Search

In this study, the recently identified human protein kinase C-theta (PKC-theta) isoform has been biochemically characterized in detail. An antiserum raised against the unique V3 domain of PKC-theta identified an 80-kDa protein in all human T-cell lines tested, in erythroleukemia K562 cells and in histiocytic lymphoma U-937 cells, but not in a B-lymphoma line (Raji) or in several melanoma, carcinoma, schwanoma or astrocytoma lines, confirming, at the protein level, its predominant expression in hematopoietic cell lines, in particular T cells. Immunoreactive PKC-theta was detected almost exclusively in the cytosolic compartment of unstimulated Jurkat T cells. Stimulation with phorbol ester, however, caused rapid translocation to the membrane. In order to compare the properties of PKC-theta with a representative member of the Ca(2+)-dependent PKC enzymes, full-length cDNAs encoding PKC-theta or PKC-alpha were transiently expressed in COS-1 cells, and recombinant enzymes were partially purified via a six-histidine peptide tag. The catalytic activity of these PKC enzymes was assayed against distinct substrates in the absence and presence of known PKC cofactors. Significant differences were found with respect to activation requirements and substrate preferences between PKC-theta and PKC-alpha. Both enzymes were stimulated by phospholipid and phorbol ester, and were active towards a PKC-derived substrate peptide corresponding to the pseudosubstrate site of PKC. In contrast to PKC-alpha, however, full activation of PKC-theta did not require Ca2+, and its basal activity towards histone H1 was not stimulated by lipid cofactors. Additionally, a myelin-basic-protein-(MBP)-derived peptide, which was readily phosphorylated by PKC-alpha, was a poor substrate for PKC-theta. Similar to PKC-alpha, transient PKC-theta overexpression in murine EL4 thymoma cells caused an approximately 2.5-fold increase in the phorbol-12-myristate-13-acetate-induced transcriptional activation of an interleukin-2 promoter-reporter gene construct. The unique expression and functional properties of PKC-theta suggest that it may play a specialized role in T-cell signaling pathways.

Two src -family protein tyrosine kinases (PTKs), p56 lck and p59 fyn , are thought to play an important role in the antigen-specific T cell receptor (TCR)/CD3-initiated signaling pathway, but their relative contribution to these events is not clearly defined. Here, we have explored the potential of catalytic RNA molecules, or ribozymes, as tools for selectively inhibiting expression of the corresponding target genes in T cells. Several lck - or fyn -specific hammerhead ribozymes were synthesized, cloned into a bacterial transcription vector, and found to display specific catalytic activity in vitro . In order to achieve stable high-level ribozyme expression in intact cells, selected ribozymes were subsequently cloned into a retroviral vector (DC-T5T) immediately downstream of a tRNA met transcription unit. Upon retroviral transduction of a human leukemic T cell line (Jurkat), two out of four chimeric tRNA: ribozymes, fyn-1 and lck-1, were stably expressed at levels of ∼10,000 or ∼25,000 copies/cell, respectively. Ribozyme expression was associated with a reduction of up to 80% ( lck ) or 61% ( fyn ) in endogenous target mRNA by comparison to the corresponding transcript levels in control clones transfected with vector alone. By contrast, expression of the corresponding target proteins was not reduced, suggesting a post-transcriptional compensatory mechanism that increases translation or stability of the p56 lck and/or p59 fyn proteins.

The p56lck and p59fyn protein tyrosine kinases are important signal transmission elements in the activation of mature T lymphocytes by ligands to the T-cell antigen receptor (TCR)/CD3 complex. The lack of either kinase results in deficient early signaling events, and pharmacological agents that block tyrosine phosphorylation prevent T-cell activation altogether. After triggering of the TCR/CD3 complex, both kinases are moderately activated and begin to phosphorylate cellular substrates, but the molecular mechanisms responsible for these changes have remained unclear. We recently found that the p72syk protein tyrosine kinase is physically associated with the TCR/CD3 complex and is rapidly tyrosine phosphorylated and activated by receptor triggering also in T cells lacking p56lck. Here we examine the regulation of p72syk and its interaction with p56lck in transfected COS-1 cells. p72syk was catalytically active and heavily phosphorylated on its putative autophosphorylation site, Tyr-518/519. Mutation of these residues to phenylalanines abolished its activity in vitro and toward cellular substrates in vivo and reduced its tyrosine phosphorylation in intact cells by approximately 90%. Coexpression of lck did not alter the catalytic activity of p72syk, but the expressed p56lck was much more active in the presence of p72syk than when expressed alone. This activation was also seen as increased phosphorylation of cellular proteins. Concomitantly, p56lck was phosphorylated at Tyr-192 in its SH2 domain, and a Phe-192 mutant p56lck was no longer phosphorylated by p72syk. Phosphate was also detected in p56lck at Tyr-192 in lymphoid cells. These findings suggest that p56lck is positively regulated by the p72syk kinase.

We recently identified Vav as a Ras-activating guanine nucleotide exchange factor (GEF) stimulated by a T-cell antigen receptor-coupled protein tyrosine kinase (PTK). Here, we describe a novel, protein kinase-independent alternative pathway of Vav activation. Phorbol ester, 1,2-diacylglycerol, or ceramide treatment of intact T cells, Vav immunoprecipitates, or partially purified Vav generated by in vitro translation or COS-1 cell transfection stimulated the Ras exchange activity of Vav in the absence of detectable tyrosine phosphorylation. GEF activity of gel-purified Vav was similarly stimulated by phorbol myristate acetate (PMA). Stimulation was resistant to PTK and protein kinase C inhibitors but was blocked by calphostin, a PMA and diacylglycerol antagonist. In vitro-translated Vav lacking its cysteine-rich domain, or mutated at a single cysteine residue within this domain (C528A), was not stimulated by PMA but was fully activated by p56lck. This correlated with increased binding of radiolabeled phorbol ester to COS-1 cells expressing wild-type, but not C528A-mutated, Vav. Thus, Vav itself is a PMA-binding and -activated Ras GEF. Recombinant interleukin-1 alpha stimulated Vav via this pathway, suggesting that diglyceride-mediated Vav activation may couple PTK-independent receptors which stimulate production of lipid second messengers to Ras in hematopoietic cells.

