Membrane-bound Merkel cell polyomavirus middle T protein constitutively activates PLCγ1 signaling through Src-family kinases.

Merkel cell polyomavirus (MCV or MCPyV) is an alphapolyomavirus causing human Merkel cell carcinoma and encodes four tumor (T) antigen proteins: large T (LT), small tumor (sT), 57 kT, and middle T (MT)/alternate LT open reading frame proteins. We show that MCV MT is generated as multiple isoforms through internal methionine translational initiation that insert into membrane lipid rafts. The membrane-localized MCV MT oligomerizes and promiscuously binds to lipid raft-associated Src family kinases (SFKs). MCV MT-SFK interaction is mediated by a Src homology (SH) 3 recognition motif as determined by surface plasmon resonance, coimmunoprecipitation, and bimolecular fluorescence complementation assays. SFK recruitment by MT leads to tyrosine phosphorylation at a SH2 recognition motif (pMTY114), allowing interaction with phospholipase C gamma 1 (PLCγ1). The secondary recruitment of PLCγ1 to the SFK-MT membrane complex promotes PLCγ1 tyrosine phosphorylation on Y783 and activates the NF-κB inflammatory signaling pathway. Mutations at either the MCV MT SH2 or SH3 recognition sites abrogate PLCγ1-dependent activation of NF-κB signaling and increase viral replication after MCV genome transfection into 293 cells. These findings reveal a conserved viral targeting of the SFK-PLCγ1 pathway by both MCV and murine polyomavirus (MuPyV) MT proteins. The molecular steps in how SFK-PLCγ1 activation is achieved, however, differ between these two viruses.

Feeding enhances fibronectin adherence of quiescent lymphocytes through non-canonical insulin signalling.

Nutritional availability during fasting and refeeding affects the temporal redistribution of lymphoid and myeloid immune cells among the circulating and tissue-resident pools. Conversely, nutritional imbalance and impaired glucose metabolism are associated with chronic inflammation, aberrant immunity and anomalous leukocyte trafficking. Despite being exposed to periodic alterations in blood insulin levels upon fasting and feeding, studies exploring the physiological effects of these hormonal changes on quiescent immune cell function and trafficking are scanty. Here, we report that oral glucose load in mice and healthy men enhances the adherence of circulating peripheral blood mononuclear cells (PBMCs) and lymphocytes to fibronectin. Adherence to fibronectin is also observed upon regular intake of breakfast following overnight fasting in healthy subjects. This glucose load-induced phenomenon is abrogated in streptozotocin-injected mice that lack insulin. Intra-vital microscopy in mice demonstrated that oral glucose feeding enhances the homing of PBMCs to injured blood vessels in vivo. Furthermore, employing flow cytometry, Western blotting and adhesion assays for PBMCs and Jurkat-T cells, we elucidate that insulin enhances fibronectin adherence of quiescent lymphocytes through non-canonical signalling involving insulin-like growth factor-1 receptor (IGF-1R) autophosphorylation, phospholipase C gamma-1 (PLCγ-1) Tyr783 phosphorylation and inside-out activation of ß-integrins respectively. Our findings uncover the physiological relevance of post-prandial insulin spikes in regulating the adherence and trafficking of circulating quiescent T-cells through fibronectin-integrin interaction.

A frequent PLCγ1 mutation in adult T-cell leukemia/lymphoma determines functional properties of the malignant cells.

