Protein kinase C activation inhibits tyrosine phosphorylation of Cbl and its recruitment of Src homology 2 domain-containing proteins.

One of the major proteins that is rapidly tyrosine phosphorylated upon stimulation of the TCR/CD3 complex is the 120-kDa product of the c-cbl protooncogene (Cbl). Upon activation, tyrosine-phosphorylated Cbl interacts with the Src homology 2 (SH2) domains of several signaling proteins, e.g., phosphatidylinositol 3-kinase (PI3-K) and CrkL. In the present study, we report that pretreatment of Jurkat T cells with PMA reduced the anti-CD3-induced tyrosine phosphorylation of Cbl and, consequently, its activation-dependent association with PI3-K and CrkL. A specific protein kinase C (PKC) inhibitor (GF-109203X) reversed the effect of PMA on tyrosine phosphorylation of Cbl and restored the activation-dependent association of Cbl with PI3-K and CrkL. We also provide evidence that PKCalpha and PKCtheta can physically associate with Cbl and are able to phosphorylate it in vitro and in vivo. Furthermore, a serine-rich motif at the C terminus of Cbl, which is critical for PMA-induced 14-3-3 binding, is also phosphorylated by PKCalpha and PKCtheta in vitro. These results suggest that, by regulating tyrosine and serine phosphorylation of Cbl, PKC is able to control the association of Cbl with signaling intermediates, such as SH2 domain-containing proteins and 14-3-3 proteins, which may consequently result in the modulation of its function.

Protein kinase C-theta (PKCtheta) distribution analysis in hematopoietic cells: proliferating T cells exhibit high proportions of PKCtheta in the particulate fraction.

A comparative analysis of protein kinase C-theta (PKCtheta) protein expression was performed in various mouse organs and tissues, freshly isolated populations of mouse and human hematopoietic cells, primary leukemias, and established cell lines of different histological origins. Results demonstrated a predominant expression of PKCtheta in lymphoid tissues and skeletal muscle. Expression levels of PKCtheta, as well as PKCalpha, delta, epsilon, zeta, and eta in the thymus, were not markedly changed during postnatal development. High levels of expression were observed in CD4(+) and CD8(+) single-positive T cells and CD4(+)CD8(+) double-positive thymocytes, while B cells were completely devoid of PKCtheta. PKCtheta was found also in platelets, but relatively low levels or no detection of PKCtheta expression were observed in neutrophils, monocytes, and macrophages. Highly proliferating leukemic T cells of established lines or primary tumors, but not freshly isolated resting peripheral blood T cells, exhibited high levels of membrane-bound PKCtheta. Increased proportions of PKCtheta in the particulate fraction was not restricted to malignant cells but correlated with the extent of proliferation of the T cells. Thus, human peripheral blood T cells that were induced to proliferate by exposure to mitogen and IL-2 expressed increased levels of PKCtheta in the particulate fraction. Significantly lower proportions of membrane-bound PKC were observed for five other isoenzymes expressed in T cells. The occurrence of PKCtheta in T, but not B, cells and its subcellular distribution in proliferating cells implicate PKCtheta in cellular mechanisms regulating the sustained proliferation of T cells.

New perspectives on PKCtheta, a member of the novel subfamily of protein kinase C.