Activation of resting T lymphocytes by ligands to the T cell receptor (TcR)/CD3 complex is initiated by phosphorylation of a number of key regulatory proteins on specific tyrosine residues. One such protein is the heterodimeric enzyme phosphatidylinositol-3-kinase (PI3K). We recently found that this enzyme is also rapidly activated following TcR/CD3 triggering and that immunoprecipitated PI3K was activated in vitro by direct tyrosine phosphorylation. Here we show that TcR/CD3-induced tyrosine phosphorylation and activation of PI3K in Jurkat T leukemia cells depend on the presence of the p56lck tyrosine kinase: in a variant of the Jurkat T cell line lacking p56lck, JCaM1, these responses were absent. We also show that p56lck directly activates PI3K purified from transfected COS-1 cells, indicating that other T cell-specific proteins are not required for the process. Finally, tryptic peptide maps show that p56lck phosphorylates three tyrosine residues in the p85 alpha subunit of PI3K and two in p110 of PI3K. Our results suggest that p56lck is required for activation of PI3K in Jurkat T cells and can itself directly activate it by phosphorylating one or several stimulatory sites.

E3 ubiquitin ligases have been placed among the essential molecules involved in the regulation of T cell functions and T cell tolerance. However, it has never been experimentally proven in vivo whether these functions indeed depend on the catalytic E3 ligase activity. The Casitas B-cell lymphoma (Cbl) family protein Cbl-b was the first E3 ubiquitin ligase directly implicated in the activation and tolerance of the peripheral T cell. In this study, we report that selective genetic inactivation of Cbl-b E3 ligase activity phenocopies the T cell responses observed when total Cbl-b is ablated, resulting in T cell hyperactivation, spontaneous autoimmunity, and impaired induction of T cell anergy in vivo. Moreover, mice carrying a Cbl-b E3 ligase-defective mutation spontaneously reject tumor cells that express human papilloma virus Ags. These data demonstrate for the first time, to our knowledge, that the catalytic function of an E3 ligase, Cbl-b, is essential for negative regulation of T cells in vivo. Thus, modulation of the E3 ligase activity of Cbl-b might be a novel modality to control T cell immunity in vaccination, cancer biology, or autoimmunity.

Two novel tyrosine kinase cDNAs were obtained from murine mast cells. These kinases, Emb and Emt, constitute a novel tyrosine kinase subfamily which may also include Tec, a kinase preferentially expressed in liver, and Dsrc28, a fruit fly kinase. Both lack hydrophobic stretches characteristic of the transmembrane domains found in growth factor receptor tyrosine kinases and carboxyl-terminal, negative regulatory tyrosine residue found in Src family kinases. In addition to the Src homology region 2 (SH2) and SH3 domains characteristic of the Src family kinases and other signaling molecules, Emb and Emt share a similar amino-terminal domain comprised mainly of two repeat segments. The emb 2.7-kb transcript was expressed in mast cells, myeloid cells and B lymphocytes while the emt 4.6-kb mRNA in mast cells, myeloid cells and T lymphocytes. The evidence for in vitro tyrosine kinase activity of Emb and Emt proteins is also provided.

Members of the protein kinase C (PKC) family of serine/threonine kinases play a key role in regulating the differentiation and growth of diverse cell types and, to date, the cloning of seven mammalian PKC genes encoding eight distinct isoforms has been reported. Here we describe the molecular cloning and deduced primary structure of a cDNA encoding a novel PKC isoform, termed PKC theta, which was isolated in the course of attempts to identify PKC genes that are expressed selectively in hematopoietic cells. Degenerate oligonucleotide primers corresponding to conserved sequence motifs, which distinguish the PKC family from other protein kinases, were employed in polymerase chain reactions (PCR) to amplify partial core sequences of putative PKC genes from a human peripheral blood lymphocyte-derived cDNA library. DNA sequencing of selected clones revealed several PKC-related sequences, including one that, on the basis of sequence comparison with known PKC isoforms, represented a novel PKC isoform. The complete cDNA sequence was determined by anchored PCR cloning and sequencing the entire coding sequence, using cDNA derived from a human leukemic T cell line (Jurkat). Included within this approximately 2.7-kilobase pair cDNA is an open reading frame of 2,118 nucleotides encoding a putative 82-kDa protein. The deduced primary structure contains consensus sequences characteristic of protein kinase catalytic domains and, based on its amino acid sequence and domain structure, is a member of the PKC family. PKC theta displays the highest homology to PKC delta, lacks the Ca(2+)-binding C2 domain and, thus, belongs to the subfamily of Ca(2+)-independent PKC enzymes which also includes the delta, epsilon, zeta, and eta isoforms. RNase protection assays and semiquantitative PCR analysis indicated that, although PKC theta transcripts are expressed ubiquitously, the highest levels are found in hematopoietic tissues and cell lines, including T cells and thymocytes. In contrast, the expression levels in the brain and testes are considerably lower, and no transcripts were detected in several human carcinoma cell lines. A rabbit antiserum raised against a unique (V3 domain) bacterially expressed PKC theta fragment immunoprecipitated specifically an 82-kDa protein from Jurkat cell lysates. Thus, PKC theta represents an additional member of the PKC family, and its predominant expression in hematopoietic cells suggests that it may play a role in signal transduction and growth regulatory pathways unique to these cells.

The Ca2+ dependent transcription factor family known as nuclear factor of activated T cells (NFAT) has been shown to be important in T-cell immune responses. Because NFAT proteins have a weak DNA-binding capacity, they cooperate with other transcription factors at composite sites within the promoters of target genes. Recently, NFAT was shown to also be important for the induction of specific genetic programs that guide the differentiation and effector or regulatory activities of CD4+ T helper subsets via the transcriptional regulation of their lineage-specific transcription factors, specifically T-bet (Th1), Gata3 (Th2), RORγt (Th17), and Foxp3 (iTregs). In addition, the NFAT family governs the transcription of several signature cytokines, including their cytokine receptors. Subsequently, the integration of these complex intracellular signal transduction cascades is considered to critically determine the crosstalk between the T-cell receptor and receptors that are activated by both the adaptive and innate immune systems to determine pathways of T helper cell differentiation and function. Here, we carefully review the critical role of the established transcriptional partners and functional outcomes of these NFAT interactions in regard to the effector responses of these clinically relevant CD4+ T helper subsets.

