C-terminal Noncatalytic Domain Research Articles

Biochemical Characterization of a Heat-Resistant κ-Carrageenase Capable of Tolerating High Temperatures up to 100 °C.

κ-Carrageenase plays a crucial role in the high-value utilization of carrageenan. Heat resistance is a key factor in the practical application of κ-carrageenase, as carrageenan exhibits gel-like properties. Previous studies have shown that the C-terminal noncatalytic domains (nonCDs) can affect the thermostability of κ-carrageenases. In this study, we expressed and characterized a κ-carrageenase, MtKC16A, which contains three nonCDs, from Microbulbifer thermotolerans. MtKC16A has the highest activity at 80 °C and pH 7.0. Surprisingly, it exhibits excellent heat resistance, with 71.58% relative activity at 100 °C and still retains over 50% residual activity after incubation at 100 °C for 60 min. Additionally, MtKC16A has been shown to have a dual substrate hydrolysis activity. It can degrade κ-carrageenan to produce highly single Nκ4 and degrade β/κ-carrageenan to produce Nκ2 and desulfated Nκ4 DA-G-DA-G4S, suggesting its potential in producing κ- and β/κ-hybrid oligosaccharides. Furthermore, we found that the unknown function domain (UNFD) in MtKC16A plays the most vital role among the three nonCDs. When this UNFD is truncated, the resulting mutants completely lose their catalytic ability at 100 °C. Finally, by introducing this UNFD to the C-terminal of another κ-carrageenase CaKC16B, we were able to improve its heat resistance at 100 °C.

Comparative Study on Enzymatic Characteristics of Two κ-Carrageenases from Carrageenan-Degrading Bacterium Catenovulum agarivorans DS2.

κ-Carrageenase plays an important role in achieving the high-value utilization of carrageenan. Factors such as the reaction temperature, thermal stability, catalytic efficiency, and product composition are key considerations for its large-scale application. Previous studies have shown that the C-terminal noncatalytic domains (nonCDs) could influence the enzymatic properties, of κ-carrageenases, providing a strategy for exploring κ-carrageenases with different properties, especially catalytic products. Accordingly, two κ-carrageenases (CaKC16A and CaKC16B), from the Catenovulum agarivorans DS2, were selected and further characterized. Bioinformatics analysis suggested that CaKC16A contained a nonCD but CaKC16B did not. CaKC16A exhibited better enzymatic properties than CaKC16B, including thermal stability, substrate affinity, and catalytic efficiency. After truncation of the nonCD of CaKC16A, its thermal stability, substrate affinity, and catalytic efficiency have significantly decreased, indicating the vital role of nonCD in maintaining a good enzymatic property. Moreover, CaKC16A degraded κ-carrageenan to produce a highly single κ-neocarratetrose, while CaKC16B produced a single κ-neocarrabiose. CaKC16A could degrade β/κ-carrageenan to produce a highly single desulfated κ-neocarrahexaose, while CaKC16B produced κ-neocarrabiose and desulfated κ-neocarratetrose. Furthermore, it was proposed that CaKC16A and CaKC16B participate in the B/KC metabolic pathway and serve different roles, providing new insight into obtaining κ-carrageenases with different properties.

Structural Optimization and Anticancer Activity of Polo-like Kinase 1 (Plk1) Polo-Box Domain (PBD) Inhibitors and Their Prodrugs.

