Type III secretion effectors of the IpaH family are E3 ubiquitin ligases

Many bacteria pathogenic for plants or animals, including Shigella spp., which is responsible for shigellosis in humans, use a type III secretion apparatus to inject effector proteins into host cells. Effectors alter cell signaling and host responses induced upon infection; however, their precise biochemical activities have been elucidated in very few cases. Utilizing Saccharomyces cerevisiae as a surrogate host, we show that the Shigella effector IpaH9.8 interrupts pheromone response signaling by promoting the proteasome-dependent destruction of the MAPKK Ste7. In vitro, IpaH9.8 displayed ubiquitin ligase activity toward ubiquitin and Ste7. Replacement of a Cys residue that is invariant among IpaH homologs of plant and animal pathogens abolished the ubiquitin ligase activity of IpaH9.8. We also present evidence that the IpaH homolog SspH1 from Salmonella enterica can ubiquitinate ubiquitin and PKN1, a previously identified SspH1 interaction partner. This study assigns a function for IpaH family members as E3 ubiquitin ligases.     

A Salmonella type III secretion effector interacts with the mammalian serine/threonine protein kinase PKN1

Essential to salmonellae pathogenesis is an export device called the type III secretion system (TTSS), which mediates the transfer of bacterial effector proteins from the bacterial cell into the host cell cytoplasm. Once inside the host cell, these effectors are then capable of altering a variety of host cellular functions in order to promote bacterial survival and colonization. SspH1 is a Salmonella enterica serovar Typhimurium TTSS effector that localizes to the mammalian nucleus and down-modulates production of proinflammatory cytokines by inhibiting nuclear factor (NF)-kappaB-dependent gene expression. To identify mammalian binding partners of SspH1 a yeast two-hybrid screen against a human spleen cDNA library was performed. It yielded a serine/threonine protein kinase called protein kinase N 1 (PKN1). The leucine-rich repeat domain of SspH1 was demonstrated to mediate this interaction and also inhibition of NF-kappaB-dependent gene expression. This suggested that PKN1 may play a role in modulation of the NF-kappaB signalling pathway. Indeed, we found that expression of constitutively active PKN1 in mammalian cells results in a decrease, while depletion of PKN1 by RNA interference causes an increase in NF-kappaB-dependent reporter gene expression. These data indicate that SspH1 may inhibit the host's inflammatory response by interacting with PKN1.     

Structure of an SspH1-PKN1 Complex Reveals the Basis for Host Substrate Recognition and Mechanism of Activation for a Bacterial E3 Ubiquitin Ligase

IpaH proteins are bacterium-specific E3 enzymes that function as type three secretion system (T3SS) effectors in Salmonella, Shigella, and other Gram-negative bacteria. IpaH enzymes recruit host substrates for ubiquitination via a leucine-rich repeat (LRR) domain, which can inhibit the catalytic domain in the absence of substrate. The basis for substrate recognition and the alleviation of autoinhibition upon substrate binding is unknown. Here, we report the X-ray structure of Salmonella SspH1 in complex with human PKN1. The LRR domain of SspH1 interacts specifically with the HR1b coiled-coil subdomain of PKN1 in a manner that sterically displaces the catalytic domain from the LRR domain, thereby activating catalytic function. SspH1 catalyzes the ubiquitination and proteasome-dependent degradation of PKN1 in cells, which attenuates androgen receptor responsiveness but not NF-κB activity. These regulatory features are conserved in other IpaH-substrate interactions. Our results explain the mechanism whereby substrate recognition and enzyme autoregulation are coupled in this class of bacterial ubiquitin ligases.     

