Protein kinase B (PKB), also known as Akt or RAC-PK, is a serine/threonine kinase that can be activated by growth factors via phosphatidylinositol 3-kinase. In this article we show that PKC but not PKC␣ and PKC␦ can co-immunoprecipitate PKB from CHO cell lysates. Association of PKB with PKC was also found in COS-1 cells transiently expressing PKB and PKC, and moreover we found that this association is mediated by the AH domain of PKB. Stimulation of COS-1 cells with plateletderived growth factor (PDGF) resulted in a decrease in the PKB-PKC interaction. The use of kinase-inactive mutants of both kinases revealed that dissociation of the complex depends upon PKB activity. Analysis of the activities of the interacting kinases showed that PDGFinduced activation of PKC was not affected by co-expression of PKB. However, both PDGF-and p110-CAAXinduced activation of PKB were significantly abolished in cells co-expressing PKC. In contrast, co-expression of a kinase-dead PKC mutant showed an increased induction of PKB activity upon PDGF treatment. Downstream signaling of PKB, such as the inhibition of glycogen synthase kinase-3, was also reduced by coexpression of PKC. A clear inhibitory effect of PKC was found on the constitutively active double PKB mutant (T308D/S473D). In summary, our results demonstrate that PKB interacts with PKC in vivo and that PKC acts as a negative regulator of PKB.Protein kinase B (PKB), 1 also referred to as c-Akt or RAC-PK is a 60-kDa serine/threonine kinase which is the cellular homologue of the viral oncogene v-Akt (1-3). So far, three isoforms of PKB have been isolated: PKB␣, PKB␤, and PKB␥ (1, 2, 4, 5). Overexpression of PKB family members has been correlated with different cancers such as breast cancer and some pancreatic and ovarian cancers (2, 6, 7). Recently, PKB has been found to yield an anti-apoptotic signal, which is crucial for cell survival in both fibroblasts and neuronal cells (8, 9). Other reports have indicated a role for PKB in the regulation of glycogen synthesis by inhibition of glycogen synthase kinase-3 (GSK-3) (10, 11). In addition, glucose uptake and metabolism in 3T3-L1 adipocytes have been shown to be regulated by PKB by mediating the translocation of the glucose transporter GLUT4 to the plasma membrane (12, 13). Moreover, a role for PKB has been described in the regulation of protein synthesis through indirect activation of the p70 ribosomal S6 kinase (p70 S6K ) (14). PKB comprises a NH 2 -terminal Akt homology (AH) domain of 148 amino acids, a catalytic domain of 264 amino acids showing high homology with cyclic AMP-dependent protein kinase A (PKA) and protein kinase C (PKC) and a short COOHterminal tail of 68 amino acids. A pleckstrin homology (PH) domain of 106 amino acids is present within the AH domain. Treatment of cells with different growth factors, insulin, or phosphatase inhibitors results in rapid activation of PKB (10,14,15). Also heat shock, hyperosmolarity stress, and intracellular cAMP elevation were shown to activate PKB in vivo (16,17). Growth facto...

No abstract

Microbial metabolites may influence the metabolic integrity of intestinal epithelial cells and induce mucosal immune responses. Therefore, we investigated the effects of the microbial metabolites butyrate, iso-valerate, and ammonium on Caco-2 cells and macrophages. Barrier functioning was determined by measuring transepithelial electrical resistance and basolateral recoveries of metabolites. The barrier function of Caco-2 cells remained intact after exposures. Basolateral recoveries ranged from 6.2% to 15.2%. Tumour necrosis factor-alpha and interleukin-10 were measured to determine immune reactions. The Caco-2 cells did not secrete both cytokines. Physiological concentrations of butyrate and iso-valerate stimulated the secretion of tumour necrosis factor-alpha and suppressed the secretion of interleukin-10 by macrophages that are not protected by an epithelial barrier. In contrast, ammonium concentrations as high as those produced by microbiotas of IBD patients suppressed the release of both cytokines when the barrier function is impaired.

Objective: We report the characterization of MCLA-117, a novel T cell-redirecting antibody for acute myeloid leukaemia (AML) treatment targeting CD3 on T cells and CLEC12A on leukaemic cells. In AML, CLEC12A is expressed on blasts and leukaemic stem cells. Methods: The functional capacity of MCLA-117 to redirect resting T cells to eradicate CLEC12A POS tumor cells was studied using human samples, including primary AML samples. Results: Within the normal hematopoietic compartment, MCLA-117 binds to cells expressing CD3 and CLEC12A but not to early myeloid progenitors or hematopoietic stem cells. MCLA-117 induces T cell activation (EC 50 = 44 ng/mL), T cell proliferation, mild pro-inflammatory cytokine release, and redirects T cells to lyse CLEC12A POS target cells (EC 50 = 68 ng/mL). MCLA-117-induced targeting of normal CD34 POS cells co-cultured with T cells spares erythrocyte and megakaryocyte differentiation as well as preserves mono-myelocytic lineage development. In primary AML patient samples with autologous T cells, MCLA-117 robustly induced AML blast killing (23-98%) at low effector-to-target ratios (1:3-1:97). Conclusion: These findings demonstrate that MCLA-117 efficiently redirects T cells to kill tumour cells while sparing the potential of the bone marrow to develop the full hematological compartment and support further clinical evaluation as a potentially potent treatment option for AML.

Melanocortins (MC), neuropeptides derived from pro-opiomelanocortin, have been implicated in enhancing neurite outgrowth via an as yet unknown mechanism. Recently, five MC receptors have been identified, three of which, the MC3-R, the MC4-R and the MCs-R, are expressed in the nervous system. In this study, a-MSH and the melanocortin analog [D-PheT]ACTH (4-10) were able to stimulate neurite outgrowth in the neuroblastoma cell line Neuro 2A. ACTH (4-10), -/2-MSH and ORG2766 were inactive. In addition, the MCa-R antagonist [D-Arg8]ACTH (4-10), inhibited the a-MSH effect, indicating that the MCa-R mediated stimulation of neurite outgrowth by o~-MSH. Indeed, the presence of MCa-R mRNA in Neuro 2A cells was demonstrated by a RNase protection assay. Heterologous expression of the MCs-R in Neuro 2A cells lead to the recruitment of a responsiveness to -/2-MSH, but did not increase the effect of a-MSH on neurite outgrowth. This finding indicated that the function of MC4-R can also be exerted by another MC receptor, suggesting that the coupling to G,, which they have in common, plays an essential role in the neurite outgrowth promoting effect. This was further substantiated by the fact that forskolin treatment per se induced neurite outgrowth in a similar fashion. These data imply that the neurotrophic properties of a-MSH are likely to result from G~-coupled MC receptor activity in neuronal cells.