BACKGROUND: Development of adult T-cell leukemia/lymphoma (ATL) involves human T-cell leukemia virus type 1 (HTLV-1) infection and accumulation of somatic mutations. The most frequently mutated gene in ATL (36 % of cases) is phospholipase C gamma1 (PLCG1). PLCG1 is also frequently mutated in other T-cell lymphomas. However, the functional consequences of the PLCG1 mutations in cancer cells have not been characterized. METHODS: We compared the activity of the wild-type PLCγ1 with that of a mutant carrying a hot-spot mutation of PLCγ1 (S345F) observed in ATL, both in cells and in cell-free assays. To analyse the impact of the mutation on cellular properties, we quantified cellular proliferation, aggregation, chemotaxis and apoptosis by live cell-imaging in an S345F+ ATL-derived cell line (KK1) and a KK1 cell line in which we reverted the mutation to the wild-type sequence using CRISPR/Cas9 and homology-directed repair. FINDINGS: The PLCγ1 S345F mutation results in an increase of basal PLC activity in vitro and in different cell types. This higher basal activity is further enhanced by upstream signalling. Reversion of the S345F mutation in the KK1 cell line resulted in reduction of the PLC activity, lower rates of proliferation and aggregation, and a marked reduction in chemotaxis towards CCL22. The PLCγ1-pathway inhibitors ibrutinib and ritonavir reduced both the PLC activity and the tested functions of KK1 cells. INTERPRETATION: Consistent with observations from clinical studies, our data provide direct evidence that activated variants of the PLCγ1 enzyme contribute to the properties of the malignant T-cell clone in ATL. FUNDING: MRC (UK) Project Grant (P028160).

Visuosocial Preference Memory, but Not Avoidance Memory, Requires PLCγ1 in the CA2 Hippocampus.

Visuosocial memory is defined as stored visual information containing social context. Primates have a powerful ability to associate visuosocial memory with episodic memory. However, the existence of visuosocial memory in mice remains unclear. Here, we design a novel vision-specific social memory test using a portrait picture or mirrored self-image and demonstrate that mice can distinguish conspecific from other species by forming a visuosocial memory. Because CA2 hippocampus has been reported as a critical brain region for social memory, we develop CA2-specific blockade of memory formation through deletion of phospholipase C gamma 1 (PLCγ1), which is a key molecule in the brain-derived neurotrophic factor (BDNF) signaling pathway. Interestingly, these mice have intact sociability but impaired social memory in three chamber test and five-trial social memory test, which is highly dependent on visual information. Finally, PLCγ1 deletion in CA2 impairs visuosocial preference memory, but not avoidance memory, whereas non-social object recognition is intact. Our study proposes that mice have visuosocial memory, just as primates and humans.

Advanced application of stimuli-responsive drug delivery system for inflammatory arthritis treatment.

Inflammatory arthritis is a major cause of disability in the elderly. This condition causes joint pain, loss of function, and deterioration of quality of life, mainly due to osteoarthritis (OA) and rheumatoid arthritis (RA). Currently, available treatment options for inflammatory arthritis include anti-inflammatory medications administered via oral, topical, or intra-articular routes, surgery, and physical rehabilitation. Novel alternative approaches to managing inflammatory arthritis, so far, remain the grand challenge owing to catastrophic financial burden and insignificant therapeutic benefit. In the view of non-targeted systemic cytotoxicity and limited bioavailability of drug therapies, a major concern is to establish stimuli-responsive drug delivery systems using nanomaterials with on-off switching potential for biomedical applications. This review summarizes the advanced applications of triggerable nanomaterials dependent on various internal stimuli (including reduction-oxidation (redox), pH, and enzymes) and external stimuli (including temperature, ultrasound (US), magnetic, photo, voltage, and mechanical friction). The review also explores the progress and challenges with the use of stimuli-responsive nanomaterials to manage inflammatory arthritis based on pathological changes, including cartilage degeneration, synovitis, and subchondral bone destruction. Exposure to appropriate stimuli induced by such histopathological alterations can trigger the release of therapeutic medications, imperative in the joint-targeted treatment of inflammatory arthritis.

Investigation of Phospholipase Cγ1 Interaction with SLP76 Using Molecular Modeling Methods for Identifying Novel Inhibitors.