Members of the protein kinase C (PKC) family of serine/threonine protein kinases have been implicated in numerous cellular responses in a large variety of cell types. Expression patterns of individual members and differences in their cofactor requirements and potential substrate specificity suggest that each isoenzyme may be involved in specific regulatory processes. The PKCtheta isoenzyme exhibits a relatively restricted expression pattern with high protein levels found predominantly in hematopoietic cells and skeletal muscle. PKCtheta was found to be expressed in T, but not B lymphocytes, and to colocalize with the T-cell antigen receptor (TCR) at the site of contact between the antigen-responding T cell and the antigen-presenting cell (APC). Colocalization of PKCtheta with the TCR was selective for this isoenzyme and occurred only upon antigen-mediated responses leading to T-cell activation and proliferation. PKCtheta was found to be involved in the regulation of transcriptional activation of early-activation genes, predominantly AP-1, and its cellular distribution and activation were found to be regulated by the 14-3-3 protein. Other findings indicated that PKCtheta can associate with the HIV negative factor (Nef) protein, suggesting that altered regulation of PKCtheta by Nef may contribute to the T-cell impairments that are characteristic of infection by HIV. PKCtheta is expressed at relatively high levels in skeletal muscle, where it is suggested to play a role in signal transduction in both the developing and mature neuromuscular junction. In addition, PKCtheta appears to be involved in the insulin-mediated response of intact skeletal muscle, as well as in experimentally induced insulin resistance of skeletal muscle. Further studies suggest that PKCtheta is expressed in endothelial cells and is involved in multiple processes essential for angiogenesis and wound healing, including the regulation of cell cycle progression, formation and maintenance of actin cytoskeleton, and formation of capillary tubes. Here, we review recent progress in the study of PKCtheta and discuss its potential role in various cellular responses.

Direct interaction between protein kinase C theta (PKC theta) and 14-3-3 tau in T cells: 14-3-3 overexpression results in inhibition of PKC theta translocation and function.

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.

Diabetes mellitus is associated with chronic hepatitis C but not chronic hepatitis B infection.

Glucose intolerance is associated with chronic liver disease, particularly cirrhosis, and overt diabetes mellitus is two to four times more common than in the general population. Little attention has been paid to the relationship between the cause of cirrhosis and the development of glucose intolerance or whether cirrhosis is a prerequisite. We found glucose intolerance to be particularly common in patients with chronic hepatitis C, and in this retrospective study we attempt to confirm this possible association. To investigate this question we reviewed the files of 128 patients with chronic hepatitis C and 40 with chronic hepatitis B and active liver disease. Demographic, laboratory, imaging and pathology data were abstracted. The mean fasting blood glucose (+/-SD) in the hepatitis C and B groups was 160 +/- 83 and 103 +/- 18 mg/dl (P < 0.0001) with 2.5% and 39.1% respectively being overtly diabetic (P < 0.00001). However, the mean age of the hepatitis C group was much higher (45.6 +/- 12.5 vs. 60.1 +/- 12.3 years, P < 0.00001). The prevalence of diabetes was much higher among the hepatitis C patients than in the general population. Cirrhosis was not more frequent in biopsies from hepatitis C diabetic patients compared with non-diabetic or hepatitis B patients. Multivariate analysis showed that type of hepatitis and age were significant and independent predictors for developing diabetes. We conclude that there appears to be an association between diabetes mellitus and chronic hepatitis C that is not present in patients with chronic hepatitis B.

Vav: function and regulation in hematopoietic cell signaling.

Vav, a 95 kDa proto-oncogene product expressed specifically in hematopoietic cells, was originally isolated as a transforming human oncogene. Vav contains an array of functional domains that are involved in interactions with other proteins and, possibly, with lipids. These include, among others, a putative guanine nucleotide exchange domain, a cysteine-rich region similar to the phorbol ester/diacylglycerol-binding domain of protein kinase C, a pleckstrin-homology domain, and Src-homology 2 and 3 (SH2 and SH3, respectively) domains. The presence of these domains, the transforming activity of the vav oncogene, and the rapid increase in tyrosine phosphorylation of Vav induced by triggering of diverse receptors indicate that it plays an important role in hematopoietic cell signaling pathways. Such a role is supported by recent studies using "knockout" mice and transiently transfected T cells, in which Vav deletion or overexpression, respectively, had marked effects on lymphocyte development or activation. The presence of a putative guanine nucleotide exchange domain, the prototype of which is found in the dbl oncogene product, implies that Vav functions as a guanine nucleotide exchange factor (GEF) for one (or more) members of the Ras-like family of small GTP-binding proteins. In support of such a role, Vav preparations were found in some (but not other) studies to mediate in vitro-specific GEF activity for Ras. Additional studies are required to identify the physiological regulators and targets of Vav, and its exact role in hematopoietic cell development and signaling.

Expression and biochemical characterization of human protein kinase C-theta.

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.