulum stress kinase PERK (PKR-like-ER kinase) and eIF2alpha and induction of their downstream targets such as Bip and GADD153. These findings suggest a widespread involvement of ER stress and unfolded protein response in the pathophysiology of Parkinson's disease.     

Hepatitis B virus large and middle glycoproteins are degraded by a proteasome pathway in glucosidase-inhibited cells but not in cells with functional glucosidase enzyme

The secretion of hepatitis B virus (HBV) large (LHBs) and middle (MHBs) envelope polypeptides from tissue cultures requires proper protein folding and is prevented by inhibitors of the endoplasmic reticulum (ER) glucosidase. Using competitive inhibitors of the ER glucosidase, here it is shown that the amounts of glycosylated and unglycosylated forms of LHBs and MHBs proteins are all greatly reduced in tissue cultures producing HBV envelope glycoproteins. In contrast, the HBV small (SHBs) protein was not affected. The reduction in secretion of LHBs and MHBs proteins appears to be mediated by proteasomal degradation pathways, since it is prevented by either lactacystin or epoxomicin, two inhibitors of proteasomal degradation. Although there is no detectable proteasomal degradation of LHBs and MHBs in cells with functional glucosidase, the implications of the nearly quantitative sensitivity of glycosylated and unglycosylated forms of LHBs and MHBs proteins, with selective sparing of SHBs protein, in cells in which glucosidase is inhibited is surprising, and its implications are discussed.     

Sec61p-independent degradation of the tail-anchored ER membrane protein Ubc6p.

Tail-anchored proteins are distinct from other membrane proteins as they are thought to insert into the endoplasmic reticulum (ER) membrane independently of Sec61p translocation pores. These pores not only mediate import but are also assumed to catalyze export of proteins in a process called ER-associated protein degradation (ERAD). In order to examine the Sec61p dependence of the export of tail-anchored proteins, we analyzed the degradation pathway of a tail-anchored ER membrane protein, the ubiquitin-conjugating enzyme 6 (Ubc6p). In contrast to other ubiquitin conjugating enzymes (Ubcs), Ubc6p is naturally short-lived. Its proteolysis is mediated specifically by the unique Ubc6p tail region. Degradation further requires the activity of Cue1p-assembled Ubc7p, and its own catalytic site cysteine. However, it occurs independently of the other ERAD components Ubc1p, Hrd1p/Der3p, Hrd3p and Der1p. In contrast to other natural ERAD substrates, proteasomal mutants accumulate a membrane-bound degradation intermediate of Ubc6p. Most interestingly, mutations in SEC61 do not reduce the turnover of full-length Ubc6p nor cause a detectable accumulation of degradation intermediates. These data are in accordance with a model in which tail-anchored proteins can be extracted from membranes independently of Sec61p.     

Growth arrest and DNA damage-inducible protein GADD34 targets protein phosphatase 1 alpha to the endoplasmic reticulum and promotes dephosphorylation of the alpha subunit of eukaryotic translation initiation factor 2

The growth arrest and DNA damage-inducible protein, GADD34, associates with protein phosphatase 1 (PP1) and promotes in vitro dephosphorylation of the alpha subunit of eukaryotic translation initiation factor 2, (eIF-2 alpha). In this report, we show that the expression of human GADD34 in cultured cells reversed eIF-2 alpha phosphorylation induced by thapsigargin and tunicamycin, agents that promote protein unfolding in the endoplasmic reticulum (ER). GADD34 expression also reversed eIF-2 alpha phosphorylation induced by okadaic acid but not that induced by another phosphatase inhibitor, calyculin A (CA), which is a result consistent with PP1 being a component of the GADD34-assembled eIF-2 alpha phosphatase. Structure-function studies identified a bipartite C-terminal domain in GADD34 that encompassed a canonical PP1-binding motif, KVRF, and a novel RARA sequence, both of which were required for PP1 binding. N-terminal deletions of GADD34 established that while PP1 binding was necessary, it was not sufficient to promote eIF-2 alpha dephosphorylation in cells. Imaging of green fluorescent protein (GFP)-GADD34 proteins showed that the N-terminal 180 residues directed the localization of GADD34 at the ER and that GADD34 targeted the alpha isoform of PP1 to the ER. These data provide new insights into the mode of action of GADD34 in assembling an ER-associated eIF-2 alpha phosphatase that regulates protein translation in mammalian cells.     

Inhibition of GADD34, the Stress-Inducible Regulatory Subunit of the Endoplasmic Reticulum Stress Response,Does Not Enhance Functional Recovery after Spinal Cord Injury

Activation of the endoplasmic reticulum stress response (ERSR) is a hallmark of various pathological diseases and/or traumatic injuries. Restoration of ER homeostasis can contribute to improvement in the functional outcome of these diseases. Using genetic and pharmacological inhibition of the PERK-CHOP arm of the ERSR, we recently demonstrated improvements in hindlimb locomotion after spinal cord injury (SCI) and implicated oligodendrocyte survival as a potential mechanism. Here, we investigated the contribution of stress-inducible PPP1R15A/GADD34, an ERSR signaling effector downstream of CHOP that dephosphorylates eIF2α, in the pathogenesis of SCI. We show that although genetic ablation of GADD34 protects oligodendrocyte precursor cells (OPCs) against ER stress-mediated cell death in vitro and results in differential ERSR attenuation in vivo after SCI, there is no improvement in hindlimb locomotor function. Guanabenz, a FDA approved antihypertensive drug, was recently shown to reduce the burden of misfolded proteins in the ER by directly targeting GADD34. Guanabenz protected OPCs from ER stress-mediated cell death in vitro and attenuated the ERSR in vivo after SCI. However, guanabenz administration failed to rescue the locomotor deficits after SCI. These data suggest that deletion of GADD34 alone is not sufficient to improve functional recovery after SCI.     

Enzymatic blockade of the ubiquitin-proteasome pathway

Ubiquitin-dependent processes control much of cellular physiology. We show that expression of a highly active, Epstein-Barr virus-derived deubiquitylating enzyme (EBV-DUB) blocks proteasomal degradation of cytosolic and ER-derived proteins by preemptive removal of ubiquitin from proteasome substrates, a treatment less toxic than the use of proteasome inhibitors. Recognition of misfolded proteins in the ER lumen, their dislocation to the cytosol, and degradation are usually tightly coupled but can be uncoupled by the EBV-DUB: a misfolded glycoprotein that originates in the ER accumulates in association with cytosolic chaperones as a deglycosylated intermediate. Our data underscore the necessity of a DUB activity for completion of the dislocation reaction and provide a new means of inhibition of proteasomal proteolysis with reduced cytotoxicity.     

GADD34 suppresses wound healing by upregulating expression of myosin IIA

Wound healing consists of sequential steps of tissue repair, and cell migration is particularly important. In order to analyze the potential function of growth arrest and DNA damage inducible protein 34 (GADD34) in tissue repair, we performed in vitro and in vivo wound healing experiments. In an in vitro scratch assay, GADD34 knockout (KO) mouse embryonic fibroblasts (MEFs) had higher migration rates than did wild type (WT) MEFs. Furthermore, the rate of wound closure was faster in GADD34 KO MEFs than in WT MEFs. Using in vivo punch biopsy assays, GADD34 KO mice had accelerated wound healing compared to WT mice. WT mice expressed higher amounts of myosin IIA in migrating macrophages and myofibroblasts than did GADD34 KO mice. These results indicate that GADD34 negatively regulates cell migration in wound healing via expression of myosin IIA.