Polo-like kinase 1 (Plk1), a mitotic kinase whose activity is widely upregulated in various human cancers, is considered an attractive target for anticancer drug discovery. Aside from the kinase domain, the C-terminal noncatalytic polo-box domain (PBD), which mediates the interaction with the enzyme's binding targets or substrates, has emerged as an alternative target for developing a new class of inhibitors. Various reported small molecule PBD inhibitors exhibit poor cellular efficacy and/or selectivity. Here, we report structure-activity relationship (SAR) studies on triazoloquinazolinone-derived inhibitors, such as 43 (a 1-thioxo-2,4-dihydrothieno[2,3-e][1,2,4]triazolo[4,3-a]pyrimidin-5(1H)-one) that effectively block Plk1, but not Plk2 and Plk3 PBDs, with improved affinity and drug-like properties. The range of prodrug moieties needed for thiol group masking of the active drugs has been expanded to increase cell permeability and mechanism-based cancer cell (L363 and HeLa) death. For example, a 5-thio-1-methyl-4-nitroimidazolyl prodrug 80, derived from 43, showed an improved cellular potency (GI50 4.1 μM). As expected, 80 effectively blocked Plk1 from localizing to centrosomes and kinetochores and consequently induced potent mitotic block and apoptotic cell death. Another prodrug 78 containing 9-fluorophenyl in place of the thiophene-containing heterocycle in 80 also induced a comparable degree of anti-Plk1 PBD effect. However, orally administered 78 was rapidly converted in the bloodstream to parent drug 15, which was shown be relatively stable toward in vivo oxidation due to its 9-fluorophenyl group in comparison to unsubstituted phenyl. Further derivatization of these inhibitors, particularly to improve the systemic prodrug stability, could lead to a new class of therapeutics against Plk1-addicted cancers.

Examination of differential glycoprotein preferences of N-acetylglucosaminyltransferase-IV isozymes a and b

The N-glycans attached to proteins contain various GlcNAc branches, the aberrant formation of which correlates with various diseases. N-Acetylglucosaminyltransferase-IVa (GnT-IVa or MGAT4A) and Gnt-IVb (or MGAT4B) are isoenzymes that catalyze the formation of the β1,4-GlcNAc branch in N-glycans. However, the functional differences between these isozymes remain unresolved. Here, using cellular and UDP-Glo enzyme assays, we discovered that GnT-IVa and GnT-IVb have distinct glycoprotein preferences both in cells and in vitro. Notably, we show that GnT-IVb acted efficiently on glycoproteins bearing an N-glycan premodified by GnT-IV. To further understand the mechanism of this reaction, we focused on the noncatalytic C-terminal lectin domain, which selectively recognizes the product glycans. Replacement of a nonconserved amino acid in the GnT-IVb lectin domain with the corresponding residue in GnT-IVa altered the glycoprotein preference of GnT-IVb to resemble that of GnT-IVa. Our findings demonstrate that the C-terminal lectin domain regulates differential substrate selectivity of GnT-IVa and GnT-IVb, highlighting a new mechanism by which N-glycan branches are formed on glycoproteins.

MT5-MMP controls APP and β-CTF/C99 metabolism through proteolytic-dependent and -independent mechanisms relevant for Alzheimer's disease.

We previously discovered the implication of membrane-type 5-matrix metalloproteinase (MT5-MMP) in Alzheimer's disease (AD) pathogenesis. Here, we shed new light on pathogenic mechanisms by which MT5-MMP controls the processing of amyloid precursor protein (APP) and the fate of amyloid beta peptide (Aβ) as well as its precursor C99, and C83. We found in human embryonic kidney cells (HEK) carrying the APP Swedish familial mutation (HEKswe) that deleting the C-terminal non-catalytic domains of MT5-MMP hampered its ability to process APP and release the soluble 95kDa form (sAPP95). Catalytically inactive MT5-MMP variants increased the levels of Aβ and promoted APP/C99 sorting in the endolysosomal system, likely through interactions of the proteinase C-terminal portion with C99. Most interestingly, the deletion of the C-terminal domain of MT5-MMP caused a strong degradation of C99 by the proteasome and prevented Aβ accumulation. These discoveries reveal new control of MT5-MMP over APP by proteolytic and non-proteolytic mechanisms driven by the C-terminal domains of the proteinase. The targeting of these non-catalytic domains of MT5-MMP could, therefore, provide new insights into the therapeutic regulation of APP-related pathology in AD.