Salmonella Type III Secretion Effector SlrP Is an E3 Ubiquitin Ligase for Mammalian Thioredoxin

Salmonella enterica encodes two virulence-related type III secretion systems in Salmonella pathogenicity islands 1 and 2, respectively. These systems mediate the translocation of protein effectors into the eukaryotic host cell, where they alter cell signaling and manipulate host cell functions. However, the precise role of most effectors remains unknown. Using a genetic screen, we identified the small, reduction/oxidation-regulatory protein thioredoxin as a mammalian binding partner of the Salmonella effector SlrP. The interaction was confirmed by affinity chromatography and coimmunoprecipitation. In vitro, SlrP was able to mediate ubiquitination of ubiquitin and thioredoxin. A Cys residue conserved in other effectors of the same family that also possess E3 ubiquitin ligase activity was essential for this catalytic function. Stable expression of SlrP in HeLa cells resulted in a significant decrease of thioredoxin activity and in an increase of cell death. The physiological significance of these results was strengthened by the finding that Salmonella was able to trigger cell death and inhibit thioredoxin activity in HeLa cells several hours post-infection. This study assigns a functional role to the Salmonella effector SlrP as a binding partner and an E3 ubiquitin ligase for mammalian thioredoxin.Protein secretion is a basic function in all groups of bacteria. Many secretion systems have been found in Gram-negative bacteria, from the relatively simple type I secretion systems to the complex type III or type IV machines or the recently described type VI systems (reviewed in Refs. 1 and 2). Many pathogenic or symbiotic Gram-negative bacteria rely on type III secretion systems (T3SS)² for their interaction with host organisms. The T3SS is a protein export pathway that spans the inner membrane, periplasmic space, outer membrane, and host cell membrane. These complex structures are related to flagella and consist of at least 20 different subunits that enable the bacteria to translocate substrates into the cytosol of the eukaryotic host cell (reviewed in Ref. 3). These systems have also been referred to as injectisomes or molecular needles (4).Proteins secreted and translocated into eukaryotic cells through T3SS are called “effectors.” These effectors have the ability to suppress host defense signaling. Effectors of plant pathogens may target salicylic acid- and abscisic acid-dependent defenses, host vesicle trafficking, or interfere with host RNA metabolism. Effectors from animal pathogens modify the cytoskeleton to facilitate bacterial entry, modulate Rho GTPases and NF-κB, inhibit the host inflammatory response, elicit death of immune cells, and disrupt both adaptative and innate immune responses (reviewed in Ref. 5).Salmonella enterica produces two distinct T3SS essential for virulence that are encoded by genes located in Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2), respectively. The SPI-1 T3SS secretes at least 16 proteins: AvrA, GogB, SipA, SipB, SipC, SipD, SlrP, SopA, SopB/SigD, SopD, SopE, SopE2, SptP, SspH1, SteA, and SteB (6–8). Six of them have been shown to regulate actin cytoskeleton dynamics (reviewed in Ref. 9). 19 SPI-2 T3SS effectors have been identified: GogB, PipB, PipB2, SifA, SifB, SopD2, SseF, SlrP, SseG, SseI/SrfH, SseJ, SseK1, SseK2, SseL, SspH1, SspH2, SteA, SteB, and SteC. However, the biochemical functions of most of them remain unknown (reviewed in Ref. 10).The conventional paradigm, supported by in vivo and in vitro studies, is that the SPI-1-encoded T3SS is required for the invasion of M cells and cultured epithelial cells (11, 12) as well as for the inflammatory response of the intestinal cells, and that the SPI-2-encoded T3SS is essential for replication and survival within macrophages and the progression of a systemic infection (13). Recent evidence suggests that the boundaries between SPI-1 and SPI-2 function are not sharply defined: some SPI-1 effectors are detected hours or days after infection and SPI-2-encoded genes may be expressed before penetration of the intestinal epithelium. In addition, as can be noticed comparing the lists of effectors above, some effectors, including SlrP, can be secreted by both T3SS.SlrP (for Salmonella leucine-rich repeat protein) was identified as a S. enterica serovar Typhimurium host range factor by signature-tagged mutagenesis (14). A mutant in this gene has no difference in virulence with the wild-type strain when infecting calves but is 6-fold attenuated for mouse virulence after oral infection. This gene is located in a 2.9-kb DNA region with features of horizontal acquisition and has similarity to yopM from Yersinia spp. and ipaH from Shigella flexneri. The predicted protein contains 10 copies of a leucine-rich repeat signature, a protein motif frequently involved in protein-protein interactions. Other members of the leucine-rich repeat family in Salmonella are the effectors SspH1 and SspH2, which share 39 and 38% amino acid identity with SlrP, respectively. Similarity in the amino-terminal region of these three proteins helped to define a translocation signal that was also found in four other T3SS effectors: SifA, SifB, SseI, and SseJ (15). Although SlrP can be delivered into mammalian cells by both T3SS, its expression seems to be induced by RtsA, one of the main regulators of SPI-1, independently of HilA or InvF (16).Although the function of SlrP was completely unknown, the presence of leucine-rich repeats in this protein suggested that it may bind eukaryotic proteins during infection. In addition, recent reports have shown an enzymatic activity, E3 ubiquitin ligase, for effectors of the same family (17, 18).In this work we demonstrate that SlrP interacts with mammalian thioredoxin-1 (Trx). We also show that SlrP is an E3 ubiquitin ligase that can use Trx as a substrate. Our results support a model in which interaction of SlrP with Trx leads to a decrease in thioredoxin activity and triggers host cell death.     