The E3 ubiquitin ligase Casitas B-lineage lymphoma (Cbl-b) is central to antigen-induced immune tolerance and regulates the CD28 dependence of T cell activation. Cbl-b undergoes ubiquitination and proteasomal degradation after adequate costimulation of T cells; however, the mechanism involved is unknown. Here, we identified protein kinase C-theta (PKC-theta) as the critical intermediary for the inactivation of Cbl-b in response to costimulation of T cells through CD28. PKC-theta associated with Cbl-b on stimulation of the T cell receptor. After costimulation of T cells through CD28, Cbl-b was ubiquitinated and degraded through a mechanism that depended on the kinase activity of PKC-theta. Consistent with this mechanism, the impaired responses of PKCtheta-deficient T cells were at least partially restored by the concomitant genetic loss of cblb. Thus, our data establish a nonredundant antagonism between PKC-theta and Cbl-b that regulates T cell activation responses.

The protein kinase C (PKC) family of serine-threonine kinases plays a central role in T lymphocyte activation. Here, we identify NR2F6, a nuclear zinc-finger orphan receptor, as a critical PKC substrate and essential regulator of CD4(+) T cell activation responses. NR2F6 potently antagonized the ability of T helper 0 (Th0) and Th17 CD4(+) T cells to induce expression of key cytokine genes such as interleukin-2 (IL-2) and IL-17. Mechanistically, NR2F6 directly interfered with the DNA binding of nuclear factor of activated T cells (NF-AT):activator protein 1 (AP-1) but not nuclear factor kappaB (NF-kappa B) and, subsequently, transcriptional activity of the NF-AT-dependent IL-17A cytokine promoter. Consistent with our model, Nr2f6-deficient mice had hyperreactive lymphocytes, developed a late-onset immunopathology, and were hypersusceptible to Th17-dependent experimental autoimmune encephalomyelitis. Our study establishes NR2F6 as a transcriptional repressor of IL-17 expression in Th17-differentiated CD4(+) T cells in vitro and in vivo.

The physiological roles of PKC alpha and PKC theta were defined in T cell immune functions downstream of the antigen receptor. To investigate the hypothesis that both PKC isotypes may have overlapping functions, we generated mice lacking both genes. We find that PKC alpha(-/-)/theta(-/-) animals have additive T cell response defects in comparison to animals carrying single mutations in these genes. Our studies demonstrate that the activities of PKC alpha and PKC theta converge to regulate both IL-2 cytokine responses and T cell intrinsic alloreactivity in vivo. Mechanistically, this PKC alpha/theta crosstalk primarily affects the NFAT transactivation pathway in T lymphocytes, as observed by decreased phosphorylation of Ser-9 on GSK3 beta, reduced nuclear translocation and DNA binding of NFAT in isolated PKC alpha(-/-)/theta(-/-) CD3(+) T cells. This additive defect proved to be of physiological relevance, because PKC alpha(-/-)/theta(-/-) mice demonstrated significantly prolonged allograft survival in heart transplantation experiments, whereas both PKC alpha(-/-) and PKC theta(-/-) mice showed only minimal graft prolongation when compared to wild type controls. While PKC theta appears to be the rate-limiting PKC isotype mediating T lymphocyte activation, we here provide genetic evidence that PKC alpha and PKC theta have overlapping functions in alloimmunoreactivity in vivo and both PKC theta and PKC alpha isotypes must be targeted to prevent organ allograft rejection.

A comparison of the efficiencies of hydrophobic interaction chromatography, ion-exchange chromatography, reversed-phase chromatography and gel permeation chromatography in the separation of tear proteins was made using a variety of different buffers. Separation of immunoglobulins, lactoferrin, albumin, PMFA (protein migrating faster than albumin) and lysozyme was accomplished by gel permeation chromatography in less than 30 min using a TSK-type SW3000 column equilibrated with ammonium acetate buffer (pH 4.1) with a high reproducibility. When gel permeation chromatography was used as a completely automated diagnostic method, only minute volumes (1.0 microliter) of tear samples were necessary for the quantitative analysis of proteins. The other three methods proved to be more suitable for the preparation of individual tear proteins but were less suitable for their quantitation.

The main function of mature T cells is to recognize and respond to foreign antigens by a complex activation process involving differentiation of the resting cell to a proliferating lymphoblast actively secreting immunoregulatory lymphokines or displaying targeted cytotoxicity, ultimately leading to recruitment of other cell types and initiation of an effective immune response. In order to understand the physiology and pathophysiology of T lymphocytes, it is necessary to decode the biochemical processes that integrate signals from antigen, cytokine, integrin and death receptors. The principal upon which our work is based is to explore and identify gene products of distinct members of the AGC family of protein serine/threonine kinases as key players mediating cell growth regulation. Given the established important role of PKC θ as regulator of T cell fate and knowing that several other PKC isotypes are also expressed in T cells at a high level, we now summarize the physiological and non-redundant functions of PKC α, β, δ, , ζ and θ isotypes in T cells. This review describes the current knowledge of the physiological and non-redundant functions of the PKC gene products in T cells.

Using yeast two-hybrid, we isolated atypical PKCzeta as a PKCtheta-interacting kinase and demonstrated that it selectively interacted with, and was phosphorylated by, PKCtheta. Importantly, however, both atypical PKCzeta and PKCiota were functionally required in TCR/CD28-mediated activation of NF-kappaB downstream of PKCtheta in Jurkat T cells albeit, activation responses of PKCzeta-deficient CD3+ T cells were comparable with wildtype controls. This normal activation thresholds of PKCzeta-/- T cells suggested that PKCiota, the closest structural relative, might play a compensatory role in TCR/CD28-induced signalling. Consistently, both PKCzeta and PKCiota resided in the plasma membrane lipid raft microdomains of Jurkat as well as primary mouse CD3+ T cells. Thus, PKCtheta, the established constituent of the immunological synapse, physically and functionally interacted with PKCzeta and PKCiota. Together, these data demonstrate that atypical PKCzeta/iota isotypes serve as direct downstream targets of PKCtheta in the signalling pathway leading to NF-kappaB activation in T lymphocytes.