Synthesis and structure-activity relationships of targeted protein degraders for the understudied kinase NEK9

Nek9 is a member of the understudied Nek family of dark kinases. Aberrant activation of Nek9 kinase signaling has been linked to poor keratinocyte differentiation phenotypes, and is a key driver of nevus comedonicus, a rare, localized form of acne. Nek9 also has essential scaffolding roles; during mitosis the non-catalytic C-terminal domain of Nek9 binds to Nek6 and Nek7, releasing them from an auto-inhibitory conformation, and enabling proper mitotic progression. Finally, Nek9 expression has been linked to cancer proliferation. SiRNA mediated Nek9 knock-down in a panel of cancer cell lines induces G1 cell cycle arrest and inhibits proliferation when p53 is also inactivated; cell lines with functional p53 are unaffected. Presently, no selective small molecule Nek9 chemical probes are available, though a subset of promiscuous kinase inhibitors have Nek9 activity. Recently described targeted protein degradation approaches have shown that degrader molecules based on multi-targeted kinase inhibitors may effect selective kinase degradation, despite binding to many targets. In this study we report the identification and SAR of potent degraders of the NEK9 kinase that represent attractive leads for further development. Future work is needed to optimize the selectivity of the compounds.

Identification of a New Heterocyclic Scaffold for Inhibitors of the Polo-Box Domain of Polo-like Kinase 1.

As a mitotic-specific target widely deregulated in various human cancers, polo-like kinase 1 (Plk1) has been extensively explored for anticancer activity and drug discovery. Although multiple catalytic domain inhibitors were tested in preclinical and clinical studies, their efficacies are limited by dose-limiting cytotoxicity, mainly from off-target cross reactivity. The C-terminal noncatalytic polo-box domain (PBD) of Plk1 has emerged as an attractive target for generating new protein-protein interaction inhibitors. Here, we identified a 1-thioxo-2,4-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-5(1H)-one scaffold that efficiently inhibits Plk1 PBD but not its related Plk2 and Plk3 PBDs. Structure-activity relationship studies led to multiple inhibitors having ≥10-fold higher inhibitory activity than the previously characterized Plk1 PBD-specific phosphopeptide, PLHSpT (Kd ∼ 450 nM). In addition, S-methyl prodrugs effectively inhibited mitotic progression and cell proliferation and their metabolic stability was determined. These data describe a novel class of small-molecule inhibitors that offer a promising avenue for future drug discovery against Plk1-addicted cancers.

Crystal structure of Arabidopsis thaliana cytidine deaminase

Cytidine deaminase (CDA) catalyzes the (deoxy)cytidine deamination to (deoxy)uridine, which involves in the catabolic and salvage pathways of pyrimidine nucleotides in plants. CDA serves as a prototype of the cytidine deaminase superfamily that contains a number of RNA editing enzymes. Arabidopsis thaliana has only one functional CDA, AtCDA1. We solved the crystal structures of AtCDA1, which is a dimeric zinc-containing enzyme and each protomer consists of an N-terminal zinc-binding catalytic domain and a C-terminal non-catalytic domain. Both domains adopt a typical α/β/α sandwich fold. In vitro biochemical assays showed that the ribose moiety of cytidine is required for ligand binding, and structural analyses revealed a conserved catalytic mechanism is adopted by AtCDA1.