The enzyme phospholipase C gamma 1 (PLCγ1) has been identified as a potential drug target of interest for various pathological conditions such as immune disorders, systemic lupus erythematosus, and cancers. Targeting its SH3 domain has been recognized as an efficient pharmacological approach for drug discovery against PLCγ1. Therefore, for the first time, a combination of various biophysical methods has been employed to shed light on the atomistic interactions between PLCγ1 and its known binding partners. Indeed, molecular modeling of PLCγ1 with SLP76 peptide and with previously reported inhibitors (ritonavir, anethole, daunorubicin, diflunisal, and rosiglitazone) facilitated the identification of the common critical residues (Gln805, Arg806, Asp808, Glu809, Asp825, Gly827, and Trp828) as well as the quantification of their interaction through binding energies calculations. These features are in agreement with previous experimental data. Such an in depth biophysical analysis of each complex provides an opportunity to identify new inhibitors through pharmacophore mapping, molecular docking and MD simulations. From such a systematic procedure, a total of seven compounds emerged as promising inhibitors, all characterized by a strong binding with PLCγ1 and a comparable or higher binding affinity to ritonavir (∆Gbind < -25 kcal/mol), one of the most potent inhibitor reported till now.

Molecular characterization and expression analysis of olive flounder (Paralichthys olivaceus) phospholipase C gamma 1 and gamma 2.

Phospholipase C gamma 1 and gamma 2 (PLCG1 and PLCG2) are influential in modulating Ca2+ and diacylglycerol, second messengers involved in tyrosine kinase-dependent signaling, including growth factor activation. Here, we used RACE (rapid amplification of cDNA ends) to clone cDNA encoding PLCG1 (PoPLCG1) and PLCG2 (PoPLCG2) in the olive flounder (Paralichthys olivaceus). The respective 1313 and 1249 amino acid sequences share high identity with human PLCG1 and PLCG2, and contain the following domains: pleckstrin homology (PH), EF-hand, catalytic X and Y, Src homology 2 (SH2), Src homology 3 (SH3), and C2. Phylogenic analysis and sequence comparison of PoPLCG1 and PoPLCG2 with other PLC isozymes showed a close relationship between the two PLCGs, supported by structural analysis. In addition, tissue expression analysis showed that PoPLCG1 was expressed predominantly in the brain, eye, and heart, whereas PoPLCG2 was expressed principally in gills, esophagus, spleen, and kidney. Following stimulation with LPS and Poly I:C, PoPLCG expression was compared with the expression of inflammatory cytokines IL-1ß, IL-6, and TNF-α via reverse transcription-PCR and real-time quantitative PCR. Our results suggest that PoPLCG isozymes perform a critical immune function in olive flounder, being active in pathogen resistance and the inflammation process.

Oocyte activation and latent HIV-1 reactivation: AMPK as a common mechanism of action linking the beginnings of life and the potential eradication of HIV-1.

In all mammalian species studied to date, the initiation of oocyte activation is orchestrated through alterations in intracellular calcium (Ca(2+)) signaling. Upon sperm binding to the oocyte plasma membrane, a sperm-associated phospholipase C (PLC) isoform, PLC zeta (PLCζ), is released into the oocyte cytoplasm. PLCζ hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) to produce diacylglycerol (DAG), which activates protein kinase C (PKC), and inositol 1,4,5-trisphosphate (IP3), which induces the release of Ca(2+) from endoplasmic reticulum (ER) Ca(2+) stores. Subsequent Ca(2+) oscillations are generated that drive oocyte activation to completion. Ca(2+) ionophores such as ionomycin have been successfully used to induce artificial human oocyte activation, facilitating fertilization during intra-cytoplasmic sperm injection (ICSI) procedures. Early studies have also demonstrated that the PKC activator phorbol 12-myristate 13-acetate (PMA) acts synergistically with Ca(2+) ionophores to induce parthenogenetic activation of mouse oocytes. Interestingly, the Ca(2+)-induced signaling cascade characterizing sperm or chemically-induced oocyte activation, i.e. the "shock and live" approach, bears a striking resemblance to the reactivation of latently infected HIV-1 viral reservoirs via the so called "shock and kill" approach, a method currently being pursued to eradicate HIV-1 from infected individuals. PMA and ionomycin combined, used as positive controls in HIV-1 latency reversal studies, have been shown to be extremely efficient in reactivating latent HIV-1 in CD4(+) memory T cells by inducing T cell activation. Similar to oocyte activation, T cell activation by PMA and ionomycin induces an increase in intracellular Ca(2+) concentrations and activation of DAG, PKC, and downstream Ca(2+)-dependent signaling pathways necessary for proviral transcription. Interestingly, AMPK, a master regulator of cell metabolism that is activated thorough the induction of cellular stress (e.g. increase in Ca(2+) concentration, reactive oxygen species generation, increase in AMP/ATP ratio) is essential for oocyte maturation, T cell activation, and mitochondrial function. In addition to the AMPK kinase LKB1, CaMKK2, a Ca(2+)/calmodulin-dependent kinase that also activates AMPK, is present in and activated on T cell activation and is also present in mouse oocytes and persists until the zygote and two-cell stages. It is our hypothesis that AMPK activation represents a central node linking T cell activation-induced latent HIV-1 reactivation and both physiological and artificial oocyte activation. We further propose the novel observation that various compounds that have been shown to reactivate latent HIV-1 (e.g. PMA, ionomycin, metformin, bryostatin, resveratrol, etc.) or activate oocytes (PMA, ionomycin, ethanol, puromycin, etc.) either alone or in combination likely do so via stress-induced activation of AMPK.

