Protein Kinase C Enzymes in the Hematopoietic and Immune Systems.

The protein kinase C (PKC) family, discovered in the late 1970s, is composed of at least 10 serine/threonine kinases, divided into three groups based on their molecular architecture and cofactor requirements. PKC enzymes have been conserved throughout evolution and are expressed in virtually all cell types; they represent critical signal transducers regulating cell activation, differentiation, proliferation, death, and effector functions. PKC family members play important roles in a diverse array of hematopoietic and immune responses. This review covers the discovery and history of this enzyme family, discusses the roles of PKC enzymes in the development and effector functions of major hematopoietic and immune cell types, and points out gaps in our knowledge, which should ignite interest and further exploration, ultimately leading to better understanding of this enzyme family and, above all, its role in the many facets of the immune system.

Simultaneous Loss of Both Atypical Protein Kinase C Genes in the Intestinal Epithelium Drives Serrated Intestinal Cancer by Impairing Immunosurveillance.

Serrated adenocarcinoma, an alternative pathway for colorectal cancer (CRC) development, accounts for 15%-30% of all CRCs and is aggressive and treatment resistant. We show that the expression of atypical protein kinase C ζ (PKCζ) and PKCλ/ι was reduced in human serrated tumors. Simultaneous inactivation of the encoding genes in the mouse intestinal epithelium resulted in spontaneous serrated tumorigenesis that progressed to advanced cancer with a strongly reactive and immunosuppressive stroma. Whereas epithelial PKCλ/ι deficiency led to immunogenic cell death and the infiltration of CD8+ T cells, which repressed tumor initiation, PKCζ loss impaired interferon and CD8+ T cell responses, which resulted in tumorigenesis. Combined treatment with a TGF-ß receptor inhibitor plus anti-PD-L1 checkpoint blockade showed synergistic curative activity. Analysis of human samples supported the relevance of these kinases in the immunosurveillance defects of human serrated CRC. These findings provide insight into avenues for the detection and treatment of this poor-prognosis subtype of CRC.

The lymphoid lineage-specific actin-uncapping protein Rltpr is essential for costimulation via CD28 and the development of regulatory T cells.

Although T cell activation can result from signaling via T cell antigen receptor (TCR) alone, physiological T cell responses require costimulation via the coreceptor CD28. Through the use of an N-ethyl-N-nitrosourea-mutagenesis screen, we identified a mutation in Rltpr. We found that Rltpr was a lymphoid cell-specific, actin-uncapping protein essential for costimulation via CD28 and the development of regulatory T cells. Engagement of TCR-CD28 at the immunological synapse resulted in the colocalization of CD28 with both wild-type and mutant Rltpr proteins. However, the connection between CD28 and protein kinase C-θ and Carma1, two key effectors of CD28 costimulation, was abrogated in T cells expressing mutant Rltpr, and CD28 costimulation did not occur in those cells. Our findings provide a more complete model of CD28 costimulation in which Rltpr has a key role.

PRKCI promotes immune suppression in ovarian cancer.

A key feature of high-grade serous ovarian carcinoma (HGSOC) is frequent amplification of the 3q26 locus harboring PRKC-ι (PRKCI). Here, we show that PRKCI is also expressed in early fallopian tube lesions, called serous tubal intraepithelial carcinoma. Transgenic mouse studies establish PRKCI as an ovarian cancer-specific oncogene. Mechanistically, we show that the oncogenic activity of PRKCI relates in part to the up-regulation of TNFα to promote an immune-suppressive tumor microenvironment characterized by an abundance of myeloid-derived suppressor cells and inhibition of cytotoxic T-cell infiltration. Furthermore, system-level and functional analyses identify YAP1 as a downstream effector in tumor progression. In human ovarian cancers, high PRKCI expression also correlates with high expression of TNFα and YAP1 and low infiltration of cytotoxic T cells. The PRKCI-YAP1 regulation of the tumor immunity provides a therapeutic strategy for highly lethal ovarian cancer.