Functional Alterations in Ciliogenesis-Associated Kinase 1 (CILK1) that Result from Mutations Linked to Juvenile Myoclonic Epilepsy

Ciliopathies are a group of human genetic disorders associated with mutations that give rise to the dysfunction of primary cilia. Ciliogenesis-associated kinase 1 (CILK1), formerly known as intestinal cell kinase (ICK), is a conserved serine and threonine kinase that restricts primary (non-motile) cilia formation and length. Mutations in CILK1 are associated with ciliopathies and are also linked to juvenile myoclonic epilepsy (JME). However, the effects of the JME-related mutations in CILK1 on kinase activity and CILK1 function are unknown. Here, we report that JME pathogenic mutations in the CILK1 N-terminal kinase domain abolish kinase activity, evidenced by the loss of phosphorylation of kinesin family member 3A (KIF3A) at Thr672, while JME mutations in the C-terminal non-catalytic domain (CTD) have little effect on KIF3A phosphorylation. Although CILK1 variants in the CTD retain catalytic activity, they nonetheless lose the ability to restrict cilia length and also gain function in promoting ciliogenesis. We show that wild type CILK1 predominantly localizes to the base of the primary cilium; in contrast, JME variants of CILK1 are distributed along the entire axoneme of the primary cilium. These results demonstrate that JME pathogenic mutations perturb CILK1 function and intracellular localization. These CILK1 variants affect the primary cilium, independent of CILK1 phosphorylation of KIF3A. Our findings suggest that CILK1 mutations linked to JME result in alterations of primary cilia formation and homeostasis.

Biochemical and structural analysis of the Klebsiella pneumoniae cytidine deaminase CDA

The emergence of drug-resistant strains of Klebsiella pneumoniae, has exacerbated the treatment and control of the disease caused by this bacterium. Cytidine deaminases (CDA) are zinc-dependent enzymes involved in the pyrimidine salvage pathway and catalyze the formation of uridine and deoxyuridine from cytidine and deoxycytidine, respectively. To illustrate the structural basis of CDA for a deeper knowledge of the molecular mechanisms underlying the salvage pathway, we reported here the biochemical and structural analysis of CDA from pathogenic K. pneumonia. KpCDA showed deaminase activity against cytidine as well as its analog cytarabine. The deaminase activity of KpCDA on cytarabine was 1.8 times higher than that on cytidine. KpCDA is composed of an N-terminal catalytic domain and a C-terminal noncatalytic domain. Zinc, which is involved in the activity of the catalytic domain, is coordinated by His102, Cys129, and Cys132, and two 1,4-dioxane molecules were present at the active sites. KpCDA exists as a dimer and shows distinct dimeric interface compared with other CDAs. Our results provide the structural features of KpCDA, and KpCDA might be a potential antibacterial target for the disease caused by K. pneumoniae.

Development of Highly Selective 1,2,3-Triazole-containing Peptidic Polo-like Kinase 1 Polo-box Domain-binding Inhibitors.

Members of the polo-like kinase (Plk) family of serine/threonine protein kinases play crucial roles in cell cycle regulation and proliferation. Of the five Plks (Plk1–5), Plk1 is recognized as an anticancer drug target. Plk1 contains multiple structural components that are important for its proper biological function. These include an N-terminal catalytic domain and a C-terminal non-catalytic polo-box domain (PBD). The PBD binds to phosphothreonine (pT) and phosphoserine-containing sequences. Blocking PBD-dependent interactions offers a potential means of down-regulating Plk1 function that is distinct from targeting its ATP-binding site. Previously, we demonstrated by tethering alkylphenyl chains from the N(π)-position of the His residue in the 5-mer PLHSpT, that we were able to access a hydrophobic “cryptic” binding pocket on the surface of the PBD, and in so doing enhance binding affinities by approximately 1000-fold. More recently, we optimized these PBD-ligand interactions using an oxime ligation-based strategy. Herein, using azide-alkyne cycloaddition reactions, we explore new triazole-containing PBD-binding antagonists. Some of these ligands retain the high PBD-binding affinity of the parent peptide, while showing desirable enhanced selectivity for the PBD of Plk1 relative to the PBDs of Plk2 and Plk3.

Structural evidence for an essential Fe-S cluster in the catalytic core domain of DNA polymerase ϵ.

