of several cell types by SV40, especially when assayed Alphonse Garcia 3 in growth-arrested cells (Martin et al., 1979). Small t also Small t also activates AP-1 in microinjected CV-1 cells in a MAP kinase-dependent fashion (Frost et al., 1994). We have reported that inhibition of protein phosphatHowever, the mechanisms supporting the role of small t ase 2A (PP2A) by expression of SV40 small t stimulates during SV40 infection are far from being completely the mitogenic MAP kinase cascade. Here, we show that understood. SV40 small t can substitute for tumor necrosis factor-αLike PP2A, the atypical calcium-independent protein (TNF-α) or serum and stimulate atypical protein kinase kinase C ζ isoform (PKC ζ) has been involved in the C ζ (PKC ζ) activity, resulting in MEK activation, cell control of mitogenic signal transduction and survival proliferation and NF-κB-dependent gene transcrip- (Berra et al., 1993(Berra et al., , 1995Gomez et al., 1995; Diaz-Meco tional activation in CV-1 and NIH 3T3 cells. Kieser et al., 1996;Powell et al., 1996). PKC effects were abrogated by co-expression of kinase-ζ also plays a pivotal role in tumor necrosis factor-α deficient PKC ζ and inhibition of phosphatidylinositol (TNF-α) activation of NF-κB (Diaz-Meco et al., 1993, 3-kinase p85α-p110 by wortmannin, LY294002 and 1994; Dominguez et al., 1993;Lozano et al., 1994), an a dominant-negative mutant of p85α. In contrast, inducible transcriptional activator that participates in the expression of kinase-inactive ERK2 inhibited small control of cell proliferation and survival, as well as in t-dependent cell growth but was unable to abolish inflammatory response and viral gene expression small t-induced NF-κB transactivation. Our results (reviewed in Bauerle and Baltimore, 1996). NF-κB has provide the first in vivo evidence for a critical regulatory been especially implicated in the transcriptional activation role of PP2A in bifunctional PKC ζ signaling pathways of the human immunodeficiency virus type 1 (HIV-1) long controlled by phosphatidylinositol 3-kinase. Constituterminal repeat (LTR) (reviewed in Gaynor, 1992). NF-κB tive activation of PKC ζ and NF-κB following inhibition is the prototype of a family of heterodimeric transcription of PP2A supports new mechanisms by which SV40 factors composed of monomers that bind to DNA and the small t promotes cell growth and transformation. By I-κB inhibitors (reviewed in Baldwin, 1996). NF-κB is establishing PP2A as a key player in the response of retained in the cytoplasm in its inactive form by the I-κB cells to growth factors and stress signals like TNF-α, α inhibitor. Upon stimulation of cells, I-κB α becomes our findings could explain why PP2A is a primary phosphorylated and dissociates from NF-κB, resulting in target utilized during SV40 infection to alter cellular the translocation of NF-κB to the nucleus, where it carries behavior.out its transactivation function. The rapid phosphorylation Keywords: NF-κB/PI 3-kinase/PKC ζ/PP2A/SV40 of I-κB α represents a possible signal for its proteo...

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Growth factor deprivation is a physiological mechanism to regulate cell death. We utilize an interleukin‐2 (IL‐2)‐dependent murine T‐cell line to identify proteins that interact with Bad upon IL‐2 stimulation or deprivation. Using the yeast two‐hybrid system, glutathione S‐transferase (GST) fusion proteins and co‐immunoprecipitation techniques, we found that Bad interacts with protein phosphatase 1α (PP1α). Serine phosphorylation of Bad is induced by IL‐2 and its dephosphorylation correlates with appearance of apoptosis. IL‐2 deprivation induces Bad dephosphorylation, suggesting the involvement of a serine phosphatase. A serine/threonine phosphatase activity, sensitive to the phosphatase inhibitor okadaic acid, was detected in Bad immunoprecipitates from IL‐2‐stimulated cells, increasing after IL‐2 deprivation. This enzymatic activity also dephosphorylates in vivo 32P‐labeled Bad. Treatment of cells with okadaic acid blocks Bad dephosphorylation and prevents cell death. Finally, Ras activation controls the catalytic activity of PP1α. These results strongly suggest that Bad is an in vitro and in vivo substrate for PP1α phosphatase and that IL‐2 deprivation‐induced apoptosis may operate by regulating Bad phosphorylation through PP1α phosphatase, whose enzymatic activity is regulated by Ras.

