Proviral integration sites for Moloney murine leukemia virus (PIM) kinases are upregulated at the protein level in response to hypoxia and have multiple protumorigenic functions, promoting cell growth, survival, and angiogenesis. However, the mechanism responsible for the induction of PIM in hypoxia remains unknown. Here, we examined factors affecting PIM kinase stability in normoxia and hypoxia. We found that PIM kinases were upregulated in hypoxia at the protein level but not at the mRNA level, confirming that PIMs were upregulated in hypoxia in a hypoxia inducible factor 1-independent manner. PIM kinases were less ubiquitinated in hypoxia than in normoxia, indicating that hypoxia reduced their proteasomal degradation. We identified the deubiquitinase ubiquitin-specific protease 28 (USP28) as a key regulator of PIM1 and PIM2 stability. The overexpression of USP28 increased PIM protein stability and total levels in both normoxia and hypoxia, and USP28-knockdown significantly increased the ubiquitination of PIM1 and PIM2. Interestingly, coimmunoprecipitation assays showed an increased interaction between PIM1/2 and USP28 in response to hypoxia, which correlated with reduced ubiquitination and increased protein stability. In a xenograft model, USP28-knockdown tumors grew more slowly than control tumors and showed significantly lower levels of PIM1 in vivo. In conclusion, USP28 blocked the ubiquitination and increased the stability of PIM1/2, particularly in hypoxia. These data provide the first insight into proteins responsible for controlling PIM protein degradation and identify USP28 as an important upstream regulator of this hypoxia-induced, protumorigenic signaling pathway.

Ehrlichia chaffeensisTRP120 effector has evolved short linear motif (SLiM) ligand mimicry to repurpose multiple evolutionarily conserved cellular signaling pathways including Wnt, Notch and Hedgehog. In this investigation, we demonstrate thatE. chaffeensisand recombinant TRP120 deactivate Hippo signaling resulting in activation of Hippo transcription coactivator Yap and target gene expression. Moreover, a homologous 6 amino acid (QDVASH) SLiM shared by TRP120 and Wnt3a/5a ligands phenocopied Yap and β-catenin activation induced byE. chaffeensis, rTRP120 and Wnt5a. Similar Hippo gene expression profiles were also stimulated byE. chaffeensis, rTRP120, SLiM and Wnt5a. Single siRNA knockdown of Hippo transcription co-activator/factors (Yap and TEAD) significantly decreasedE. chaffeensisinfection. Yap activation was abolished in THP-1 Wnt Frizzled-5 (Fzd5) receptor knockout cells (KO), demonstrating Fzd5 receptor dependence. In addition, TRP120 Wnt-SLiM antibody blocked Hippo deactivation (Yap activation). Expression of anti-apoptotic Hippo target geneSLC2A1(encodes glucose transporter 1; GLUT1) was upregulated byE. chaffeensisand corresponded to increased levels of GLUT1. Conversely, siRNA knockdown ofSLC2A1significantly inhibited infection. Higher GLUT1 levels correlated with increased BCL-xL and decreased Bax levels. Moreover, blocking Yap activation with the inhibitor Verteporfin induced apoptosis that corresponded to significant reductions in levels of GLUT1 and BCL-xL, and activation of Bax and Caspase-3 and -9. This study identifies a novel shared Wnt/Hippo SLiM ligand mimetic and demonstrates thatE. chaffeensisdeactivates the Hippo pathway to engage the anti-apoptotic Yap-GLUT1-BCL-xL axis.

Ehrlichia chaffeensishas evolved multiple strategies to evade innate defenses of the mononuclear phagocyte. Recently, we reported theE. chaffeensisTRP120 effector functions as a Notch ligand mimetic and a ubiquitin ligase that degrades the nuclear tumor suppressor, F-box and WD repeat domain-containing 7 (FBW7), a negative regulator of Notch. The Notch receptor intracellular domain (NICD) is known to inhibit apoptosis primarily by interacting with X-linked inhibitor of apoptosis protein (XIAP) to prevent degradation. In this study, we determinedE. chaffeensisactivation of Notch signaling increases XIAP levels, thereby inhibiting intrinsic apoptosis. Increased NICD and XIAP levels were detected duringE. chaffeensisinfection and after TRP120 Notch ligand mimetic peptide treatment. Conversely, XIAP levels were reduced in the presence of Notch inhibitor DAPT. Cytoplasmic colocalization of NICD and XIAP was observed during infection and a direct interaction was confirmed by co-immunoprecipitation. Procaspase levels increased temporally during infection, consistent with increased XIAP levels; however, knockdown of XIAP during infection significantly increased apoptosis and Caspase-3, -7 and -9 levels. Further, treatment with SM-164, a second mitochondrial activator of caspases (Smac/DIABLO) antagonist, resulted in decreased procaspase levels and increased caspase activation, induced apoptosis, and significantly decreased infection. In addition, iRNA knockdown of XIAP also decreased infection and significantly increased apoptosis. Moreover, ectopic expression of TRP120 HECT Ub ligase catalytically defective mutant in HeLa cells decreased NICD and XIAP levels and increased caspase activation compared to WT. This investigation reveals a mechanism wherebyE. chaffeensisrepurposes Notch signaling to stabilize XIAP and inhibit apoptosis.

As an obligately intracellular bacterial pathogen that selectively infects the mononuclear phagocyte, Ehrlichia chaffeensis has evolved sophisticated mechanisms to subvert innate immune defenses. While the bacterium accomplishes this through a variety of mechanisms, a rapidly expanding body of evidence has revealed that E. chaffeensis has evolved survival strategies that are directed by the versatile, intrinsically disordered, 120 kDa tandem repeat protein (TRP120) effector. E. chaffeensis establishes infection by manipulating multiple evolutionarily conserved cellular signaling pathways through effector-host interactions to subvert innate immune defenses. TRP120 activates these pathways using multiple functionally distinct, repetitive, eukaryote-mimicking short linear motifs (SLiMs) located within the tandem repeat domain that have evolved in nihilo. Functionally, the best characterized TRP120 SLiMs mimic eukaryotic ligands (SLiM-icry) to engage pathway-specific host receptors and activate cellular signaling, thereby repurposing these pathways to promote infection. Moreover, E. chaffeensis TRP120 contains SLiMs that are targets of post-translational modifications such as SUMOylation in addition to many other validated SLiMs that are curated in the eukaryotic linear motif (ELM) database. This review will explore the extracellular and intracellular roles TRP120 SLiM-icry plays during infection - mediated through a variety of SLiMs - that enable E. chaffeensis to subvert mononuclear phagocyte innate defenses.