The phosphoproteome of human Jurkat T cell clones upon costimulation with anti-CD3/anti-CD28 antibodies.

Phosphorylation is a reversible post-translational modification, playing a vital role in protein function. In T cells, protein phosphorylation is the key mechanism regulating T cell receptor-driven signaling pathways. In order to gain insights into the phosphoproteome evolution of T cell activation, we performed a large-scale quantitative phosphoproteomics study of Jurkat E6.1 (wild type) and Jurkat gamma1 (Phospholipase gamma1 null) cell clones upon costimulation with anti-CD3 and anti-CD28 antibodies at times ranging from 15min to as long as 120min. In total, we identified 5585 phosphopeptides belonging to 2008 phosphoproteins from both cell clones. We detected 130 and 114 novel phosphopeptides in Jurkat E6.1 and Jurkat gamma1 clones, respectively. A significantly lower number of proteins containing regulated phosphorylation sites were identified in Jurkat gamma1 in comparison to Jurkat E6.1, reflecting the vital role of Phospholipase gamma1 in T cell signaling. Several new phosphorylation sites from lymphocyte-specific protein tyrosine kinase (Lck) were identified. Of these, serine-121 showed significant changes in JE6.1 while only small changes in the Jgamma1 clone. Our data may contribute to the current human T cell phosphoproteome and provide a better understanding on T cell receptor signaling. Data are available via ProteomeXchange with identifier PXD002871.

Phospholipase C gamma 1 is a potential prognostic biomarker for patients with locally advanced and resectable oral squamous cell carcinoma.

The aim of this study was to investigate the prognostic and predictive values of phospholipase C gamma 1 (PLCG1) expression in patients with locally advanced and resectable oral squamous cell carcinoma (OSCC), who were treated in a prospective, randomized, phase 3 trial evaluating standard treatment with surgery and postoperative radiation preceded or not by induction docetaxel, cisplatin, and 5-fluorouracil (TPF). Immunohistochemical staining for PLCG1 was performed on the biopsies of 232 out of 256 OSCC patients at clinical stage III/IVA; the PLCG1 positive score was determined by immunoreactive scoring system. The survival analysis was performed by Kaplan-Meier method; hazard ratios were calculated using the Cox proportional hazards model. Patients with a low PLCG1 expression had a significantly better overall survival (P=0.022), and a trend towards better disease-free survival (P=0.087), loco-regional recurrence-free survival (P=0.058), distant metastasis-free survival (P=0.053), and a high response rate to TPF induction chemotherapy with regard to clinical response (P=0.052) and pathological response (P=0.061), compared to those with high PLCG1 expression. Our results suggest that PLCG1 expression could be used as a prognostic biomarker for patients with advanced OSCC; however, it was not an adequate predictive biomarker for TPF induction chemotherapy.