Extracellular Nucleotides and Histamine Suppress TLR3- and RIG-I-Mediated Release of Antiviral IFNs from Human Airway Epithelial Cells.

Respiratory viruses stimulate the release of antiviral IFNs from the airway epithelium. Previous studies have shown that asthmatic patients show diminished release of type I and type III IFNs from bronchial epithelia. However, the mechanism of this suppression is not understood. In this study, we report that extracellular nucleotides and histamine, which are elevated in asthmatic airways, strongly inhibit release of type I and type III IFNs from human bronchial airway epithelial cells (AECs). Specifically, ATP, UTP, and histamine all inhibited the release of type I and type III IFNs from AECs induced by activation of TLR3, retinoic acid-inducible gene I (RIG-I), or cyclic GMP-AMP synthase-STING. This inhibition was at least partly mediated by Gq signaling through purinergic P2Y2 and H1 receptors, but it did not involve store-operated calcium entry. Pharmacological blockade of protein kinase C partially reversed inhibition of IFN production. Conversely, direct activation of protein kinase C with phorbol esters strongly inhibited TLR3- and RIG-I-mediated IFN production. Inhibition of type I and type III IFNs by ATP, UTP, histamine, and the proteinase-activated receptor 2 (PAR2) receptor agonist SLIGKV also occurred in differentiated AECs grown at an air-liquid interface, indicating that the suppression is conserved following mucociliary differentiation. Importantly, histamine and, more strikingly, ATP inhibited type I IFN release from human airway cells infected with live influenza A virus or rhinovirus 1B. These results reveal an important role for extracellular nucleotides and histamine in attenuating the induction of type I and III IFNs from AECs and help explain the molecular basis of the suppression of IFN responses in asthmatic patients.

A cascade of protein kinase C isozymes promotes cytoskeletal polarization in T cells.

Polarization of the T cell microtubule-organizing center (MTOC) toward the antigen-presenting cell (APC) is driven by the accumulation of diacylglycerol (DAG) at the immunological synapse (IS). The mechanisms that couple DAG to the MTOC are not known. By single-cell photoactivation of the T cell antigen receptor (TCR), we found that three distinct isoforms of protein kinase C (PKC) were recruited by DAG to the IS in two steps. PKC-ɛ and PKC-η accumulated first in a broad region of membrane, whereas PKC-θ arrived later in a smaller zone. Functional experiments indicated that PKC-θ was required for MTOC reorientation and that PKC-ɛ and PKC-η operated redundantly to promote the recruitment of PKC-θ and subsequent polarization responses. Our results establish a previously uncharacterized role for PKC proteins in T cell polarity.

A motif in the V3 domain of the kinase PKC-θ determines its localization in the immunological synapse and functions in T cells via association with CD28.

Protein kinase C-θ (PKC-θ) translocates to the center of the immunological synapse, but the underlying mechanism and its importance in T cell activation are unknown. Here we found that the V3 domain of PKC-θ was necessary and sufficient for localization to the immunological synapse mediated by association with the coreceptor CD28 and dependent on the kinase Lck. We identified a conserved proline-rich motif in V3 required for association with CD28 and immunological synapse localization. We found association with CD28 to be essential for PKC-θ-mediated downstream signaling and the differentiation of T helper type 2 cells (T(H)2 cells) and interleukin 17-producing helper T cells (T(H)17 cells) but not of T helper type 1 cells (T(H)1 cells). Ectopic expression of V3 sequestered PKC-θ from the immunological synapse and interfered with its functions. Our results identify a unique mode of CD28 signaling, establish a molecular basis for the immunological synapse localization of PKC-θ and indicate V3-based 'decoys' may be therapeutic modalities for T cell-mediated inflammatory diseases.

The kinase GLK controls autoimmunity and NF-κB signaling by activating the kinase PKC-θ in T cells.