DNA polymerase ϵ (Pol ϵ), the major leading-strand DNA polymerase in eukaryotes, has a catalytic subunit (Pol2) and three non-catalytic subunits. The N-terminal half of Pol2 (Pol2CORE) exhibits both polymerase and exonuclease activity. It has been suggested that both the non-catalytic C-terminal domain of Pol2 (with the two cysteine motifs CysA and CysB) and Pol2CORE (with the CysX cysteine motif) are likely to coordinate an Fe–S cluster. Here, we present two new crystal structures of Pol2CORE with an Fe–S cluster bound to the CysX motif, supported by an anomalous signal at that position. Furthermore we show that purified four-subunit Pol ϵ, Pol ϵ CysAMUT (C2111S/C2133S), and Pol ϵ CysBMUT (C2167S/C2181S) all have an Fe–S cluster that is not present in Pol ϵ CysXMUT (C665S/C668S). Pol ϵ CysAMUT and Pol ϵ CysBMUT behave similarly to wild-type Pol ϵ in in vitro assays, but Pol ϵ CysXMUT has severely compromised DNA polymerase activity that is not the result of an excessive exonuclease activity. Tetrad analyses show that haploid yeast strains carrying CysXMUT are inviable. In conclusion, Pol ϵ has a single Fe–S cluster bound at the base of the P-domain, and this Fe–S cluster is essential for cell viability and polymerase activity.

Diversity between mammalian tolloid proteinases: Oligomerisation and non-catalytic domains influence activity and specificity.

The mammalian tolloid family of metalloproteinases is essential for tissue patterning and extracellular matrix assembly. The four members of the family: bone morphogenetic protein-1 (BMP-1), mammalian tolloid (mTLD), tolloid-like (TLL)-1 and TLL-2 differ in their substrate specificity and activity levels, despite sharing similar domain organization. We have previously described a model of substrate exclusion by dimerisation to explain differences in the activities of monomeric BMP-1 and dimers of mTLD and TLL-1. Here we show that TLL-2, the least active member of the tolloid family, is predominantly monomeric in solution, therefore it appears unlikely that substrate exclusion via dimerisation is a mechanism for regulating TLL-2 activity. X-ray scattering and electron microscopy structural and biophysical analyses reveal an elongated shape for the monomer and flexibility in the absence of calcium. Furthermore, we show that TLL-2 can cleave chordin in vitro, similar to other mammalian tolloids, but truncated forms of TLL-2 mimicking BMP-1 are unable to cleave chordin. However, both the N- and C-terminal non-catalytic domains from all mammalian tolloids bind chordin with high affinity. The mechanisms underlying substrate specificity and activity in the tolloid family are complex with variation between family members and depend on both multimerisation and substrate interaction.

Mechanistic basis of Nek7 activation through Nek9 binding and induced dimerization

Mitotic spindle assembly requires the regulated activities of protein kinases such as Nek7 and Nek9. Nek7 is autoinhibited by the protrusion of Tyr97 into the active site and activated by the Nek9 non-catalytic C-terminal domain (CTD). CTD binding apparently releases autoinhibition because mutation of Tyr97 to phenylalanine increases Nek7 activity independently of Nek9. Here we find that self-association of the Nek9-CTD is needed for Nek7 activation. We map the minimal Nek7 binding region of Nek9 to residues 810–828. A crystal structure of Nek7Y97F bound to Nek9810–828 reveals a binding site on the C-lobe of the Nek7 kinase domain. Nek7Y97F crystallizes as a back-to-back dimer between kinase domain N-lobes, in which the specific contacts within the interface are coupled to the conformation of residue 97. Hence, we propose that the Nek9-CTD activates Nek7 through promoting back-to-back dimerization that releases the autoinhibitory tyrosine residue, a mechanism conserved in unrelated kinase families.

Loss of Nek11 Prevents G2/M Arrest and Promotes Cell Death in HCT116 Colorectal Cancer Cells Exposed to Therapeutic DNA Damaging Agents.

