Molecular cloning and biochemical studies identified protein kinase C (PKC) enzymes as members of a distinct family of serine/threonine protein kinases, playing critical roles in the regulation of cellular differentiation and proliferation of diverse cell types (reviewed in reference 36). In an attempt to find PKC isoforms that are involved in growth control and/or activation of T lymphocytes, we have identified PKC-(5), whose human gene locus was recently mapped to chromosome 10p15 (15). PKC-is characterized by a unique tissue distribution, i.e., in skeletal muscle, lymphoid organs, and hematopoietic cell lines, particularly T cells (4,5,10,34,39,53), and by isoenzyme-specific activation requirements and substrate preferences in vitro (4). PKC-undergoes cytosol-to-membrane translocation in T cells stimulated with phorbol esters (4), implying that this isoform is likely to be involved in T-cell activation pathways. The unique expression and functional properties of PKC-suggest that it may play a specialized role in T-cell signaling pathways.T-cell activation results in the expression of interleukin-2 (IL-2), an autocrine growth factor that is a critical stimulus for the growth and differentiation of B and T lymphocytes. Pharmacological and biochemical studies indicate that activation of two major signaling pathways, one of which can be triggered by phorbol esters (such as phorbol 12-myristate 13-acetate [PMA]) and the other of which can be triggered by Ca 2ϩ ionophores, is required for induction of IL-2 (reviewed in reference 51). A substantial amount of work over the past several years has shown the requirement of cooperative interactions of several transcription factors, including AP-1, NF-B, NF-AT, and NF-IL2A (Oct-1), with the minimal inducible promoter/enhancer region of the IL-2 gene (11). Several lines of evidence point to AP-1 as a critical transcription factor for IL-2 regulation. AP-1 is a dimer of different members of the Fos (c-Fos, FosB, Fra-1, Fra-2, and FosB2) and Jun (c-Jun, JunB, and JunD) family of proteins (1). AP-1 thereby interacts with the IL-2 regulatory region directly (25,26,33,47) and also indirectly as a component of NF-AT and NF-IL2 (37, 50). AP-1 activity is regulated by de novo synthesis of Jun and Fos proteins, as well as by posttranslational modifications such as phosphorylation and dephosphorylation (1,8,9,30,43,48). Two potential AP-1-binding sites have been identified in the mouse and human IL-2 enhancer region at Ϫ150 bp (proximal AP-1) and Ϫ180 bp (distal AP-1). These elements show sequence similarity to the consensus AP-1 enhancer sequence and have been studied by deletional, mutational, and gel shift analyses (14,18,25,40). Most of these data support an important role for AP-1 in IL-2 transcription, especially as a result of the interaction with the proximal enhancer site (25).PKC has been implicated in the activation of AP-1 in T lymphocytes, as demonstrated by studies involving PKC-specific pharmacological inhibitors (24, 28) or PKC down-regulation by chronic phorbol este...

Soluble tumour necrosis factor receptors (sTNF‐Rs) play a role as modulators of the biological function of tumour necrosis factor‐α (TNF‐α) in an agonist/antagonist pattern. In various pathologic states the production and release of sTNF‐Rs may mediate host response and determine the course and outcome of disease by interacting with TNF‐α and competing with cell surface receptors. The determination of sTNF‐Rs in body fluids such as plasma or serum is a new tool to gain information about immune processes and provides valuable insight into a variety of pathological conditions. Regarding its immediate clinical use, sTNF‐Rs levels show high accuracy in the follow‐up and prognosis of various diseases. In HIV infection and sepsis, sTNF‐Rs concentrations strongly correlate with the clinical stage and the progression of disease and can be of predictive value. Determination of sTNF‐Rs also gives useful information for monitoring cancer and autoimmune diseases. The information provided is often even superior to that obtained with classical disease markers, probably due to the direct involvement of the “TNF system” in the pathogenetic mechanisms in these patients. The available data imply that the measurement of sTNF‐Rs, especially of the sTNF‐R 75kD type, is a useful adjunct for quantification of the Th1‐type immune response, similar to other immune activation markers such as neopterin and β2‐microglobulin. Endogenous sTNF‐Rs concentrations appear to reflect the activation state of the TNF‐α/TNF receptor system.

