Protein kinase D (PKD) is an antigen receptor-activated serine kinase localized at either the plasma membrane or the cytosol of lymphocytes. To probe PKD function at these different locations, transgenesis was used to target active PKD either to the membrane or cytosol of pre-T cells. In recombinase gene null pre-T cells, membrane and cytosolic active PKD both induced differentiation reminiscent of beta selection: downregulation of CD25 and upregulation of CD2 and CD5. Active PKDs also induced pre-T cell proliferation, although this response was not universal to all thymocyte subsets. There were two striking differences between the actions of the differentially localized PKDs. Membrane but not cytosolic PKD could induce expression of CD8 and CD4 in recombinase null mice; cytosolic but not membrane PKD suppressed Vbeta to DJbeta rearrangements of the TCRbeta chain locus in wild-type T cells. PKD function is thus determined by its intracellular location and cell context.

Stimulation of the T cell receptor activates the ERK1/2 and p38 mitogen-activated protein kinase (MAPK) cascades. We demonstrate that TCR stimulation also activates the mitogen-and stress-activated kinases (MSK) downstream of ERK1/2 and p38 in both a T cell line and primary peripheral T cells. MSK1/2-knockout mice were found to have normal numbers of T cells in the thymus, and development of these cells appeared unaffected. Using naive T cells and T lymphoblasts from MSK1/2-knockout mice, it was found that MSK was the kinase responsible for phosphorylation of the transcription factor CREB in response to TCR stimulation. Phosphorylation of CREB by MSK has been linked to the transcription of nur77, nor1 and c-fos downstream of MAPK signalling in various cell types. In T cells, the TCR-dependent transcription of these genes was found to require a MAPK-dependent but MSK-independent signalling pathway. Nevertheless, the number of T cells present in the spleens of MSK1/2-knockout mice and the IL-2-induced proliferation of these cells was reduced compared to wild-type mice. This correlated to a reduction in the TCR-induced up-regulation of the IL-2 receptor CD25 and a requirement for MSK in IL-2-induced CREB phosphorylation.

The newly cloned and characterized hepatitis GB virus-C (HGBV-C), which is the same virus as the independently discovered hepatitis G virus, has a global distribution, is transmitted parenterally, and causes chronic viremia. The pathological consequences of infection with HGBV-C are uncertain, and its hepatocarcinogenic potential is unknown. We used a case-control format to compare the prevalence of HGBV-C infection in 167 southern African blacks with hepatocellular carcinoma (HCC) and 167 race-, age-, and sex-matched hospital-based control subjects, and to test for possible interactive effects between this virus and hepatitis B and C viruses in the development of the tumor. The presence of HGBV-C ribonucleic acid was detected in serum samples by reverse transcription, amplification of the resulting complementary deoxyribonucleic acid by the polymerase chain reaction (PCR), and Southern hybridization using a probe from the NS3/helicase region of the genome. Serum samples were also tested for the presence of hepatitis B virus surface antigen, antibodies to hepatitis C virus, and hepatitis C virus ribonucleic acid. Individuals infected with HGBV-C did not have an increased relative risk of developing HCC (relative risk 0.9; 95% confidence limits 0.5, 1.7). Moreover, co-infection with HGBV-C did not further increase the risk of tumor development in patients who were chronically infected with hepatitis B and/or C viruses. HGBV-C is unrelated to hepatocellular carcinoma development in black Africans.

This study explores the links between the GTPase RhoA and the serine kinase protein kinase D (PKD) during thymocyte development. The rationale is that RhoA and PKD regulate common biological responses during T cell development, but there is nothing known about their interdependence. In fibroblasts, Rho function is required for activation of PKD catalytic activity. However, the data show that activation of Rho is neither sufficient nor essential for PKD activation in T cells. One alternative explanation for the apparent convergence of PKD and Rho signaling in T cells is that PKD responses might be Rho-dependent. To address this latter possibility, we probed the Rho requirements for the actions of constitutively active PKD mutants in pre-T cells of transgenic mice. Active PKD can localize to either the plasma membrane or the cytosol, and we therefore compared the Rho requirements for the actions of membrane-or cytosol-localized PKD. Here we show that membrane-localized PKD regulation of pre-T cell differentiation is Rho-dependent, but the actions of cytosol-localized PKD are not. These studies demonstrate that a Rho requirement for PKD activation is not ubiquitous. Moreover, links between PKD and Rho are determined by the cellular location of PKD.Protein kinase D1 is a member of a conserved family of serine/threonine kinases that includes PKD2 and PKC/PKD3 (1, 2). PKD1 contains multiple domains with the N-terminal region composed of two cysteine-rich motifs, which display high affinity for diacylgylcerol (DAG) 4 or tumor-promoting phorbol esters and a pleckstrin homology domain (3, 4). The C-terminal region contains the serine/threonine catalytic kinase domain that shares homology with the calmodulin-dependent kinases. PKDs are expressed ubiquitously and can be activated by a wide variety of physiological stimuli, including growth factors, neuropeptides, and oxidative stress (4). PKDs are particularly abundant in lymphocytes where they are selectively activated by triggering of antigen receptors (5, 6). The activation of PKD in lymphocytes requires DAG binding and protein kinase C (PKC)-mediated phosphorylation of two serine residues (Ser-744 and 748) within the activation loop of the catalytic domain (1, 6).Recently, it has been shown that PKD has the capacity to regulate the functional activity of ␤1 integrins via regulation of the Rap1 GTPase (7). As well, a recently proposed function for PKD in lymphocytes is the phosphorylation of class II histone deacetylases (8 -10). The activation of PKD by antigen receptors is a sustained response and is also associated with changes in PKD intracellular location (11). In the initial phase of lymphocyte activation there is a rapid translocation of PKD to the plasma membrane. However, during sustained activation, PKD relocates to the cytosol where it remains active for a prolonged period of time (11). The function of PKD at these different locations has been probed in an in vivo model using active PKD mutants targeted to either the plasma membrane or the cytosol of pre-T ...