Polymorphisms in RAS Guanyl-releasing Protein 3 are Associated with Chronic Liver Disease and Hepatocellular Carcinoma in a Korean Population

RAS guanyl-releasing protein 3 (RasGRP3), a member of the Ras subfamily of GTPases, functions as a guanosine triphosphate (GTP)/guanosine diphosphate (GDP)-regulated switch that cycles between inactive GDP- and active GTP-bound states during signal transduction. Various growth factors enhance hepatocellular carcinoma (HCC) proliferation via activation of the Ras/Raf-1/ extracellular signal-regulated kinase (ERK) pathway, which depends on RasGRP3 activation. We investigated the relationship between polymorphisms in RasGRP3 and progression of hepatitis B virus (HBV)-infected HCC in a Korean population. Nineteen RasGRP3 SNPs were genotyped in 206 patients with chronic liver disease (CLD) and 86 patients with HCC. Our results revealed that the T allele of the rs7597095 SNP and the C allele of the rs7592762 SNP increased susceptibility to HCC (OR=1.55, p=0.04 and OR=1.81~2.61, p=0.01~0.03, respectively). Moreover, patients who possessed the haplotype (ht) 1 ( A-T-C-G) or diplotype (dt) 1 ( ht1/ht1) variations had increased susceptibility to HCC (OR=1.79 ~2.78, p=0.01~0.03). In addition, we identified an association between haplotype1 (ht1) and the age of HCC onset; the age of HCC onset are earlier in ht1 +/+ than ht1 +/- or ht1 -/- (HR=0.42~0.66, p=0.006~0.015). Thus, our data suggest that RasGRP3 SNPs are significantly associated with an increased risk of developing HCC.

Inositol 5'-phosphatase, SHIP1 interacts with phospholipase C-gamma1 and modulates EGF-induced PLC activity

Phospholipase C-gamma1, containing two SH2 and one SH3 domains which participate in the interaction between signaling molecules, plays a significant role in the growth factor-induced signal transduction. However, the role of the SH domains in the growth factor-induced PLC-gamma1 regulation is unclear. By peptide-mass fingerprinting analysis, we have identified SHIP1 as the binding protein for the SH3 domain of PLC-gamma1. SHIP1 was co-immunoprecipitated with PLC-gamma1 and potentiated EGF-induced PLC-gamma1 activation. However, inositol 5'-phosphatase activity of SHIP1 was not required for the potentiation of EGF-induced PLC-gamma1 activation. Taken together, these results suggest that SHIP1 may function as an adaptor protein which can potentiate EGF-induced PLC-gamma1 activation without regards to its inositol 5'-phosphatase activity.

Effect of Phospholipase C-gamma1 Overexpression on the Protein Level of Waf1, PCNA, and Cyclin B1 Following Ultraviolet C Irradiation.

PURPOSE: It has been demonstrated that PLC-gamma1 is overexpressed in many tumor cells, and that overexpression of Phospholipase C (PLC)-gamma1 is associated with tumor progression. In order to understand the effect of the PLC-gamma1 overexpression on the regulation of cell cycle regulators following DNA damage, we analyzed the expression level of PCNA, cyclin B1, and p21 Waf1 after ultraviolet C (UVC) irradiation in PLC-gamma1-transfected PC12 cells. MATERIALS AND METHODS: PC12 and 3Y1 cells, transfected with empty vector or rat PLC-gamma1 cDNA, were used for this study. Following UVC irradiation, cell cycle progression was analyzed by flow cytometry and protein expression was detected by Western blotting. RESULTS: Waf1 protein was markedly down-regulated, whereas PCNA and cyclin B1 was up-regulated in PLC-gamma1 overexpressed-cells as compared to the vector transfected-cells. When the cells were irradiated with UVC, PCNA was slightly increased within 3-hours of the UV irradiation and then was markedly decreased in Vector/ PC12 cells, while it remained high until 37 hour after UVC in PLC-gamma1/PC12 cells. In contrast, cyclin B1 was gradually decreased following UVC irradiation in both cells. CONCLUSION: The overexpression of PLC-gamma1 affects the expression level of PCNA after UVC irradiation. We proposed that the overexpression of PLC-gamma1 may contribute to the UV-induced genomic instability by up-regulating the expression of PCNA.