Protein kinase C-θ (PKC-θ) is required for activation of the transcription factor NF-κB induced by signaling via the T cell antigen receptor (TCR); however, the direct activator of PKC-θ is unknown. We report that the kinase GLK (MAP4K3) directly activated PKC-θ during TCR signaling. TCR signaling activated GLK by inducing its direct interaction with the upstream adaptor SLP-76. GLK-deficient mice had impaired immune responses and were resistant to experimental autoimmune encephalomyelitis. Consistent with that, people with systemic lupus erythematosus had considerable enhanced GLK expression and activation of PKC-θ and the kinase IKK in T cells, and the frequency of GLK-overexpressing T cells was directly correlated with disease severity. Thus, GLK is a direct activator of PKC-θ, and activation of GLK-PKC-θ-IKK could be used as new diagnostic biomarkers and therapeutic targets for systemic lupus erythematosus.

Pattern recognition receptor signaling in human dendritic cells is enhanced by ICOS ligand and modulated by the Crohn's disease ICOSLG risk allele.

Inflammatory bowel disease (IBD) is characterized by dysregulated intestinal immune homeostasis and cytokine secretion. Multiple loci are associated with IBD, but a functional explanation is missing for most. Here we found that pattern-recognition receptor (PRR)-induced cytokine secretion was diminished in human monocyte-derived dendritic cells (MDDC) from rs7282490 ICOSLG GG risk carriers. Homotypic interactions between the costimulatory molecule ICOS and the ICOS ligand on MDDCs amplified nucleotide-binding oligomerization domain 2 (NOD2)-initiated cytokine secretion. This amplification required arginine residues in the ICOSL cytoplasmic tail that recruited the adaptor protein RACK1 and the kinases PKC and JNK leading to PKC, MAPK, and NF-κB activation. MDDC from rs7282490 GG risk-carriers had reduced ICOSL expression and PRR-initiated signaling and this loss-of-function ICOSLG risk allele associated with an ileal Crohn's disease phenotype, similar to polymorphisms in NOD2. Taken together, ICOSL amplifies PRR-initiated outcomes, which might contribute to immune homeostasis.

Latency reversal agents modulate HIV antigen processing and presentation to CD8 T cells.

Latency reversal agents (LRA) variably induce HIV re-expression in CD4 T cells but reservoirs are not cleared. Whether HIV epitope presentation is similar between latency reversal and initial infection of CD4 T cells is unknown yet crucial to define immune responses able to detect HIV-infected CD4 T cells after latency reversal. HIV peptides displayed by MHC comes from the intracellular degradation of proteins by proteasomes and post-proteasomal peptidases but the impact of LRAs on antigen processing is not known. Here we show that HDAC inhibitors (HDCAi) reduced cytosolic proteolytic activities while PKC agonists (PKCa) increased them to a lesser extent than that induced by TCR activation. During the cytosolic degradation of long HIV peptides in LRA-treated CD4 T cells extracts, HDACi and PKCa modulated degradation patterns of peptides and altered the production of HIV epitopes in often opposite ways. Beyond known HIV epitopes, HDACi narrowed the coverage of HIV antigenic fragments by 8-11aa degradation peptides while PKCa broadened it. LRAs altered HIV infection kinetics and modulated CD8 T cell activation in an epitope- and time-dependent manner. Interestingly the efficiency of endogenous epitope processing and presentation to CD8 T cells was increased by PKCa Ingenol at early time points despite low levels of antigens. LRA-induced modulations of antigen processing should be considered and exploited to enhance and broaden HIV peptide presentation by CD4 T cells and to improve immune recognition after latency reversal. This property of LRAs, if confirmed with other antigens, might be exploited to improve immune detection of diseased cells beyond HIV.

Galectin-9 and VISTA Expression Define Terminally Exhausted T Cells in HIV-1 Infection.