The role of RAR1 in Agrobacterium-mediated plant transformation

RAR1 is identified as a critical protein involved in plant innate immunity. We investigated the role of RAR1 in Agrobacterium-mediated plant transformation based on the previous findings that accessory proteins associated with the E3 ligase complex such as SGT1, which tightly interacts with RAR1, play a role in the transformation process. RAR1 gene silencing in Nicotiana benthamiana and Arabidopsis rar1 mutant analysis suggested that RAR1 is required for early stages of Agrobacterium-mediated plant transformation. This finding further illustrates that RAR1, along with SGT1, that serve as a HSP90 co-chaperone is important for Agrobacterium-mediated plant transformation.     

Host Cell-catalyzed S-Palmitoylation Mediates Golgi Targeting of the Legionella Ubiquitin Ligase GobX

The facultative intracellular pathogen Legionella pneumophila, the causative agent of Legionnaires disease, infects and replicates within human alveolar macrophages. L. pneumophila delivers almost 300 effector proteins into the besieged host cell that alter signaling cascades and create conditions that favor intracellular bacterial survival. In order for the effectors to accomplish their intracellular mission, their activity needs to be specifically directed toward the correct host cell protein or target organelle. Here, we show that the L. pneumophila effector GobX possesses E3 ubiquitin ligase activity that is mediated by a central region homologous to mammalian U-box domains. Furthermore, we demonstrate that GobX exploits host cell S-palmitoylation to specifically localize to Golgi membranes. The hydrophobic palmitate moiety is covalently attached to a cysteine residue at position 175, which is part of an amphipathic α-helix within the C-terminal region of GobX. Site-directed mutagenesis of cysteine 175 or residues on the hydrophobic face of the amphipathic helix strongly attenuated palmitoylation and Golgi localization of GobX. Together, our study provides evidence that the L. pneumophila effector GobX exploits two post-translational modification pathways of host cells, ubiquitination and S-palmitoylation.     

Recognition and ubiquitination of Salmonella type III effector SopA by a ubiquitin E3 ligase, HsRMA1

Salmonella translocate bacterial effectors into host cells to confer bacterial entry and survival. It is not known how the host cells cope with the influx of these effectors. We report here that the Salmonella effector, SopA, interacts with host HsRMA1, a ubiquitin E3 ligase with a previously unknown function. SopA is ubiquitinated and degraded by the HsRMA1-mediated ubiquitination pathway. A sopA mutant escapes out of the Salmonella-containing vacuoles less frequently to the cytosol than wild type Salmonella in HeLa cells in a HsRMA1-dependent manner. Our data suggest that efficient bacterial escape into the cytosol of epithelial cells requires HsRMA1-mediated SopA ubiquitination and contributes to Salmonella-induced enteropathogenicity.     