The Nek11 kinase is a potential mediator of the DNA damage response whose expression is upregulated in early stage colorectal cancers (CRCs). Here, using RNAi-mediated depletion, we examined the role of Nek11 in HCT116 WT and p53-null CRC cells exposed to ionizing radiation (IR) or the chemotherapeutic drug, irinotecan. We demonstrate that depletion of Nek11 prevents the G2/M arrest induced by these genotoxic agents and promotes p53-dependent apoptosis both in the presence and absence of DNA damage. Interestingly, Nek11 depletion also led to long-term loss of cell viability that was independent of p53 and exacerbated following IR exposure. CRC cells express four splice variants of Nek11 (L/S/C/D). These are predominantly cytoplasmic, but undergo nucleocytoplasmic shuttling mediated through adjacent nuclear import and export signals in the C-terminal non-catalytic domain. In HCT116 cells, Nek11S in particular has an important role in the DNA damage response. These data provide strong evidence that Nek11 contributes to the response of CRC cells to genotoxic agents and is essential for survival either with or without exposure to DNA damage.

Bimodal Interaction of Mammalian Polo-Like Kinase 1 and a Centrosomal Scaffold, Cep192, in the Regulation of Bipolar Spindle Formation.

Serving as microtubule-organizing centers, centrosomes play a key role in forming bipolar spindles. The mechanism of how centrosomes promote bipolar spindle assembly in various organisms remains largely unknown. A recent study with Xenopus laevis egg extracts suggested that the Plk1 ortholog Plx1 interacts with the phospho-T46 (p-T46) motif of Xenopus Cep192 (xCep192) to form an xCep192-mediated xAurA-Plx1 cascade that is critical for bipolar spindle formation. Here, we demonstrated that in cultured human cells, Cep192 recruits AurA and Plk1 in a cooperative manner, and this event is important for the reciprocal activation of AurA and Plk1. Strikingly, Plk1 interacted with Cep192 through either the p-T44 (analogous to Xenopus p-T46) or the newly identified p-S995 motif via its C-terminal noncatalytic polo-box domain. The interaction between Plk1 and the p-T44 motif was prevalent in the presence of Cep192-bound AurA, whereas the interaction of Plk1 with the p-T995 motif was preferred in the absence of AurA binding. Notably, the loss of p-T44- and p-S995-dependent Cep192-Plk1 interactions induced an additive defect in recruiting Plk1 and γ-tubulin to centrosomes, which ultimately led to a failure in proper bipolar spindle formation and mitotic progression. Thus, we propose that Plk1 promotes centrosome-based bipolar spindle formation by forming two functionally nonredundant complexes with Cep192.

Structural and biochemical characterization of the KLHL3–WNK kinase interaction important in blood pressure regulation

WNK1 [with no lysine (K)] and WNK4 regulate blood pressure by controlling the activity of ion co-transporters in the kidney. Groundbreaking work has revealed that the ubiquitylation and hence levels of WNK isoforms are controlled by a Cullin-RING E3 ubiquitin ligase complex (CRL3KLHL3) that utilizes CUL3 (Cullin3) and its substrate adaptor, KLHL3 (Kelch-like protein 3). Loss-of-function mutations in either CUL3 or KLHL3 cause the hereditary high blood pressure disease Gordon's syndrome by stabilizing WNK isoforms. KLHL3 binds to a highly conserved degron motif located within the C-terminal non-catalytic domain of WNK isoforms. This interaction is essential for ubiquitylation by CRL3KLHL3 and disease-causing mutations in WNK4 and KLHL3 exert their effects on blood pressure by disrupting this interaction. In the present study, we report on the crystal structure of the KLHL3 Kelch domain in complex with the WNK4 degron motif. This reveals an intricate web of interactions between conserved residues on the surface of the Kelch domain β-propeller and the WNK4 degron motif. Importantly, many of the disease-causing mutations inhibit binding by disrupting critical interface contacts. We also present the structure of the WNK4 degron motif in complex with KLHL2 that has also been reported to bind WNK4. This confirms that KLHL2 interacts with WNK kinases in a similar manner to KLHL3, but strikingly different to how another KLHL protein, KEAP1 (Kelch-like enoyl-CoA hydratase-associated protein 1), binds to its substrate NRF2 (nuclear factor-erythroid 2-related factor 2). The present study provides further insights into how Kelch-like adaptor proteins recognize their substrates and provides a structural basis for how mutations in WNK4 and KLHL3 lead to hypertension.