Effect of Phospholipase C-gamma1 Overexpression on the Protein Level of Waf1, PCNA, and Cyclin B1 Following Ultraviolet C Irradiation / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: It has been demonstrated that PLC-gamma1 is overexpressed in many tumor cells, and that overexpression of Phospholipase C (PLC)-gamma1 is associated with tumor progression. In order to understand the effect of the PLC-gamma1 overexpression on the regulation of cell cycle regulators following DNA damage, we analyzed the expression level of PCNA, cyclin B1, and p21 Waf1 after ultraviolet C (UVC) irradiation in PLC-gamma1-transfected PC12 cells. MATERIALS AND METHODS: PC12 and 3Y1 cells, transfected with empty vector or rat PLC-gamma1 cDNA, were used for this study. Following UVC irradiation, cell cycle progression was analyzed by flow cytometry and protein expression was detected by Western blotting. RESULTS: Waf1 protein was markedly down-regulated, whereas PCNA and cyclin B1 was up-regulated in PLC-gamma1 overexpressed-cells as compared to the vector transfected-cells. When the cells were irradiated with UVC, PCNA was slightly increased within 3-hours of the UV irradiation and then was markedly decreased in Vector/ PC12 cells, while it remained high until 37 hour after UVC in PLC-gamma1/PC12 cells. In contrast, cyclin B1 was gradually decreased following UVC irradiation in both cells. CONCLUSION: The overexpression of PLC-gamma1 affects the expression level of PCNA after UVC irradiation. We proposed that the overexpression of PLC-gamma1 may contribute to the UV-induced genomic instability by up-regulating the expression of PCNA.

Expression of PLC- 1 in Human Middle Ear Cholesteatoma / 대한이비인후과학회지

BACKGROUND: EGF and TGF-gamma are believed to mediate their pleiotrophic actions by binding to and activating cell surface receptors with an intrinsic protein-tyrosine kinase. Protein-tyrosine kinase phosphorylation has been considered involved in intrinsic signal transduction, proliferation and transformation of the cells. Phospholipase C-gamma1 is well characterized substrate for tyrosine kinase. OBJECTIVE: The purpose of this study is to elucidate the distribution of PLC-gamma1 in normal meatal skin and cholesteatoma matrix. MATERIALS AND METHODS: 8 cholesteatoma specimens were obtained from operated patients for immunohistochemistry and western blot analysis. RESULTS: On immunohistochemistry, PLC-gamma1 was detected only in basal layer of the deep meatal skin, but was readily detectable in both the basal and suprabasal layer in cholesteatoma matrix. By western blot analysis, considerable higher levels of PLC-gamma1 protein were detectable in cholesteatoma matrix compared with the deep meatal skin. CONCLUSION: Overexpression of PLC-gamma1 in cholesteatoma suggests a possible derangement of enhanced growth signal transduction in keratinocytes.

Alteration of Phospholipase C Activity in Human Gastric Cancer Tissues

Phospholipase C (PLC) plays a pivotal role in transmembrane signal transduction pathway for cellular proliferation differentiation and growth. Thus far, there have been few reports in which PLC activity was investigated in human malignant neoplastic tissues. In the present study, we evaluated PLC activity in 23 human gastric cancer tissues and normal mucosal tissues to investigate whether alteration of PLC activity is associated with gastric cancer. The amount of [14C] diacylglycerol, one of the earliest products of inositol phospholipid hydrolysis by PLC, was measured by thin layer chromatography. Also, expression of PLC-gamma1, which is one of the most important PLC isozymes,was examined by immunohistochemistry using specific monoclonal antibody directed against PLC-gamma1. The results are summarized as follows. PLC activity in all 23 gastric cancer tissues (1.35+/-1.04 units/mg of protein) was significantly higher than normal mucosal tissues (0.28+/-0.21 units/mg of protein) (P0.05). PLC-gamma1 immunoreactivity was detected in all of 23 cases studied. The intensity and extent of PLC-gamma1 immunoreactivity was not correlated with PLC enzyme activity, although stronger intensity was demonstrated in malignant cells in comparison to normal gland epithelial cells. The present study provides the first evidence of significant elevation of PLC activity in human stomach cancer tissues. Our results strongly suggest that PLC might be involved in tumorigenesis and/or progression(uncontrolled continuous cycling of cells) of human gastric cancer. Further studies are needed to elucidate the role of elevated PLC activity in cancer tissues.