We report significant upregulation of Galectin-9 (Gal-9) and VISTA on both CD4+ and CD8+ T cells in HIV-infected human patients. Gal-9 and VISTA expression was associated with impaired T cells effector functions. Although Gal-9 was coexpressed with other coinhibitory receptors such as TIGIT, CD160, CD39, and VISTA, it was simultaneously coexpressed with PD-1. Coexpression of Gal-9 with PD-1 was associated with a more terminally exhausted T cell phenotype in HIV-1 patients. This was marked by higher expression of EOMES, blimp1, and Glut1 in Gal-9+ versus Gal-9- T cells, which is consistent with an exhausted T cell phenotype. Gal-9+ T cells exhibited the phenotype characteristics of effector T cells (CD45RA+, CD45RO-/lo, CD62L-, CD27lo) with higher T-bet expression. A positive correlation between the plasma viral load with the plasma Gal-9 levels in treatment-naive HIV patients and an inverse correlation between CD4 count with the frequency of CD4+Gal-9+ T cells were observed. Increased percentages of Gal-9+ T cells was evident in HIV-treated patients. Enhanced expression of Gal-9 on T cells following PMA stimulation via protein kinase C suggests persistent TCR stimulation as a potential contributing factor in Gal-9 upregulation in HIV patients. This was supported by the constant degranulation of Gal-9+ T cells. Moreover, CD44 clustering by Gal-9 may influence cytoskeleton rearrangement and coclustering of CD3, which likely impact initiation of signal transduction via TCR. Our preliminary data also confirm upregulation of Gal-9 on T cells in hepatitis B virus and HPV infections. These results demonstrate a novel role for Gal-9 and VISTA in HIV pathogenesis.

Protein Kinase C-η Deficiency Does Not Impair Antiviral Immunity and CD8+ T Cell Activation.

We reported that protein kinase C-η (PKCη) forms a novel (to our knowledge) signaling complex with the checkpoint inhibitory protein CTLA-4 in regulatory T cells (Tregs). This complex is required for the contact-dependent suppressive activity of Tregs, including suppression of antitumor immunity. However, the importance of PKCη in protective immunity mediated by T effector cells remains unclear. We used mice with germline or conditional Treg-specific deletion of Prkch, the PKCη-encoding gene, to explore CD8+ T cell-dependent antiviral immunity using the lymphocytic choriomeningitis virus Armstrong strain acute infection model as well as the in vitro activation of murine or human CD8+ T cells. Five days following infection, germline Prkch -/- mice displayed enhanced viral clearance compared with control mice. Similarly, Prkch Treg-specific conditional knockout mice also showed improved viral clearance and displayed enhanced expression of granzyme B and IFN-γ by both virus-specific and total CD8+ T cells, demonstrating that enhanced viral clearance in germline Prkch -/- mice is caused by PKCη deficiency in Tregs and the resulting functional defect of Prkch -/- Tregs. In addition, purified Prkch -/- mouse CD8+ T cells as well as PRKCH knockdown human CD8+ T cells displayed intact, or even enhanced, T cell activation in vitro as measured by proliferation and expression of granzyme B and IFN-γ. Thus, global PKCη deletion does not impair overall CD8+ T cell-mediated immunity, including antiviral immunity, implying that selective pharmacological PKCη inhibition could be safely used in vivo to inhibit undesired contact-dependent suppression by Tregs and, thus, enhance tumor-specific and, likely, virus-specific immunity.

Polarized secretion of lysosomes at the B cell synapse couples antigen extraction to processing and presentation.

Engagement of the B cell receptor (BCR) by surface-tethered antigens (Ag) leads to formation of a synapse that promotes Ag uptake for presentation onto major histocompatibility complex class II (MHCII) molecules. We have highlighted the membrane trafficking events and associated molecular mechanisms involved in Ag extraction and processing at the B cell synapse. MHCII-containing lysosomes are recruited to the synapse where they locally undergo exocytosis, allowing synapse acidification and the extracellular release of hydrolases that promote the extraction of the immobilized Ag. Lysosome recruitment and secretion results from the polarization of the microtubule-organizing center (MTOC), which relies on the cell division cycle (Cdc42)-downstream effector, atypical protein kinase C (aPKCζ). aPKCζ is phosphorylated upon BCR engagement, associates to lysosomal vesicles, and is required for their polarized secretion at the B cell synapse. Regulation of B lymphocyte polarity therefore emerges as a central mechanism that couples Ag extraction to Ag processing and presentation.