Distinct Expression Patterns of ICK/MAK/MOK Protein Kinases in the Intestine Implicate Functional Diversity

ICK/MRK (intestinal cell kinase/MAK-related kinase), MAK (male germ cell-associated kinase), and MOK (MAPK/MAK/MRK-overlapping kinase) are closely related serine/threonine protein kinases in the protein kinome. The biological functions and regulatory mechanisms of the ICK/MAK/MOK family are still largely elusive. Despite significant similarities in their catalytic domains, they diverge markedly in the sequence and structural organization of their C-terminal non-catalytic domains, raising the question as to whether they have distinct, overlapping, or redundant biological functions. In order to gain insights into their biological activities and lay a fundamental groundwork for functional studies, we investigated the spatio-temporal distribution patterns and the expression dynamics of ICK/MAK/MOK protein kinases in the intestine. We found that ICK/MAK/MOK proteins display divergent expression patterns along the duodenum-to-colon axis and during postnatal murine development. Furthermore, they are differentially partitioned between intestinal epithelium and mesenchyme. A significant increase in the protein level of ICK, but not MAK, was induced in human primary colon cancer specimens. ICK protein level was up-regulated whereas MOK protein level was down-regulated in mouse intestinal adenomas as compared with their adjacent normal intestinal mucosa. These data suggest distinct roles for ICK/MAK/MOK protein kinases in the regulation of intestinal neoplasia. Taken together, our findings demonstrate that the expressions of ICK/MAK/MOK proteins in the intestinal tract can be differentially and dynamically regulated, implicating a significant functional diversity within this group of protein kinases.

PD-1 Decreases PTEN Protein Stability While Increasing PTEN Phosphatase Activity by Inhibiting CK2.