Ubiquitinylation of α-synuclein by carboxyl terminus Hsp70-interacting protein (CHIP) is regulated by Bcl-2-associated athanogene 5 (BAG5)

Parkinson's disease (PD) is a common neurodegenerative condition in which abnormalities in protein homeostasis, or proteostasis, may lead to accumulation of the protein α-synuclein (α-syn). Mutations within or multiplications of the gene encoding α-syn are known to cause genetic forms of PD and polymorphisms in the gene are recently established risk factors for idiopathic PD. α-syn is a major component of Lewy bodies, the intracellular proteinaceous inclusions which are pathological hallmarks of most forms of PD. Recent evidence demonstrates that α-syn can self associate into soluble oligomeric species and implicates these α-syn oligomers in cell death. We have previously shown that carboxyl terminus of Hsp70-interacting protein (CHIP), a co-chaperone molecule with E3 ubiquitin ligase activity, may reduce the levels of toxic α-syn oligomers. Here we demonstrate that α-syn is ubiquitinylated by CHIP both in vitro and in cells. We find that the products from ubiquitinylation by CHIP include both monoubiquitinylated and polyubiquitinylated forms of α-syn. We also demonstrate that CHIP and α-syn exist within a protein complex with the co-chaperone bcl-2-associated athanogene 5 (BAG5) in brain. The interaction of CHIP with BAG5 is mediated by Hsp70 which binds to the tetratricopeptide repeat domain of CHIP and the BAG domains of BAG5. The Hsp70-mediated association of BAG5 with CHIP results in inhibition of CHIP E3 ubiquitin ligase activity and subsequently reduces α-syn ubiquitinylation. Furthermore, we use a luciferase-based protein-fragment complementation assay of α-syn oligomerization to investigate regulation of α-syn oligomers by CHIP in living cells. We demonstrate that BAG5 mitigates the ability of CHIP to reduce α-syn oligomerization and that non-ubiquitinylated α-syn has an increased propensity for oligomerization. Thus, our results identify CHIP as an E3 ubiquitin ligase of α-syn and suggest a novel function for BAG5 as a modulator of CHIP E3 ubiquitin ligase activity with implications for CHIP-mediated regulation of α-syn oligomerization.     

PINK1 phosphorylates ubiquitin to activate Parkin E3 ubiquitin ligase activity

PINK1 kinase activates the E3 ubiquitin ligase Parkin to induce selective autophagy of damaged mitochondria. However, it has been unclear how PINK1 activates and recruits Parkin to mitochondria. Although PINK1 phosphorylates Parkin, other PINK1 substrates appear to activate Parkin, as the mutation of all serine and threonine residues conserved between Drosophila and human, including Parkin S65, did not wholly impair Parkin translocation to mitochondria. Using mass spectrometry, we discovered that endogenous PINK1 phosphorylated ubiquitin at serine 65, homologous to the site phosphorylated by PINK1 in Parkin’s ubiquitin-like domain. Recombinant TcPINK1 directly phosphorylated ubiquitin and phospho-ubiquitin activated Parkin E3 ubiquitin ligase activity in cell-free assays. In cells, the phosphomimetic ubiquitin mutant S65D bound and activated Parkin. Furthermore, expression of ubiquitin S65A, a mutant that cannot be phosphorylated by PINK1, inhibited Parkin translocation to damaged mitochondria. These results explain a feed-forward mechanism of PINK1-mediated initiation of Parkin E3 ligase activity.     