Using model tumor T cell lines, protein kinase C (PKC) α has been implicated in IL-2 cytokine promoter activation in response to Ag receptor stimulation. In this study, for the first time, PKCα null mutant mice are analyzed and display normal T and B lymphocyte development. Peripheral CD3+ PKCα-deficient T cells show unimpaired activation-induced IL-2 cytokine secretion, surface expression of CD25, CD44, and CD69, as well as transactivation of the critical transcription factors NF-AT, NF-κB, AP-1, and STAT5 in vitro. Nevertheless, CD3/CD28 Ab- and MHC alloantigen-induced T cell proliferation and IFN-γ production are severely impaired in PKCα−/− CD3+ T cells. Consistently, PKCα-deficient CD3+ T cells from OVA-immunized PKCα-deficient mice exhibit markedly reduced recall proliferation to OVA in in vitro cultures. In vivo, PKCα-deficient mice give diminished OVA-specific IgG2a and IgG2b responses following OVA immunization experiments. In contrast, OVA-specific IgM and IgG1 responses and splenic PKCα−/− B cell proliferation are unimpaired. Our genetic data, thus, define PKCα as the physiological and nonredundant PKC isotype in signaling pathways that are necessary for T cell-dependent IFN-γ production and IgG2a/2b Ab responses.

We observe that protein kinase C (PKC) is phosphorylated on the activation loop at threonine 538 (Thr-538) before T cell activation. Our results are inconsistent with the conclusions of Lee et al. (Reports, 1 April 2005, p. 114) that the Thr-538 phosphorylation of PKC is regulated by T cell receptor activation. Other mechanisms, such as autophosphorylation of Thr-219, might orchestrate the cellular function of PKC in T cells.

Protein kinase C theta (PKC theta) is unique among PKC isozymes in its translocation to the center of the immune synapse in T cells and its unique downstream signaling. Here we show that the hematopoietic protein tyrosine phosphatase (HePTP) also accumulates in the immune synapse in a PKC theta-dependent manner upon antigen recognition by T cells and is phosphorylated by PKC theta at Ser-225, which is required for lipid raft translocation. Immune synapse translocation was completely absent in antigen-specific T cells from PKC theta-/- mice. In intact T cells, HePTP-S225A enhanced T-cell receptor (TCR)-induced NFAT/AP-1 transactivation, while the acidic substitution mutant was as efficient as wild-type HePTP. We conclude that HePTP is phosphorylated in the immune synapse by PKC theta and thereby targeted to lipid rafts to temper TCR signaling. This represents a novel mechanism for the active immune synapse recruitment and activation of a phosphatase in TCR signaling.

Hydrophobic vitamins are transported in human plasma and extravascular fluids by carrier proteins. No specific protein has been described so far for vitamin E, which plays a crucial role in protecting against oxidative damage and disease. We report here the purification of a 75-kDa glycoprotein with vitamin E-binding properties by stepwise chromatography of lipoprotein-depleted human plasma and monitoring of vitamin E (alpha-tocopherol)-binding activity. Partial sequencing identified this protein as afamin, a previously described member of the albumin gene family with four or five potential N-glycosylation sites. Glycosylation analysis indicated that >90% of the glycans were sialylated biantennary complex structures. The vitamin E-binding properties were confirmed using recombinantly expressed afamin. Qualitative and quantitative analysis of plasma and extravascular fluids revealed an abundant presence of this protein not only in plasma (59.8+/-13.3 microg/mL) but also in extravascular fluids such as follicular (34.4+/-12.7 microg/mL) and cerebrospinal (0.28+/-0.16 microg/mL) fluids, suggesting potential roles for afamin in fertility and neuroprotection. Afamin is partly (13%) bound to plasma lipoproteins. Afamin and vitamin E concentrations significantly correlate in follicular and cerebrospinal fluids but not in plasma. The vitamin E association of afamin in follicular fluid was directly demonstrated by gel filtration chromatography and immunoprecipitation which complements the in vitro findings for purified native and recombinant afamin.

PKCepsilon has been strongly linked to cell activation and proliferation in many cell types, including leukemic T-cell lines. In particularly, an essential role of PKCepsilon has been established in the IKK-beta/I-kappaB/NF-kappaB transactivation cascade. To study the physiological function of PKCepsilon in primary T-cells, we used our newly established PKCepsilon null mice. Unexpectedly, however, we did not reveal any defect in the development and function of CD3+ T-cells. Proliferative responses as well as IL-2 cytokine secretion of PKCepsilon-deficient T-cells induced by allogenic MHC, plate-bound anti-CD3 antibodies (with or without anti-CD28 costimulation), or mitogenic stimuli such as phorbol ester and Ca2+ ionophore were comparable with wild-type controls. Consistently, after CD3/CD28 engagement, deficiency of PKCepsilon did not impair NF-kappaB transactivation as well as CD25, CD44 and CD69 induction. Thus, PKCepsilon-deficient T-cells had similar physiological thresholds for activation in vitro. This finding suggests that PKCepsilon plays a redundant role in TCR-induced regulation of T-cell proliferation.

PKCbeta has been established to be essential in B cell receptor (BCR) signaling. Additionally, a critical role of PKCbeta in TCR/CD28-stimulated regulation of IL-2 gene transcription but also exocytotic IL-2 secretion was observed in leukemic T cell lines. To now study the physiological function of PKCbeta in primary CD3(+) T cells, we used our established PKCbeta null mice. Unexpectantly, we did not reveal any defect in the development and function of T cells. Proliferative responses as well as IL-2 cytokine secretion of PKCbeta-deficient CD3(+) T cells induced by allogenic MHC, plate-bound anti-CD3 antibodies (with or without anti-CD28 costimulation), or mitogenic stimuli such as phorbol ester and Ca(2+) ionophore were comparable with wild-type controls. Thus, PKCbeta-deficient T cells had similar physiological thresholds for activation in vitro. These findings suggest that PKCbeta plays a redundant role in TCR-induced regulation of IL-2 cytokine production and T cell proliferation.

