A Protein Kinase Induced by Pseudorabies Virus in Infected Cells

This thesis describes studies of the phosphorylation of proteins during the infection of baby hamster kidney fibroblasts (BHK cells) with the swine herpes virus, pseudorabies virus, with particular emphasis on the protein kinase activites in infected cells. Protein kinases present in the cytosol of BHK cells infected with pseudorabies virus were compared with those present in the cytosol of uninfected cells. The cytosol was first subjected to chromatography on DEAE-cellulose and fractions analysed for protein kinase activities. In the fractions from uninfected cells the activities were found to correspond to the following previously characterized protein kinases: i) cyclic AMP-dependent protein kinase type I; ii) cyclic AMP-dependent protein kinase type II; iii) protein kinase C; iv) casein kinase I; v) casein kinase II; vi) a less clearly defined protamine kinase which is probably the proteolytic fragment of protein kinase C. In BHK cells infected with pseudorabies virus the protein kinases listed above were present in amounts comparable to those in uninfected cells. However, a new protein kinase activity was detected in infected cells, appearing about 4 h after infection and increasing during the following 6 h, at least. This protein kinase designated virus-induced protein kinase (ViPK), was the subject of subsequent studies. The new protein kinase was further purified either by Blue A dye-ligand chromatography or by high-performance size-exclusion and ion- exchange chromatography. The latter procedure resulted in 100-fold purification of the enzyme. The partially-purified preparations were used to characterize the new protein kinase. It had an apparent molecular weight of 68,000 on the basis of size-exclusion chromatography, and had a sedimentation coefficient of 4.3S. It catalysed the phosphorylation of serine residues of basic proteins in vitro, with protamine a better substrate than mixed histones; and used ATP (apparent Km=60 muM), but not GTP, as phosphate donor. Molecules that can serve as effectors for other protein kinases (cyclic AMP, cyclic GMP, Ca2+ + calmodulin, Ca2+ + phospholipid, double-stranded RNA, and heparin) did not significantly alter the activity of this enzyme. A striking characteristic of the protein kinase was a high KC1 concentration optimum with the persistence of activity up to 800 mM KC1, at least. These characteristics distinguish it from other protein kinases found in BHK cells or reported to be present in other eukaryotic cells. Further studies attempted to relate the new protein kinase activity to proteins which become phosphorylated during viral infection. The results obtained show that the major virion phosphoproteins were not phosphorylated by the new protein kinase in vitro, even though that activity was also present in the virion. An enzyme activity, present in BHK cells and a similar activity from virus particles, both with characteristics consistent with those of casein kinase II, phosphorylated these proteins in vitro. Thus, it is possible that these viral proteins synthesized de novo in infected cells can be recognized as substrates by cellular casein kinase II. Analysis of viral non-structural proteins showed that one of the proteins tested, viral DNase, served as a substrate iin vitro for a partially-purified preparation of the new protein kinase. Studies of the phosphorylation of ribosomal protein S6 in vitro by protein kinase activities from infected cells showed that the new protein kinase and also some other cellular protein kinases (cyclic AMP-dependent protein kinases and protein kinase C) can catalyse this reaction. Specific phosphorylation of S6 by the new protein kinase was only observed in certain conditions, e. g. 600 mM KC1 or 7-10 mM spermine. Phosphopeptide maps of S6 following phosphorylation in vivo and in vitro by the new kinase were similar. However, the enzyme catalysed the incorporation of only two phosphate groups per molecule of S6 while the stoichiometry of phosphorylation in vivo was up to five phosphate groups per S6 molecule. This finding does not support the possibility that the new protein kinase is responsible for the complete phosphorylation of ribosomal protein S6 observed in vivo. One of the cellular kinases, protein kinase C appears to be a better candidate, on the basis of the results of studies of stoichiometry and phosphopeptides

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)