Abstract 2145Maintenance of peripheral tolerance is essential for homeostasis of the immune system. The newer pathway of the B7:CD28 family consisting of the receptor PD-1 (CD279) and its ligands, PD-L1 (B7-H1; CD274) and PD-L2 (B7-DC; CD273), plays a vital role in the induction and maintenance of T cell anergy and peripheral tolerance. This pathway also regulates the balance between stimulatory and inhibitory signals needed for effective immunity and maintenance of T cell homeostasis but can also compromise anti-tumor and anti-viral immunity. The precise mechanisms via which PD-1 mediates its inhibitory effects in T cells remain elusive. One of the critical signaling pathways targeted by PD-1 is the PI3K/Akt pathway, via which PD-1 mediates inhibitory effects on T cell proliferation, cytokine production and glucose uptake. In contrast to CTLA-4, which affects PI3/Akt signaling by targeting Akt through PP2A activation, PD-1 targets the PI3K/Akt pathway by directly inhibiting activation of PI3K. It has been proposed that PD-1 inhibits PI3K by upregulating PTEN (phosphatase and tensin homologue deleted on chromosome 10), a phosphoinositole 3,4,5-trisphosphatase important for antagonizing PI3K signaling, but the mechanism remains unclear. In the present study we sought to dissect the effects of PD-1 mediated signaling on the regulation of PTEN expression and function. We used highly purified primary human CD4+ T cells and magnetic beads conjugated with monoclonal antibodies (mAbs) against CD3 and CD28 with or without agonist mAb against PD-1. As determined by assessment of PTEN lipid phosphatase activity, stimulation of T cells via TCR/CD3, CD28 and PD-1 resulted in prominent increase of PTEN phosphatase activity compared to T cells stimulated via TCR/CD3 and CD28 without PD-1 signals. Surprisingly, although T cells receiving PD-1 signals displayed increased PTEN phosphatase activity they expressed lower levels of PTEN compared to T cells stimulated via TCR/CD3 and CD28, as determined by both western blot and by flow cytometry. In other cell types, the abundance of PTEN can be regulated transcriptionally and through phosphorylation-dependent modulation of protein stability. Assessment of PTEN mRNA by real time qPCR indicated that PTEN mRNA levels were comparable between T cells stimulated via TCR/CD3/CD28 and T cells stimulated via TCR/CD3/CD28 and PD-1. PTEN possesses a C-terminal non-catalytic regulatory domain, which plays an important role in its biological activity. A cluster of serine/threonine residues within this region is phosphorylated by the serine/threonine kinase casein kinase 2 (CK2). CK2-mediated phosphorylation stabilizes PTEN by preventing its ubiquitin-dependent degradation while decreasing PTEN phosphatase activity. In contrast, impaired phosphorylation in this region results in decreased PTEN protein stability but increased phosphatase activity. Because PD-1 signaling resulted in reduced PTEN protein expression but increased phosphatase activity, we examined whether PD-1 might alter CK2 activity and CK2-mediated phosphorylation of PTEN. In CD4+ T cells stimulated via TCR/CD3 and CD28, CK2 activation was robust and resulted in sustained phosphorylation on PTEN S380/T382/383 cluster. In contrast, T cells receiving PD-1 signals displayed impaired CK2 activity and significantly diminished phosphorylation of PTEN in the S380/T382/383 cluster. To determine whether downregulation of PTEN protein expression via PD-1 was mediated via proteasome-dependent degradation we used the proteasome inhibitor MG132. Stimulation of T cells via TCR/CD3, CD28 and PD-1 in the presence of MG132 prevented PD-1-mediated decrease of PTEN and resulted in reduced PTEN activity. Our results identify CK2 as a new target of PD-1-mediated signaling and reveal an unexpected mechanism via which PD-1 regulates PTEN phosphatase activity and inhibition of the PI3K/Akt pathway. Disclosures:No relevant conflicts of interest to declare.

Lipoprotein lipase is frequently overexpressed or translocated in cervical squamous cell carcinoma and promotes invasiveness through the non-catalytic C terminus

Background:We studied the biological significance of genes involved in a novel t(8;12)(p21.3;p13.31) reciprocal translocation identified in cervical squamous cell carcinoma (SCC) cells.Methods:The rearranged genes were identified by breakpoint mapping, long-range PCR and sequencing. We investigated gene expression in vivo using reverse-transcription PCR and tissue microarrays, and studied the phenotypic consequences of forced gene overexpression.Results:The rearrangement involved lipoprotein lipase (LPL) and peroxisome biogenesis factor-5 (PEX5). Whereas LPL–PEX5 was expressed at low levels and contained a premature stop codon, PEX5–LPL was highly expressed and encoded a full-length chimeric protein (including the majority of the LPL coding region). Consistent with these findings, PEX5 was constitutively expressed in normal cervical squamous cells, whereas LPL expression was negligible. The LPL gene was rearranged in 1 out of 151 cervical SCCs, whereas wild-type LPL overexpression was common, being detected in 10 out of 28 tissue samples and 4 out of 10 cell lines. Forced overexpression of wild-type LPL and PEX5–LPL fusion transcripts resulted in increased invasiveness in cervical SCC cells, attributable to the C-terminal non-catalytic domain of LPL, which was retained in the fusion transcripts.Conclusion:This is the first demonstration of an expressed fusion gene in cervical SCC. Overexpressed wild-type or translocated LPL is a candidate for targeted therapy.