The ubiquitously expressed Na(+)/H(+) exchanger (NHE1) plays an important role in the regulation of the intracellular pH. Induction of NHE activity by phorbol esters and inhibition of growth factor-mediated stimulation of the NHE by protein kinase C (PKC) inhibitors suggest an implication of PKCs in the regulation of the NHE. Expression of PKC isotype-specific dominant negative and constitutively active mutants or downregulation of PKC by isotype-specific antisense oligonucleotides revealed that stimulation by epidermal growth factor (EGF) or phorbol ester of the NHE in NIH3T3 cells is a PKC(alpha)-specific effect. Elevation of cytoplasmic calcium by a Ca(2+) ionophore or thapsigargin causes a growth factor-independent stimulation of the NHE predominantly mediated by calcium/calmodulin kinase II. It is concluded that in NIH3T3 cells overexpressing the EGF receptor (EGFR6 cells), EGF requires cPKC(alpha) for the activation of the NHE, while calcium/calmodulin-dependent kinases are essential in thapsigargin induced stimulation of the NHE.

Protein glycosylation plays an important role in protein stability, folding, and secretion but presents a major challenge for protein crystallization. Crystallization of highly glycosylated proteins is often difficult because of conformational and chemical inhomogeneity of the glycan decorations. Therefore, it is almost always necessary to modify the material to obtain a conformational homogenous protein that can crystallize. (1) We have explored several avenues in the case of the human 87 kDa glycoprotein afamin. Afamin (AFM), a member of the albumin gene family, is mainly expressed in the liver and secreted into the bloodstream. (2) Elevated afamin plasma concentrations are associated with major diseases such as metabolic syndrome and cancer;(3) however, pathophysiological functions are largely unknown. Therefore, we are pursuing the crystal structure of various forms of recombinantly expressed human afamin (rhAFM) for structure guided exploration and analysis of its function. Multiple variants of rhAFM are pursued: 1) fully glycosylated wild-type rhAFM (expressed in CHO cell lines); 2) rhAFM complexed with Fab fragments of two anti-AFM mAbs, 3) enzymatic deglycosylation of rhAFM with PGNaseF, 4) partially glycosylated rhAFM (expressed in glycosylation-deficient Lec1-CHO cells); and 5) glycosylation-free rhAFM, obtained from HEK293 cells virally transfected with a cDNA mutant lacking all 5 glycosylation sites by replacement of ASN with ASP. Yields of up to 2 mg/mL glycosilation free mutant rhAFM in HEK273 cells have been comparable to native rhAFM. C-terminal His6-tagged rhAFM was captured by Ni-IMAC from serum-free cell culture supernatants. All variants and Fab-AFM complexes were polished via SEC yielding pure products per SDS-PAGE and immunoblot analysis. Crystals have been obtained from Fab fragments and crystalline spherulites which are useful for microseeding from Fab-AFM complexes.

The apoE gene has been identified as a major susceptibility locus for late-onset Alzheimer's disease (LOAD). The epsilon4 allele greatly reduces age of onset of LOAD as compared to the wild-type 3 allele. The molecular mechanism(s) underlying the association has not yet been fully elucidated. The apoE protein has been shown to physically interact with the Abeta region of the Amyloid Precursor Protein (APP), but also with the ectodomain of the APP holoprotein itself. In this study we have used apoE fusion proteins containing either the ER retention sequence KDEL or trans-Golgi network (TGN) signal sequence in order to define potential apoE-mediated alterations in APP protein processing. Co-expression and pulse-chase experiments showed that a functional apoE:APP interaction occurs intracellularly which directly affects maturation and subsequently the secretion kinetics of APP. In addition, an epsilon4 allele-specific induction of Abeta production has been demonstrated. apoE3 resulted in increased Abeta production only when targeted to the ER, as observed in cells transfected with an apoE3KDEL fusion protein as well as following treatment with brefeldin A. The findings suggest that in cells that express both apoE and APP, such as astrocytes and microglia, a functional apoE:APP interaction may occur which modulates APP processing and Abeta production.

Protein kinase C theta (PKC theta) is known to induce NF-kappa B, an essential transcriptional element in T cell receptor/CD28-mediated interleukin-2 production but also T cell survival. Here we provide evidence that PKC theta is physically and functionally coupled to Akt1 in this signaling pathway. First, T cell receptor/CD3 ligation was sufficient to induce activation as well as plasma membrane recruitment of PKC theta. Second, PKC theta selectively cooperated with Akt1, known to act downstream of CD28 co-receptor signaling, in activating a NF-kappa B reporter in T cells. Third, Akt1 function was shown to be required for PKC theta-mediated NF-kappa B transactivation. Fourth, PKC theta co-immunoprecipitated with Akt1; however, neither Akt1 nor PKC theta served as a prominent substrate for each other in vitro as well as in intact T cells. Finally, plasma membrane targeting of PKC theta and Akt1 exerted synergistic transactivation of the I-kappa B kinase beta/inhibitor of NF-kappa B/NF-kappa B signaling cascade independent of T cell activation. Taken together, these findings suggest a direct cross-talk between PKC theta and Akt1 in Jurkat T cells.

ICln is a ubiquitously expressed eukaryotic protein. Expression of the protein in Xenopus laevis oocytes, the knocking-down of the protein in fibroblasts, or the reconstitution of the protein in lipid bilayer led to the assumption that this protein is an ionic channel or a significant part thereof. However, other possible roles for ICln in potential regulatory mechanisms have been postulated, as diverse as regulator of cell morphology by interacting with the Skb1 protein and/or interaction with core spliceosomal proteins. Here we show that ICln is able to interact with SnRNP core proteins SmD1, SmD2, SmD3, SmX5 and SmB/B'.

Although for a few malignancies, such as childhood leukaemia, Hodgkin’s disease, and testicular cancer, impressive progress has been achieved, cancer mortality as a whole has remained almost unchanged during the last two decades despite enormous worldwide efforts. This is particularly surprising in view of the tremendous progress that has been achieved during the last 20–25 years with regard to our understanding of the molecular mechanisms underlying the regulation of normal growth and dysregulation of malignant growth. Major scientific breakthroughs — the discovery of oncogenes, tumour suppressor genes, mechanisms of angiogenesis, and apoptosis — have occurred during the last two to three decades, but these have not led to any major progress in cancer treatment.