Expression of transforming Ha-Ras L61 in NIH3T3 cells causes profound morphological alterations which include a disassembly of actin stress fibers. The Ras-induced dissolution of actin stress fibers is blocked by the specific PKC inhibitor GF109203X at concentrations which inhibit the activity of the atypical aPKC isotypes λ and ζ, whereas lower concentrations of the inhibitor which block conventional and novel PKC isotypes are ineffective. Coexpression of transforming Ha-Ras L61 with kinase-defective, dominant-negative (DN) mutants of aPKC-λ and aPKC-ζ, as well as antisense constructs encoding RNA-directed against isotype-specific 5′ sequences of the corresponding mRNA, abrogates the Ha-Ras–induced reorganization of the actin cytoskeleton. Expression of a kinase-defective, DN mutant of cPKC-α was unable to counteract Ras with regard to the dissolution of actin stress fibers. Transfection of cells with constructs encoding constitutively active (CA) mutants of atypical aPKC-λ and aPKC-ζ lead to a disassembly of stress fibers independent of oncogenic Ha-Ras. Coexpression of (DN) Rac-1 N17 and addition of the phosphatidylinositol 3′-kinase (PI3K) inhibitors wortmannin and LY294002 are in agreement with a tentative model suggesting that, in the signaling pathway from Ha-Ras to the cytoskeleton aPKC-λ acts upstream of PI3K and Rac-1, whereas aPKC-ζ functions downstream of PI3K and Rac-1.This model is supported by studies demonstrating that cotransfection with plasmids encoding L61Ras and either aPKC-λ or aPKC-ζ results in a stimulation of the kinase activity of both enzymes. Furthermore, the Ras-mediated activation of PKC-ζ was abrogated by coexpression of DN Rac-1 N17.

In this study, the recently identified human protein kinase C-6 (PKC-8) isoform has been biochemically characterized in detail. An antiserum raised against the unique V3 domain of PKC-O 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-8 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-O with a representative member of the Ca2+-dependent PKC enzymes, full-length cDNAs encoding PKC-8 or PKC-a 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-8 and PKC-a. 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-a, however, full activation of PKC-8 did not require Ca2+, and its basal activity towards histone H1 was not stimulated by lipid cofactors. Additionally, a myelinbasic-protein-(MBP)-derived peptide, which was readily phosphorylated by PKC-a, was a poor substrate for PKC-8. Similar to PKC-a, transient PKC-8 overexpression in murine EL4 thymoma cells caused an approximately 2.5-fold increase in the phorbo1-12-myristate-13-acetate-induced transcriptional activation of an interleukin-2 promoter-reporter gene construct. The unique expression and functional properties of PKC-8 suggest that it may play a specialized role in T-cell signaling pathways.Molecular cloning and biochemical studies identified protein kinase C (PKC) enzymes as members of a distinct family of serinehhreonine protein kinases that constitutes, at present, ten mammalian members, i.e. a, pI, P I , y, 6, E , [, q, O and z/A. These phospholipid-dependent, diacylglycerolactivated enzymes are thought to play key roles in the regulation of many cellular functions, including proliferation and differentiation (reviewed in [l]). PKC enzymes can be grouped into three subfamilies based on their domain structure. The first subfamily includes the Ca2+-dependent a, p and 'J isoforms [2-61. These enzymes contain, in addition to the catalytic C3 domain that is shared by all members of the family, a phospholipid-binding and diacylglycerob'phorbolester-binding C1 domain and a Ca"-binding C2 domain.PKC isoforms 6, E , q [7-111 and O [12-141 contain the C1

No abstract

Tryptophan levels decrease during normal pregnancy and the decrease may be related to immune activation phenomena.