A Secreted Effector Protein of Ustilago maydis Guides Maize Leaf Cells to Form Tumors

The biotrophic smut fungus Ustilago maydis infects all aerial organs of maize (Zea mays) and induces tumors in the plant tissues. U. maydis deploys many effector proteins to manipulate its host. Previously, deletion analysis demonstrated that several effectors have important functions in inducing tumor expansion specifically in maize leaves. Here, we present the functional characterization of the effector See1 (Seedling efficient effector1). See1 is required for the reactivation of plant DNA synthesis, which is crucial for tumor progression in leaf cells. By contrast, See1 does not affect tumor formation in immature tassel floral tissues, where maize cell proliferation occurs independent of fungal infection. See1 interacts with a maize homolog of SGT1 (Suppressor of G2 allele of skp1), a factor acting in cell cycle progression in yeast (Saccharomyces cerevisiae) and an important component of plant and human innate immunity. See1 interferes with the MAPK-triggered phosphorylation of maize SGT1 at a monocot-specific phosphorylation site. We propose that See1 interferes with SGT1 activity, resulting in both modulation of immune responses and reactivation of DNA synthesis in leaf cells. This identifies See1 as a fungal effector that directly and specifically contributes to the formation of leaf tumors in maize.     

SGT1 encodes an essential component of the yeast kinetochore assembly pathway and a novel subunit of the SCF ubiquitin ligase complex.

We have identified SGT1 as a dosage suppressor of skp1-4, a mutation causing defects in yeast kinetochore function. Sgt1p physically associates with Skp1p in vivo and in vitro. SGT1 is an essential gene, and different sgt1 conditional mutants arrest with either a G1 or G2 DNA content. Genetic and phenotypic analyses of sgt1-3 (G2 allele) mutants support an essential role in kinetochore function. Sgt1p is required for assembling the yeast kinetochore complex, CBF3, via activation of Ctf13p. Sgt1p also associates with SCF (Skp1p/Cdc53p/F box protein) ubiquitin ligase. sgt1-5 (G1 allele) mutants are defective in Sic1p turnover in vivo and Cln1p ubiquitination in vitro. Human SGT1 rescues an sgt1 null mutation, suggesting that the function of SGT1 is conserved in evolution.     

ESCRT-Independent Budding of HIV-1 Gag Virus-Like Particles from Saccharomyces cerevisiae Spheroplasts

Heterologous expression of HIV-1 Gag in a variety of host cells results in its packaging into virus-like particles (VLPs) that are subsequently released into the extracellular milieu. This phenomenon represents a useful tool for probing cellular factors required for viral budding and has contributed to the discovery of roles for ubiquitin ligases and the endosomal sorting complexes required for transport (ESCRTs) in viral budding. These factors are highly conserved throughout eukaryotes and have been studied extensively in the yeast Saccharomyces cerevisiae, a model eukaryote previously utilized as a host for the production of VLPs. We used heterologous expression of HIV Gag in yeast spheroplasts to examine the role of ESCRTs and associated factors (Rsp5, a HECT ubiquitin ligase of the Nedd4 family; Bro1, a homolog of Alix; and Vps4, the AAA-ATPase required for ESCRT function in all contexts/organisms investigated) in the generation of VLPs. Our data reveal: 1) characterized Gag-ESCRT interaction motifs (late domains) are not required for VLP budding, 2) loss of function alleles of the essential HECT ubiquitin ligase Rsp5 do not display defects in VLP formation, and 3) ESCRT function is not required for VLP formation from spheroplasts. These results suggest that the egress of HIV Gag from yeast cells is distinct from the most commonly described mode of exit from mammalian cells, instead mimicking ESCRT-independent VLP formation observed in a subset of mammalian cells. As such, budding of Gag from yeast cells appears to represent ESCRT-independent budding relevant to viral replication in at least some situations. Thus the myriad of genetic and biochemical tools available in the yeast system may be of utility in the study of this aspect of viral budding.     