Recent studies have identified protein kinase Ctheta; (PKCtheta), a member of the Ca(2+)-independent PKC family, as an essential component of the T-cell synapse that cooperates with calcineurin to activate the interleukin-2 (IL-2) gene. Several selective functions of PKCtheta involved in the activation and survival of T cells are reviewed herein. Among these, the nuclear factor-kappaB (NF-kappaB) signaling cascade appears to be the most critical target of PKCtheta in the T-cell receptor/CD28 costimulatory pathway that leads to T-cell activation.

Lipoprotein(a) (Lp(a)) is a major independent risk factor for atherothrombotic disease in humans. The physiological function(s) of Lp(a) as well as the precise mechanism(s) by which high plasma levels of Lp(a) increase risk are unknown. Binding of apolipoprotein(a) (apo(a)) to fibrin(ogen) and other components of the blood clotting cascade has been demonstrated in vitro, but the domains in fibrin(ogen) critical for interaction are undefined. We used apo(a) kringle IV subtypes to screen a human liver cDNA library by the yeast GAL4 two-hybrid interaction trap system. Among positive clones that emerged from the screen, clones were identified as fibrinogen beta- and gamma-chains. Peptide-based pull-down experiments confirmed that the emerging peptide motif, conserved in the carboxyl-terminal globular domains of the fibrinogen beta and gamma modules specifically interacts with apo(a)/Lp(a) in human plasma as well as in cell culture supernatants of HepG2 and Chinese hamster ovary cells, ectopically expressing apo(a)/Lp(a). The influence of lysine in the fibrinogen peptides and of lysine binding sites in apo(a) for the interaction was evaluated by binding experiments with apo(a) mutants and a mutated fibrin(ogen) peptid. This confirmed the lysine binding sites in kringle IV type 10 of apo(a) as the major fibrin(ogen) binding site but also demonstrated lysine-independent interactions.

Sufficient gene transfer into CNS-derived cells is the most crucial step to develop strategies for gene therapy. In this study liposome-mediated gene transfer using a β-galactosidase (β-GAL) reporter gene was performed in vitro (C6 glioma cells, NT2 neuronal precursor cells, 3T3 fibroblasts, primary glial cells) and in vivo. Using Trypan blue exclusion staining, optimal lipid concentration was observed in the range of 10-12 μg/mL. Under optimal conditions (80,000 cells/16 mm well, incubation overnight, lipid/DNA ratio = 1:18) a high transfection rate was achieved (

Recent studies have documented direct interactions between 14-3-3 proteins and several oncogene and proto-oncogene products involved in signal transduction pathways. Studies on the effects of 14-3-3 proteins on protein kinase C (PKC) activity in vitro have reported conflicting results, and previous attempts to demonstrate a direct association between PKC and 14-3-3 were unsuccessful. Here, we examined potential physical and functional interactions between PKC theta, a Ca(2+)-independent PKC enzyme which is expressed selectively in T lymphocytes, and the 14-3-3 tau isoform in vitro and in intact T cells. PKC theta and 14-3-3 tau coimmunoprecipitated from Jurkat T cells, and recombinant 14-3-3 tau interacted directly with purified PKC theta in vitro. Transient overexpression of 14-3-3 tau suppressed stimulation of the interleukin 2 (IL-2) promoter mediated by cotransfected wild-type or constitutively active PKC theta, as well as by endogenous PKC in ionomycin- and/or phorbol ester-stimulated cells. This did not represent a general inhibition of activation events, since PKC-independent (but Ca(2+)-dependent) activation of an IL-4 promoter element was not inhibited by 14-3-3 tau under similar conditions. Overexpression of wild-type 14-3-3 tau also inhibited phorbol ester-induced PKC theta translocation from the cytosol to the membrane in Jurkat cells, while a membrane-targeted form of 14-3-3 tau caused increased localization of PKC theta in the particulate fraction in unstimulated cells. Membrane-targeted 14-3-3 tau was more effective than wild-type 14-3-3 tau in suppressing PKC theta-dependent IL-2 promoter activity, suggesting that 14-3-3 tau inhibits the function of PKC theta not only by preventing its translocation to the membrane but also by associating with it. The interaction between 14-3-3 and PKC theta may represent an important general mechanism for regulating PKC-dependent signals and, more specifically, PKC theta-mediated functions during T-cell activation.

T-lymphocyte stimulation requires activation of several protein kinases, including the major phorbol ester receptor protein kinase C (PKC), ultimately leading to induction of lymphokines, such as interleukin-2 (IL-2). The revelant PKC isoforms which are involved in the activation cascades of nuclear transcription factors involved in IL-2 production have not yet been clearly defined. We have examined the potential role of two representative PKC isoforms in the induction of the IL-2 gene, i.e., PKC-alpha and PKC-theta, the latter being expressed predominantly in hematopoietic cell lines, particularly T cells. Similar to that of PKC-alpha, PKC-theta overexpression in murine EL4 thymoma cells caused a significant increase in phorbol 12-myristate 13-acetate (PMA)-induced transcriptional activation of full-length IL-2-chloramphenicol acetyltransferase (CAT) and NF-AT-CAT but not of NF-IL2A-CAT or NF-kappaB promoter-CAT reporter gene constructs. Importantly, the critical AP-1 enhancer element was differentially modulated by these two distinct PKC isoenzymes, since only PKC-theta but not PKC-alpha overexpression resulted in an approximately 2.8-fold increase in AP-1-collagenase promoter CAT expression in comparison with the vector control. Deletion of the AP-1 enhancer site in the collagenase promoter rendered it unresponsive to PKC-theta. Expression of a constitutively active mutant PKC-theta A148E (but not PKC-alpha A25E) was sufficient to induce activation of AP-1 transcription factor complex in the absence of PMA stimulation. Conversely, a catalytically inactive PKC-theta K409R (but not PKC-alpha K368R) mutant abrogated endogenous PMA-mediated activation of AP-1 transcriptional complex. Dominant negative mutant Ha-RasS17N completely inhibited the PKC-O A148E-induced signal, PKC-O. Expression of a constitutively active mutant PKC-O A148E (but not PKC-alpha A25E) was sufficient to induce activation of AP-1 transcription factor complex in the absence of PMA stimulation. Conversely, a catalytically inactive PKC-O K409R (but not PKC-alpha K368R) mutant abrogated endogenous PMA-mediated activation of AP-1 transcriptional complex. Dominant negative mutant Ha-enRasS17N completely inhibited in the PKC-O A148E-induced signal, identifying PKC-theta as a specific constituent upstream of or parallel to Ras in the signaling cascade leading to AP transcriptional activation.