Multiple roles of phosphoinositide-specific phospholipase C isozymes

Phosphoinositide-specific phospholipase C is an effector molecule in the signal transduction process. It generates two second messengers, inositol-1,4,5-trisphosphate and diacylglycerol from phosphatidylinositol 4,5-bisphosphate. Currently, thirteen mammal PLC isozymes have been identified, and they are divided into six groups: PLC-beta, -gamma, -delta, -epsilon, -zeta and -eta. Sequence analysis studies demonstrated that each isozyme has more than one alternative splicing variant. PLC isozymes contain the X and Y domains that are responsible for catalytic activity. Several other domains including the PH domain, the C2 domain and EF hand motifs are involved in various biological functions of PLC isozymes as signaling proteins. The distribution of PLC isozymes is tissue and organ specific. Recent studies on isolated cells and knockout mice depleted of PLC isozymes have revealed their distinct phenotypes. Given the specificity in distribution and cellular localization, it is clear that each PLC isozyme bears a unique function in the modulation of physiological responses. In this review, we discuss the structural organization, enzymatic properties and molecular diversity of PLC splicing variants and study functional and physiological roles of each isozyme.open19320

NMR backbone assignments of the tyrosine kinase domain of human fibroblast growth factor receptor 3 in apo state and in complex with inhibitor PD173074

Fibroblast growth factors receptors (FGFR) are transmembrane protein tyrosine kinases involved in many cellular process, including growth, differentiation and angiogenesis. Dysregulation of FGFR enzymatic activity is associated with developmental disorders and cancers; therefore FGFRs have become attractive targets for drug discovery, with a number of agents in late-stage clinical trials. Here, we present the backbone resonance assignments of FGFR3 tyrosine kinase domain in the ligand-free form and in complex with the canonical FGFR kinase inhibitor PD173074. Analysis of chemical shift changes upon inhibitor binding highlights a characteristic pattern of allosteric network perturbations that is of relevance for future drug discovery activities aimed at development of conformationally-selective FGFR inhibitors

Novel PLCG2 Mutation in a Patient With APLAID and Cutis Laxa.

Background: The auto-inflammation and phospholipase Cγ2 (PLCγ2)-associated antibody deficiency and immune dysregulation (APLAID) syndrome is a rare primary immunodeficiency caused by a gain-of-function mutation S707Y in the PLCG2 gene previously described in two patients from one family. The APLAID patients presented with early-onset blistering skin lesions, posterior uveitis, inflammatory bowel disease (IBD) and recurrent sinopulmonary infections caused by a humoral defect, but lacked circulating autoantibodies and had no cold-induced urticaria, contrary to the patients with the related PLAID syndrome. Case: We describe a new APLAID patient who presented with vesiculopustular rash in the 1st weeks of life, followed by IBD, posterior uveitis, recurrent chest infections, interstitial pneumonitis, and also had sensorineural deafness and cutis laxa. Her disease has been refractory to most treatments, including IL1 blockers and a trial with ruxolitinib has been attempted. Results: In this patient, we found a unique de novo heterozygous missense L848P mutation in the PLCG2 gene, predicted to affect the PLCγ2 structure. Similarly to S707Y, the L848P mutation led to the increased basal and EGF-stimulated PLCγ2 activity in vitro. Whole blood assays showed reduced production of IFN-γ and IL-17 in response to polyclonal T-cell stimulation and reduced production of IL-10 and IL-1β after LPS stimulation. Reduced IL-1β levels and the lack of clinical response to treatment with IL-1 blockers argue against NLRP3 inflammasome hyperactivation being the main mechanism mediating the APLAID pathogenesis. Conclusion: Our findings indicate that L848P is novel a gain-of-function mutation that leads to PLCγ2 activation and suggest cutis laxa as a possible clinical manifestations of the APLAID syndrome

A Hypermorphic Missense Mutation in PLCG2, Encoding Phospholipase Cγ2, Causes a Dominantly Inherited Autoinflammatory Disease with Immunodeficiency