Role of novel protein kinase C in neuroendocrine-immune regulatory network in haemocytes of Litopenaeus vannamei: An in vitro approach.

Shrimp lack adaptive immune systems and mainly rely on the cellular and humoral defences, involving the haemocytes (functionally analogous to vertebrate leukocytes) in non-self matter recognition, elimination, and in downstream coagulation. Furthermore, the linkage between stress-induced catecholamine (CA), a class of biogenic amines (BAs), releasing and immunological responses has been detected in shrimp. Varied isotypes of protein kinase C (PKC) regulate multiple cellular processes following their specific location and distribution within the cells, and a novel PKC identified in Litopenaeus vannamei (termed as LvnPKC) is proposed to mediate signaling transduction of immunocompetence and BA biosynthesis. In the present study, we analyzed the effects of the LvnPKC-silenced haemocytes by co-incubating with its dsRNA on the immune responses specific to prophenoloxidase (proPO) and antioxidant systems as well as phagocytic activity. In addition, the capability of haemocytes to produce BAs was assessed. The results revealed that LvnPKC-silenced haemocytes can induce interference in phenoloxidase and superoxide dismutase activities, respiratory bursts, and phagocytic activity; meanwhile, the disturbed gene expressions of proPO activating enzyme, proPOII, lipopolysaccharide- and ß-1,3-glucan-binding protein, and cytosolic manganese superoxide dismutase were detected. The same deviated pattern was observed in tyrosine, dopamine, and norepinephrine levels, and in dopamine ß-hydroxylase (DBH) activity and gene expressions of tyrosine hydroxylase, DOPA decarboxylase, and DBH involving in BA biosynthesis. Taken together, these results suggest that the immunocompetence and BA biosynthesis of haemocytes can be mediated via LvPKC signaling transduction, which proved the presence of a neuroendocrine-immune regulatory network in haemocytes.

Mammalian target of rapamycin protein complex 2 regulates differentiation of Th1 and Th2 cell subsets via distinct signaling pathways.

Many functions of the mammalian target of rapamycin (mTOR) complex 1 (mTORC1) have been defined, but relatively little is known about the biology of an alternative mTOR complex, mTORC2. We showed that conditional deletion of rictor, an essential subunit of mTORC2, impaired differentiation into T helper 1 (Th1) and Th2 cells without diversion into FoxP3(+) status or substantial effect on Th17 cell differentiation. mTORC2 promoted phosphorylation of protein kinase B (PKB, or Akt) and PKC, Akt activity, and nuclear NF-kappaB transcription factors in response to T cell activation. Complementation with active Akt restored only T-bet transcription factor expression and Th1 cell differentiation, whereas activated PKC-theta reverted only GATA3 transcription factor and the Th2 cell defect of mTORC2 mutant cells. Collectively, the data uncover vital mTOR-PKC and mTOR-Akt connections in T cell differentiation and reveal distinct pathways by which mTORC2 regulates development of Th1 and Th2 cell subsets.

Involvement of a novel protein kinase C (nPKC) in immunocompetence in hemocytes of white shrimp, Litopenaeus vannamei.