The Shigella Type Three Secretion System Effector OspG Directly and Specifically Binds to Host Ubiquitin for Activation

The genus Shigella infects human gut epithelial cells to cause diarrhea and gastrointestinal disorders. Like many other Gram-negative bacterial pathogens, the virulence of Shigella spp. relies on a conserved type three secretion system that delivers a handful of effector proteins into host cells to manipulate various host cell physiology. However, many of the Shigella type III effectors remain functionally uncharacterized. Here we observe that OspG, one of the Shigella effectors, interacted with ubiquitin conjugates and poly-ubiquitin chains of either K48 or K63 linkage in eukaryotic host cells. Purified OspG protein formed a stable complex with ubiquitin but showed no interactions with other ubiquitin-like proteins. OspG binding to ubiquitin required the carboxyl terminal helical region in OspG and the canonical I44-centered hydrophobic surface in ubiquitin. OspG and OspG-homologous effectors, NleH1/2 from enteropathogenic E coli (EPEC), contain sub-domains I-VII of eukaryotic serine/threonine kinase. GST-tagged OspG and NleH1/2 could undergo autophosphorylation, the former of which was significantly stimulated by ubiquitin binding. Ubiquitin binding was also required for OspG functioning in attenuating host NF-κB signaling. Our data illustrate a new mechanism that bacterial pathogen like Shigella exploits ubiquitin binding to activate its secreted virulence effector for its functioning in host eukaryotic cells.     

CUL2LRR1, TRAIP and p97 control CMG helicase disassembly in the mammalian cell cycle

The eukaryotic replisome is disassembled in each cell cycle, dependent upon ubiquitylation of the CMG helicase. Studies of Saccharomyces cerevisiae, Caenorhabditis elegans and Xenopus laevis have revealed surprising evolutionary diversity in the ubiquitin ligases that control CMG ubiquitylation, but regulated disassembly of the mammalian replisome has yet to be explored. Here, we describe a model system for studying the ubiquitylation and chromatin extraction of the mammalian CMG replisome, based on mouse embryonic stem cells. We show that the ubiquitin ligase CUL2^LRR1 is required for ubiquitylation of the CMG‐MCM7 subunit during S‐phase, leading to disassembly by the p97 ATPase. Moreover, a second pathway of CMG disassembly is activated during mitosis, dependent upon the TRAIP ubiquitin ligase that is mutated in primordial dwarfism and mis‐regulated in various cancers. These findings indicate that replisome disassembly in diverse metazoa is regulated by a conserved pair of ubiquitin ligases, distinct from those present in other eukaryotes.     

Host Factors Required for Modulation of Phagosome Biogenesis and Proliferation of Francisella tularensis within the Cytosol

Francisella tularensis is a highly infectious facultative intracellular bacterium that can be transmitted between mammals by arthropod vectors. Similar to many other intracellular bacteria that replicate within the cytosol, such as Listeria, Shigella, Burkholderia, and Rickettsia, the virulence of F. tularensis depends on its ability to modulate biogenesis of its phagosome and to escape into the host cell cytosol where it proliferates. Recent studies have identified the F. tularensis genes required for modulation of phagosome biogenesis and escape into the host cell cytosol within human and arthropod-derived cells. However, the arthropod and mammalian host factors required for intracellular proliferation of F. tularensis are not known. We have utilized a forward genetic approach employing genome-wide RNAi screen in Drosophila melanogaster-derived cells. Screening a library of ∼21,300 RNAi, we have identified at least 186 host factors required for intracellular bacterial proliferation. We silenced twelve mammalian homologues by RNAi in HEK293T cells and identified three conserved factors, the PI4 kinase PI4KCA, the ubiquitin hydrolase USP22, and the ubiquitin ligase CDC27, which are also required for replication in human cells. The PI4KCA and USP22 mammalian factors are not required for modulation of phagosome biogenesis or phagosomal escape but are required for proliferation within the cytosol. In contrast, the CDC27 ubiquitin ligase is required for evading lysosomal fusion and for phagosomal escape into the cytosol. Although F. tularensis interacts with the autophagy pathway during late stages of proliferation in mouse macrophages, this does not occur in human cells. Our data suggest that F. tularensis utilizes host ubiquitin turnover in distinct mechanisms during the phagosomal and cytosolic phases and phosphoinositide metabolism is essential for cytosolic proliferation of F. tularensis. Our data will facilitate deciphering molecular ecology, patho-adaptation of F. tularensis to the arthropod vector and its role in bacterial ecology and patho-evolution to infect mammals.     