To increase the inherently weak immunogenicity of synthetic peptide vaccines, we used recombinant DNA techniques to generate chimeras between immunogenic determinants of human immunodeficiency virus type 1 (HIV-1) gp120 and antibody Fab fragments reactive with surface structures displayed specifically on human antigen-presenting cells (APCs), including surface immunoglobulin D (sIgD) and class II major histocompatibility complex (MHC) molecules. Hybridomas producing anti-human MHC class II (HLA-DR) or surface immunoglobulin D monoclonal antibodies (MAbs) that recognize nonpolymorphic determinants were used to clone chimeric Fab gene fragments by employing an established procedure to generate antigen-binding Fab libraries in phagemid vector pComb3. Molecular and immunochemical analysis indicated that the expected chimeric Fab fragments expressing the HIV-1 epitopes were correctly cloned and expressed in Escherichia coli and retained the binding specificity of the native (hybridoma-derived) MAb. The chimeric Fab fragments targeted the linked HIV-1-derived antigenic determinants to the surface of human APCs in vitro, as evidenced by fluorescence-activated cell sorter analysis. Furthermore, such recombinant immunotargeted HIV-1 peptide antigens demonstrated improved immunogenicity over equivalent nonimmunotargeted control antigens, as shown by their ability to stimulate interleukin-2 production by CD4+ T-helper cells from human donors exposed to HIV-1 antigens. These data suggest that immunotargeting of recombinant peptide antigens via the attached Fab fragments facilitates uptake by human APCs with subsequent access to the MHC class II processing pathway, thereby validating the immunotargeting concept for such recombinant subunit vaccines in an in vitro human system.

Previous studies from our laboratory and those of others suggested the possibility that the T-cell antigen receptor alpha (TCR alpha) chain from some T cells can be released in a soluble form and can have antigen-specific immunoregulatory activity. We have analyzed this phenomenon by in vitro transcription and translation (IVTT) of a cDNA encoding a TCR alpha chain (A1.1 TCR alpha) suspected of having such activity. We found that TCR alpha, but not TCR beta, protein produced in this way showed antigen-specific regulatory activity in an in vitro immune-response assay. Protein derived from truncated forms of the A1.1 TCR alpha cDNA had activity providing that, in addition to the variable (V) and joining (J) regions of the alpha chain (VJ alpha), at least the first 25 amino acids of the alpha chain of the constant (C) region (C alpha) were present. Addition of an irrelevant protein sequence to the VJ alpha failed to impart activity to the molecule, suggesting that the C alpha requirement is not simply for stabilization of the resulting protein. These results are discussed in the context of other recent studies on the immunoregulatory activity of soluble TCR alpha molecules, and the possible physiological relevance of these observations is considered.

Members of the protein kinase C (PKC) family of serine/threonine kinases play critical roles in the regulation of cellular differentiation and proliferation of diverse cell types. In an attempt to find PKC isoforms that are involved in growth control and/or activation of T lymphocytes, we have used a human peripheral blood lymphocyte-derived cDNA library to identify a novel PKC isoform, termed PKC-{theta}. The human PKC-{theta} cDNA (human gene symbol PRKCQ) was characterized and found to encode an {approximately} 80-kDa protein, expressed predominantly in lymphoid tissues and hematopoietic cell lines, in particular T cells. A murine homolog gene derived from skin cDNA library was found to be expressed predominantly in skeletal muscle. Molecular cloning and biochemical studies identified PKC enzymes as members of a distinct family that constitutes, at the gene level, nine mammalian members, i.e., {alpha}, {beta}, {gamma}, {delta}, {epsilon}, {zeta}, {theta}, and {lambda}, of which three have already been assigned to individual human chromosomes. Thus, PKC-{alpha} was mapped to H17q22-q24, PKC-{Beta} to H16p12-q11.1, and PKC-{gamma} to H19q13.4. The chromosomal location of other members is yet unknown. 13 refs., 1 fig.

Activation of resting T lymphocytes by ligands to the T-cell antigen receptor (TCR)/CD3 complex is initiated by rapid tyrosine phosphorylation of cellular proteins. Protein-tyrosine kinases (PTKs) of the src family are known to be important, but the mechanism of their recruitment and their interactions with PTKs of other families are incompletely understood. We show that a member of another family of PTKs, the p72syk kinase, is constitutively bound to the TCR/CD3 complex and becomes tyrosine phosphorylated and activated within 1 min after TCR/CD3 stimulation. This activation did not depend on the presence of p56lck in T cells and in transfected COS cells. In both cases, however, the phosphorylation of cellular substrates was augmented by src family PTKs. We propose that p72syk may act as an immediate receptor-activated kinase upstream of the related p70zap PTK and the src family PTKs p56lck and p59fyn in T cells and that these src family PTKs act as signal amplifiers.

We observe that protein kinase C θ (PKCθ) is phosphorylated on the activation loop at threonine 538 (Thr-538) before T cell activation. Our results are inconsistent with the conclusions of Lee et al . (Reports, 1 April 2005, p. 114) that the Thr-538 phosphorylation of PKCθ is regulated by T cell receptor activation. Other mechanisms, such as autophosphorylation of Thr-219, might orchestrate the cellular function of PKCθ in T cells.

Background Protein kinase C (PKC) has become a major focus among cell biologists interested in second-messenger signal transduction and much has been learned about differences in the cellular localization and function of its different isotypes. In this study we systematically address the genomic locations and gene structures of the human PKC gene module.