SummaryTheileria is an intracellular parasite that causes lymphoproliferative disorders in cattle, and infection of leucocytes induces a transformed phenotype similar to tumour cells, but the mechanisms by which the parasite induces this phenotype are not understood. Here, we show that infected B lymphocytes display constitutive phosphoinositide 3-kinase (PI3-K) activity, which appears to be necessary for proliferation, but not survival. Importantly, we demonstrate that one mechanism by which PI3-K mediates the proliferation of infected B lymphocytes is through the induction of a granulocyte±monocyte colony-stimulating factor (GM-CSF)-dependent autocrine loop. PI3-K induction of GM-CSF appears to be at the transcriptional level and, consistently, we demonstrate that PI3-K is also involved in the constitutive induction of AP-1 and NF-kB, which characterizes Theileria-infected leucocytes. Taken together, our results highlight a novel strategy exploited by the intracellular parasite Theileria to induce continued proliferation of its host leucocyte.

Abstract. Villin is a calcium-regulated actin-binding protein that caps, severs, and bundles actin filaments in vitro. This 92,500-D protein is a major constituent of the actin bundles within the microvilli of the brush border surface of intestinal and kidney proximal tubule cells. Villin is a very early marker of cells involved in absorption and its expression is highly increased during intestinal cell differentiation.The amino acid sequence deduced from the cDNA sequence revealed that human villin is composed of three domains. The first two domains appear as the result of a duplication: their structural organization is similar. We can then define a basic unit in which a slightly hydrophilic motif is followed by three hydrophobic motifs, similar between themselves and regularly spaced. The duplicated domain is highly homologous to three other actin-severing proteins and this basic structure represents the whole molecule in severin and fragmin, while two basic units compose gelsolin.The third domain which is carboxy terminal is villin specific: it is unique among actin modulating proteins so far known. It could account for its actin-binding properties (dual regulation by calcium of severing and bundling activities). We propose that it may also be related to the subcellular localization of villin in different epithelial cell types.

Protein phosphatase types 1 (PP1) and 2A (PP2A) represent two major families of serine/threonine protein phosphatases that have been implicated in the regulation of many cellular processes, including cell growth and apoptosis in mammalian cells. PP1 and PP2A proteins are composed of oligomeric complexes comprising a catalytic structure (PP1c or PP2AC) containing the enzymatic activity and at least one more interacting subunit. The binding of different subunits to a catalytic structure generates a broad variety of holoenzymes. We showed here that casein kinase 2␣ (Ck2␣) and simian virus 40 small t antigen share a putative common ␤-strand structure required for PP2A1 trimeric holoenzyme binding. We have also characterized DPT-sh1, a short basic peptide from Ck2␣ that interacted only in vitro with the PP2A-A subunit and behaves as a nontoxic penetrating shuttle in several cultivated human cell lines and chick embryos. In addition, DPT-sh1 specifically accumulated in human red cells infected with Plasmodium falciparum malaria parasites. We therefore designed bipartite peptides containing DPT-sh1 and PP1-or PP2A-interacting sequences. We found that DPT-5, a DPT-sh1-derived peptide containing a short sequence identified in CD28 antigen, interacts with PP2A-B␣, and DPT-7, another DPT-sh1-derived peptide containing a short sequence identified in Bad as a PP1 catalytic consensus docking motif, induce apoptosis in cultivated cell lines. These results clearly indicate that the rational design of PP1/PP2A interacting peptides is a pertinent strategy to deregulate intracellular survival pathways.In eukaryotic cells, biological activity of nearly 30% of proteins is modulated by phosphorylation. Reversible protein phosphorylation regulates multiple cellular processes, including metabolism, signal transduction pathways, cell cycle progression, oncogenic transformation, cell differentiation, and apoptosis (Garcia et al., , 2003. Protein phosphorylation regulates the activities of protein kinases and protein phosphatases themselves (Hunter, 2000). Type 1 (PP1) and type 2A (PP2A) Ser/Thr protein phosphatases are major regulators of cell dephosphorylation. Binding of PP1 catalytic subunit (PP1c) to specific regulatory subunits generates a large family of PP1 holoenzymes (Cohen, 2002). For PP2AThis work was supported in part by the Institut-Pasteur (PTR-136 and DVPI 27147-27188) and by the Association pour la Recherche sur le Cancer grant 4437 (to A.G.) and grant 4812 (to S.A.S.). F.D. was supported by a fellowship grant from Institut-Pasteur (DVPI). J.G. is a recipient of a Ministere de la Recherche et Technologie fellowship from the French Government. V.J.Y. was supported by a Marie Curie IntraEuropean fellowship within the 6th European Community Framework Programme (contract MEIF-2003-501887