Lipoprotein receptors, such as LRP, have been shown to assemble multiprotein complexes containing intracellular signaling molecules; however, in vivo, their signaling function is poorly understood. Using a novel LRP receptor fusion construct, a type I transmembrane protein chimera, termed sIgG-LRP (bearing the intracellular COOH-terminal tail of human LRP as recombinant fusion to a transmembrane region plus the extracellular IgG-F c domain), we here investigated LRP signal transduction specificity in intact cells. First and similar to activated ␣2-macroglobulin as agonist of endogenous LRP, expression of sIgG-LRP demonstrated significant apoptosis protection. Second and similar to ␣2-macroglobulin-induced endogenous LRP, sIgG-LRP is sufficient to negatively modulate mitogen-induced Elk-1 and cJun (but not NF-B) transcriptional activity. Third, expression of sIgG-LRP also impaired cJun transactivation mediated by constitutive active mutants of Rac-1 and MEKK-1. Fourth and unexpectedly, sIgG-LRP expression was found to be associated with a marked enhancement of mitogen-induced cJun amino-terminal kinase (JNK) activation. Fifth, confocal microscopic examination and subcellular fractionation demonstrated that sIgG-LRP and JNK co-localize in transfected cells. Therefore, sIgG-LRP expression was found to significantly impair activation-induced translocation of JNK into the nucleus. Taken together, we here demonstrate that sIgG-LRP protein sequesters activated JNK into the plasma membrane compartment in intact cells, inhibiting nuclear activation of the JNK-dependent transcription factors Elk-1 and cJun. Low density lipoprotein receptor-related protein (LRP)1 is one member of the LDL receptor family that also includes the LDL receptor, the very low density lipoprotein receptor, megalin, LRP5, LRP6, and apoER2 receptor (see Ref. 1 LRP is expressed abundantly in neurons and microglia of the central nervous system (3, 4). Disruption of the LRP gene in mice blocks development of LRP Ϫ/Ϫ embryos around the implantation (5). However, the complex phenotype of the few malformed LRP-deficient embryos that survive until E10 (6), similar to the very low density lipoprotein ApoER2 receptor double knockout phenotype (7), postulated some LRP receptor signaling function(s). Consistently, LRP and several of its ligands, including ␣2-macroglobulin, tissue plasminogen activator (tPA), apoE-containing lipoproteins, and the amyloid precursor protein (APP) (8, 9), have been implicated in various cellular functions including the neuropathogenesis of Alzheimer's disease (see Ref. 10 for review).Based on yeast two-hybrid and co-immunoprecipitation analysis, lipoprotein receptors assemble intracellular multiprotein complexes containing the adapter and scaffold proteins Dab-1 (7), FE65 (11) and Shc (12, 13), the non receptor tyrosine kinases Src and Fyn (14), and the JNK-interacting proteins (JIP-1 & 2) (15, 16), which act as molecular scaffolds for the JNK signaling pathway (see Ref. 17 for review). Quite similarly, such intracellular signali...

The predominant expression of protein kinase C (PKC) theta in T cells (J. Biol. Chem. 1993. 268: 4997-5004), its isoenzyme-specific ability to stimulate AP-1 transcriptional activity (Mol. Cell. Biol. 1996. 16: 1842-1850) and the recent discovery of its selective and antigen-dependent colocalization with the contact region between T cells and antigen-presenting cells (Nature 1997. 385: 83-89) suggest that, among the PKC family members, PKCtheta plays a specialized role in T cell activation. By investigating the downstream effectors of PKCtheta we now demonstrate a direct and isoenzyme-specific contribution of PKCtheta to c-Jun-N-terminal kinase/stress-activated protein kinase (JNK/SAPK) but not extracellular regulated kinase (ERK) activation. Expression of a constitutively active (CA) form of PKCtheta (but not CA-PKCalpha, epsilon and lambda/iota) resulted in strong activation of JNK/SAPK and expression of a dominant-negative form of PKCtheta interfered with the endogenous activation signal for JNK/SAPK. Importantly, Ca2+ ionophore and CA-PKCtheta (but not CA-PKCalpha, epsilon and lambda/iota) caused synergistic activation of the IL-2 promoter. Together, these data establish that PKCtheta is required for activation of JNK/SAPK signaling leading to IL-2 promoter transcription in T lymphocytes.