An open reading frame upstream of the Methanococcus vannielii L12 gene has been detected. The beginning of this open reading frame agrees with the N-terminal region of a protein (MvaL10) which has been isolated from the 50 S ribosomal subunit of M. vannielii and sequenced. The length of this gene is 1008 nucleotides, coding for 336 amino acids. Excellent sequence similarities were found to the L10-like ribosomal proteins from Halobacterium halobium and man. The N-terminal part of the MvaL10 protein shows significant sequence similarities to the E. coli L10 protein. MvaL10 is more than twice as long as E. coli L10 but is of length similar to those of the homologous halobacterial and human proteins. Interestingly, the C-terminal region of MvaL10 shows exceptionally high similarity to the C-terminal sequence of the MvaL12 protein. This is not the case for the E. coli proteins but was also observed for the human, Halobacterium and Sulfolobus proteins.

Summary 50S ribosomes from Escherichia coli mutants lacking r-protein EL1 have been reconstituted with total r-proteins from Methanococcus vannielli , which had been radioactively labelled (either in vivo or in vitro ). A single protein was incorporated, designated ML6 according to the nomenclature of Schmid and Bock ( Schmid, G. and Bock, A . (1982) Zbl. Bakt. Hyg., I. Abt. Orig., C 3, 347–353), indicating that M. vannielli r-protein ML6 is structurally equivalent to E. coli r-protein L1. Protein ML6 was purified to homogeneity and an antiserum was raised which was used to localize protein ML6 by immuno electron microscopy on the surface of 50S subunits from M. vannielii and from mutant ribosomes, that had been reconstituted with ML6. In both cases antibody binding was to the broader lateral protuberance, in the same region where EL 1 has been located in E. coli wild type. Finally it was shown in a poly(U) dependent poly (Phe) in vitro translation system that reconstitution of the mutant 50S subunits with protein ML6 fully restored their activity to wild type level, thereby establishing also the functional equivalence of r-protein ML6 and EL1. We have thus for the first time constructed in vitro a chimeric ribosomal subunit, containing rRNA's and r-proteins from an eubacterium ( E. coli ) and a single r-protein from an archaebacterium ( M. vannielii ). The data suggest that important regions of r-proteins are, as is the case with rRNA's, also conserved between members of these two primary lineages.

Members of the MAGUK family proteins cluster receptors and intracellular signaling molecules at the neuronal synapse. We report that genetic inactivation of the MAGUK family protein CARD11/Carma1/Bimp3 results in a complete block in T and B cell immunity. CARD11 is essential for antigen receptor- and PKC-mediated proliferation and cytokine production in T and B cells due to a selective defect in JNK and NFκB activation. Moreover, B cell proliferation and JNK activation were impaired upon stimulation of TLR4 with lipopolysaccharide, indicating that CARD11 is involved in both the innate and adaptive immune systems. Our results show that the same family of molecules are critical regulators of neuronal synapses and immune receptor signaling.

Protein kinase (PK) Ctheta and Akt/PKBalpha cooperate in T cell receptor/CD28-induced T cell signaling. We here demonstrate the recruitment of endogenous Akt1 and PKCtheta to lipid rafts in CD3-stimulated T cells. Further we show that Myr-PKCtheta mediates translocation of endogenous Akt1 to the plasma membrane as well as to lipid rafts, most likely explained by the observed complex formation of both protein kinases. In addition, in peripheral mouse T cells, the PKC inhibitor Gö6850 could partially block Akt1 activation in CD3-induced signaling, placing PKC isotype(s) upstream of Akt1. However, T cells derived from PKCtheta knockout mice were not impaired in CD3- or phorbol ester-induced Akt1 activity. Taken together, the results of this study give new insights into the functional link of Akt1 and PKCtheta in T cell signaling, demonstrating the co-recruitment of the two kinases and showing a novel pathway leading to Akt1 transactivation where PKC isotype(s) are involved but PKCtheta is not essential.

Interleukin-17A (IL-17A) is the signature cytokine produced by Th17 CD4+ T cells and has been tightly linked to autoimmune pathogenesis. In particular, the transcription factors NFAT and RORγt are known to activate Il17a transcription, although the detailed mechanism of action remains incompletely understood. Here, we show that the nuclear orphan receptor NR2F6 can attenuate the capacity of NFAT to bind to critical regions of the Il17a gene promoter. In addition, because NR2F6 binds to defined hormone response elements (HREs) within the Il17a locus, it interferes with the ability of RORγt to access the DNA. Consistently, NFAT and RORγt binding within the Il17a locus were enhanced in Nr2f6-deficient CD4+ Th17 cells but decreased in Nr2f6-overexpressing transgenic CD4+ Th17 cells. Taken together, our findings uncover an example of antagonistic regulation of Il17a transcription through the direct reciprocal actions of NR2F6 versus NFAT and RORγt., Highlights ► NR2F6 controls IL-17A expression in Th17 cells and attenuates EAE. ► NR2F6 counteracts NFAT and RORγt binding to the Il17a promoter. ► NR2F6 is released from the Il17a promoter upon TCR stimulation. ► NR2F6 acts as a gatekeeper for the transcriptional regulation of Il17a in Th17 cells.

It is well known that protein kinase C (PKC) plays an important role in regulation of TCR cell surface expression levels. However, eight different PKC isotypes are present in T cells, and to date the particular isotype(s) involved in TCR down-regulation remains to be identified. The aim of this study was to identify the PKC isotype(s) involved in TCR down-regulation and to elucidate the mechanism by which they induce TCR down-regulation. To accomplish this, we studied TCR down-regulation in the human T cell line Jurkat, in primary human T cells, or in the mouse T cell line DO11.10 in which we either overexpressed constitutive active or dominant-negative forms of various PKC isotypes. In addition, we studied TCR down-regulation in PKC knockout mice and by using small interfering RNA-mediated knockdown of specific PKC isotypes. We found that PKCα and PKCθ were the only PKC isotypes able to induce significant TCR down-regulation. Both isotypes mediated TCR down-regulation via the TCR recycling pathway that strictly depends on Ser126 and the di-leucine-based receptor-sorting motif of the CD3γ chain. Finally, we found that PKCθ was mainly implicated in down-regulation of directly engaged TCR, whereas PKCα was involved in down-regulation of nonengaged TCR.