Complementary (c)DNA encoding novel protein kinase C (PKC) messenger (m)RNA of the white shrimp Litopenaeus vannamei, consisted of 2454-bp cDNA containing an open reading frame (ORF) of 2232 bp, belonging to the novel (n)PKC family of proteins characterized by their containing two phorbol ester/diacylglycerol-binding domains (C1 domain), a C2 domain, and a catalytic domain of the serine/threonine kinase, designated LvnPKC. A comparison of amino acid sequences showed that LvnPKC was closely related to arthropod nPKC. LvnPKC cDNA was detected in all tested tissues with a real-time PCR including the hepatopancreas, gills, muscles, subcuticular epithelium, abdominal nerve, thoracic nerve, brain, the stomach, heart, and especially in hemocytes and the intestines. Moreover, significantly upregulated LvnPKC expression was only observed in the eyestalk, brain, and hepatopancreas of shrimp transferred from 28 °C to 18 °C for 30 min. Induction of LvnPKC expression in hemocytes of L. vannamei injected with Vibrio alginolyticus at 105 cfu shrimp-1 was detected earlier than in those injected with 103 cfu shrimp-1. Shrimp received LvnPKC-dsRNA for 1 days specifically depleted the expression of LvnPKC mRNA in hemocytes compared those of diethylpyrocarbonate water treatment. After that, significantly decreased expressions of lipopolysaccharide - and ß-1,3-glucan-binding protein, prophenoloxidase-activating enzyme, peroxinectin, prophenoloxidase I, and prophenoloxidase II in the prophenoloxidase-activating system; lysozyme and cytosolic manganese superoxide dismutase and mitochondrial manganese superoxide dismutase in the antioxidant system were observed. We therefore concluded that LvnPKC is involved in immune defense of L. vannamei exposed to hypothermal stress or infected with V. alginolyticus.

Golgi-Associated Protein Kinase C-ε Is Delivered to Phagocytic Cups: Role of Phosphatidylinositol 4-Phosphate.

Protein kinase C-ε (PKC-ε) at phagocytic cups mediates the membrane fusion necessary for efficient IgG-mediated phagocytosis. The C1B and pseudosubstrate (εPS) domains are necessary and sufficient for this concentration. C1B binds diacylglycerol; the docking partner for εPS is unknown. Liposome assays revealed that the εPS binds phosphatidylinositol 4-phosphate (PI4P) and PI(3,5)P2 Wortmannin, but not LY294002, inhibits PKC-ε concentration at cups and significantly reduces the rate of phagocytosis. As Wortmannin inhibits PI4 kinase, we hypothesized that PI4P mediates the PKC-ε concentration at cups and the rate of phagocytosis. PKC-ε colocalizes with the trans-Golgi network (TGN) PI4P reporter, P4M, suggesting it is tethered at the TGN. Real-time imaging of GFP-PKC-ε-expressing macrophages revealed a loss of Golgi-associated PKC-ε during phagocytosis, consistent with a Golgi-to-phagosome translocation. Treatment with PIK93, a PI4 kinase inhibitor, reduces PKC-ε at both the TGN and the cup, decreases phagocytosis, and prevents the increase in capacitance that accompanies membrane fusion. Finally, expression of the Golgi-directed PI4P phosphatase, hSac1-K2A, recapitulates the PIK93 phenotype, confirming that Golgi-associated PI4P is critical for efficient phagocytosis. Together these data are consistent with a model in which PKC-ε is tethered to the TGN via an εPS-PI4P interaction. The TGN-associated pool of PKC-ε concentrates at the phagocytic cup where it mediates the membrane fusion necessary for phagocytosis. The novelty of these data lies in the demonstration that εPS binds PI4P and PI(3,5)P2 and that PI4P is necessary for PKC-ε localization at the TGN, its translocation to the phagocytic cup, and the membrane fusion required for efficient Fc [γ] receptor-mediated phagocytosis.

Complement C5a Functions as a Master Switch for the pH Balance in Neutrophils Exerting Fundamental Immunometabolic Effects.

During sepsis, excessive activation of the complement system with generation of the anaphylatoxin C5a results in profound disturbances in crucial neutrophil functions. Moreover, because neutrophil activity is highly dependent on intracellular pH (pHi), we propose a direct mechanistic link between complement activation and neutrophil pHi In this article, we demonstrate that in vitro exposure of human neutrophils to C5a significantly increased pHi by selective activation of the sodium/hydrogen exchanger. Upstream signaling of C5a-mediated intracellular alkalinization was dependent on C5aR1, intracellular calcium, protein kinase C, and calmodulin, and downstream signaling regulated the release of antibacterial myeloperoxidase and lactoferrin. Notably, the pH shift caused by C5a increased the glucose uptake and activated glycolytic flux in neutrophils, resulting in a significant release of lactate. Furthermore, C5a induced acidification of the extracellular micromilieu. In experimental murine sepsis, pHi of blood neutrophils was analogously alkalinized, which could be normalized by C5aR1 inhibition. In the clinical setting of sepsis, neutrophils from patients with septic shock likewise exhibited a significantly increased pHi These data suggest a novel role for the anaphylatoxin C5a as a master switch of the delicate pHi balance in neutrophils resulting in profound inflammatory and metabolic changes that contribute to hyperlactatemia during sepsis.