Protein kinase C (PKC) was initially identified as a serine/threonine protein kinase dependent on calcium and phospholipids and shown to be involved in intracellular signaling pathways. PKC isoforms have been classified into four groups: Ca(2+)-dependent conventional PKC alpha, beta I, beta II, gamma; Ca(2+)-independent, novel PKC delta, epsilon, eta, phi; atypical PKC zeta, lambda, iota which are not activated by Ca2+ or diacylglycerol, and the recently discovered PKCmu. We reported that activation of the zeta PKC isoform is an important step in interleukin-2 (IL-2)-mediated proliferation (Gómez, J., Pitton, C., García, A., Martínez, A., Silva, A. and Rebollo, A., Exp. Cell Res. 1995. 218: 105.). zeta PKC is also required for mitogenic activation of fibroblasts and for the maturation pathway activated by insulin and Ras. Contradictory results have been reported regarding the subcellular redistribution of zeta PKC upon activation. We report here, using confocal microscopy, that IL-2 induces expression, translocation and association of zeta PKC to a structure coincident with the actin cytoskeleton. Furthermore, we show that zeta PKC has a role in maintaining the integrity of the actin cytoskeletal structure in IL-2-stimulated cells. On the contrary, zeta PKC is not involved in the actin cytoskeleton organization when cells are maintained in IL-4, confirming our previous results showing that IL-4-induced signal transduction is PKC independent.

In contrast to the purely enhancer-dependent effect of cytokines such as TNF on the activity of the HIV regulatory region (LTR), we observed that okadaic acid (OKA) activates HIV transcription through both the enhancer, responding to the factor NF-kappa B, and the promoter domain of the LTR. The inducibility of HIV LTR-driven luciferase expression constructs in lymphoblastoid cells stimulated by OKA depended on both functional Sp1 binding elements and the ability of the TATA box to bind the protein TBP. In both transformed and normal lymphocytes, OKA stimulation induced intense phosphorylation of the constitutively expressed Sp1 protein in the nucleus, a property of OKA not shared by TNF, phorbol ester, or PHA and interleukin 2. Responsiveness of LTR constructs deleted of kappa B elements to HIV Tat expression was increased upon OKA but not TNF stimulation. Our results suggest that SP1 phosphorylation induced by OKA, a selective inhibitor of the serine-threonine phosphatase PP2A, facilitates the formation of a transcription complex involving general transcription factors, HIV Tat, and Sp1 proteins. The formation of this complex would increase, independently of an in synergy with NF-kappa B, the low basal activity of the HIV LTR observed in normal T lymphocytes.