Whole-exome sequencing was performed in a family affected by dominantly inherited inflammatory disease characterized by recurrent blistering skin lesions, bronchiolitis, arthralgia, ocular inflammation, enterocolitis, absence of autoantibodies, and mild immunodeficiency. Exome data from three samples, including the affected father and daughter and unaffected mother, were filtered for the exclusion of reported variants, along with benign variants, as determined by PolyPhen-2. A total of eight transcripts were identified as possible candidate genes. We confirmed a variant, c.2120C>A (p.Ser707Tyr), within PLCG2 as the only de novo variant that was present in two affected family members and not present in four unaffected members. PLCG2 encodes phospholipase Cγ2 (PLCγ2), an enzyme with a critical regulatory role in various immune and inflammatory pathways. The p.Ser707Tyr substitution is located in an autoinhibitory SH2 domain that is crucial for PLCγ2 activation. Overexpression of the altered p.Ser707Tyr protein and ex vivo experiments using affected individuals’ leukocytes showed clearly enhanced PLCγ2 activity, suggesting increased intracellular signaling in the PLCγ2-mediated pathway. Recently, our laboratory identified in individuals with cold-induced urticaria and immune dysregulation PLCG2 exon-skipping mutations resulting in protein products with constitutive phospholipase activity but with reduced intracellular signaling at physiological temperatures. In contrast, the p.Ser707Tyr substitution in PLCγ2 causes a distinct inflammatory phenotype that is not provoked by cold temperatures and that has different end-organ involvement and increased intracellular signaling at physiological temperatures. Our results highlight the utility of exome-sequencing technology in finding causal mutations in nuclear families with dominantly inherited traits otherwise intractable by linkage analysis

Crystal structure of the human, FIC-Domain containing protein HYPE and implications for its functions

Protein AMPylation, the transfer of AMP from ATP to protein targets, has been recognized as a new mechanism of host-cell disruption by some bacterial effectors that typically contain a FIC-domain. Eukaryotic genomes also encode one FIC-domain protein, HYPE, which has remained poorly characterized. Here we describe the structure of human HYPE, solved by X-ray crystallography, representing the first structure of a eukaryotic FIC-domain protein. We demonstrate that HYPE forms stable dimers with structurally and functionally integrated FIC-domains and with TPR-motifs exposed for protein-protein interactions. As HYPE also uniquely possesses a transmembrane helix, dimerization is likely to affect its positioning and function in the membrane vicinity. The low rate of autoAMPylation of the wild-type HYPE could be due to autoinhibition, consistent with the mechanism proposed for a number of putative FIC AMPylators. Our findings also provide a basis to further consider possible alternative cofactors of HYPE and distinct modes of target-recognition

Disease Variants of FGFR3 Reveal Molecular Basis for the Recognition and Additional Roles for Cdc37 in Hsp90 Chaperone System

Receptor tyrosine kinase FGFR3 is involved in many signaling networks and is frequently mutated in developmental disorders and cancer. The Hsp90/ Cdc37 chaperone system is essential for function of normal and neoplastic cells. Here we uncover the mechanistic inter-relationships between these pro- teins by combining approaches including NMR, HDX-MS, and SAXS. We show that several disease- linked mutations convert FGFR3 to a stronger client, where the determinant underpinning client strength involves an allosteric network through the N-lobe and at the lobe interface. We determine the architec- ture of the client kinase/Cdc37 complex and dem on- strate, together with site-speci?c information, that binding of Cdc37 to unrelated kinases induces a common, extensive conformational remodeling of the kinase N-lobe, beyond localized changes and in- teractions within the binary complex. As further shown for FGFR3, this processing by Cdc37 deacti- vates the kinase and presents it, in a speci ?c orienta- tion established in the complex, for direct recognition by Hsp90

FLIM FRET Technology for Drug Discovery: Automated Multiwell-Plate High-Content Analysis, Multiplexed Readouts and Application in Situ**

A fluorescence lifetime imaging (FLIM) technology platform intended to read out changes in Förster resonance energy transfer (FRET) efficiency is presented for the study of protein interactions across the drug-discovery pipeline. FLIM provides a robust, inherently ratiometric imaging modality for drug discovery that could allow the same sensor constructs to be translated from automated cell-based assays through small transparent organisms such as zebrafish to mammals. To this end, an automated FLIM multiwell-plate reader is described for high content analysis of fixed and live cells, tomographic FLIM in zebrafish and FLIM FRET of live cells via confocal endomicroscopy. For cell-based assays, an exemplar application reading out protein aggregation using FLIM FRET is presented, and the potential for multiple simultaneous FLIM (FRET) readouts in microscopy is illustrated