Prkcz null mice show normal learning and memory.

Protein kinase M-ζ (PKM-ζ) is a constitutively active form of atypical protein kinase C that is exclusively expressed in the brain and implicated in the maintenance of long-term memory. Most studies that support a role for PKM-ζ in memory maintenance have used pharmacological PKM-ζ inhibitors such as the myristoylated zeta inhibitory peptide (ZIP) or chelerythrine. Here we use a genetic approach and target exon 9 of the Prkcz gene to generate mice that lack both protein kinase C-ζ (PKC-ζ) and PKM-ζ (Prkcz(-/-) mice). Prkcz(-/-) mice showed normal behaviour in a cage environment and in baseline tests of motor function and sensory perception, but displayed reduced anxiety-like behaviour. Notably, Prkcz(-/-) mice did not show deficits in learning or memory in tests of cued fear conditioning, novel object recognition, object location recognition, conditioned place preference for cocaine, or motor learning, when compared with wild-type littermates. ZIP injection into the nucleus accumbens reduced expression of cocaine-conditioned place preference in Prkcz(-/-) mice. In vitro, ZIP and scrambled ZIP inhibited PKM-ζ, PKC-ι and PKC-ζ with similar inhibition constant (K(i)) values. Chelerythrine was a weak inhibitor of PKM-ζ (K(i) = 76 µM). Our findings show that absence of PKM-ζ does not impair learning and memory in mice, and that ZIP can erase reward memory even when PKM-ζ is not present.

Leveraging Siglec-8 endocytic mechanisms to kill human eosinophils and malignant mast cells.

BACKGROUND: Sialic acid-binding immunoglobulin-like lectin (Siglec)-8 is a cell-surface protein expressed selectively on human eosinophils, mast cells, and basophils, making it an ideal target for the treatment of diseases involving these cell types. However, the effective delivery of therapeutic agents to these cells requires an understanding of the dynamics of Siglec-8 surface expression. OBJECTIVES: We sought to determine whether Siglec-8 is endocytosed in human eosinophils and malignant mast cells, identify mechanisms underlying its endocytosis, and demonstrate whether a toxin can be targeted to Siglec-8-bearing cells to kill these cells. METHODS: Siglec-8 surface dynamics were examined by flow cytometry using peripheral blood eosinophils, mast cell lines, and Siglec-8-transduced cells in the presence of inhibitors targeting components of endocytic pathways. Siglec-8 intracellular trafficking was followed by confocal microscopy. The ribosome-inhibiting protein saporin was conjugated to a Siglec-8-specific antibody to examine the targeting of an agent to these cells through Siglec-8 endocytosis. RESULTS: Siglec-8 endocytosis required actin rearrangement, tyrosine kinase and protein kinase C activities, and both clathrin and lipid rafts. Internalized Siglec-8 localized to the lysosomal compartment. Maximal endocytosis in Siglec-8-transduced HEK293T cells required an intact immunoreceptor tyrosine-based inhibitory motif. Siglec-8 was also shuttled to the surface via a distinct pathway. Sialidase treatment of eosinophils revealed that Siglec-8 is partially masked by sialylated cis ligands. Targeting saporin to Siglec-8 consistently caused extensive cell death in eosinophils and the human mast cell leukemia cell line HMC-1.2. CONCLUSIONS: Therapeutic payloads can be targeted selectively to eosinophils and malignant mast cells by exploiting this Siglec-8 endocytic pathway.