The Hsc/Hsp70 co-chaperone network controls antigen aggregation and presentation during maturation of professional antigen presenting cells

The maturation of mouse macrophages and dendritic cells involves the transient deposition of ubiquitylated proteins in the form of dendritic cell aggresome-like induced structures (DALIS). Transient DALIS formation was used here as a paradigm to study how mammalian cells influence the formation and disassembly of protein aggregates through alterations of their proteostasis machinery. Co-chaperones that modulate the interplay of Hsc70 and Hsp70 with the ubiquitin-proteasome system (UPS) and the autophagosome-lysosome pathway emerged as key regulators of this process. The chaperone-associated ubiquitin ligase CHIP and the ubiquitin-domain protein BAG-1 are essential for DALIS formation in mouse macrophages and bone-marrow derived dendritic cells (BMDCs). CHIP also cooperates with BAG-3 and the autophagic ubiquitin adaptor p62 in the clearance of DALIS through chaperone-assisted selective autophagy (CASA). On the other hand, the co-chaperone HspBP1 inhibits the activity of CHIP and thereby attenuates antigen sequestration. Through a modulation of DALIS formation CHIP, BAG-1 and HspBP1 alter MHC class I mediated antigen presentation in mouse BMDCs. Our data show that the Hsc/Hsp70 co-chaperone network controls transient protein aggregation during maturation of professional antigen presenting cells and in this way regulates the immune response. Similar mechanisms may modulate the formation of aggresomes and aggresome-like induced structures (ALIS) in other mammalian cell types.     

The CUL7/F-box and WD Repeat Domain Containing 8 (CUL7/Fbxw8) Ubiquitin Ligase Promotes Degradation of Hematopoietic Progenitor Kinase 1

HPK1, a member of mammalian Ste20-like serine/threonine kinases, is lost in >95% pancreatic cancer through proteasome-mediated degradation. However, the mechanism of HPK1 loss has not been defined. The aims of this study are to identify the ubiquitin ligase and to examine the mechanisms that targets HPK1 degradation. We found that the CUL7/Fbxw8 ubiquitin ligase targeted HPK1 for degradation via the 26 S proteasome. The ubiquitination of HPK1 required its kinase activity and autophosphorylation. Wild-type protein phosphatase 4 (PP4), but not the phosphatase-dead PP4 mutant, PP4-RL, inhibits the interaction of Fbxw8 with HPK1 and Fbxw8-mediated ubiquitination of HPK1. In addition, we showed that Thr-355 of HPK1 is a key PP4 dephosphorylation site, through which CUL7/Fbxw8 ubiquitin ligase and PP4 regulates HPK1 stability. Knockdown of Fbxw8 restores endogenous HPK1 protein expression and inhibits cell proliferation of pancreatic cancer cells. Our study demonstrated that targeted degradation of HPK1 by the CUL7/Fbxw8 ubiquitin ligase constitutes a negative-feedback loop to restrain the activity of HPK1 and that CUL7/Fbxw8 ubiquitin ligase promotes pancreatic cancer cell proliferation. CUL7/Fbxw8 ubiquitin ligase-mediated HPK1 degradation revealed a direct link and novel role of CUL7/Fbxw8 ubiquitin ligase in the MAPK pathway, which plays a critical role in cell proliferation and differentiation.