Alphonse Garcia

Protein phosphatase 2A is a critical regulator of protein kinase C ζ signaling targeted by SV40 small t to promote cell growth and NF‐κB activation

We have reported that inhibition of protein phosphatase 2A (PP2A) by expression of SV40 small t stimulates the mitogenic MAP kinase cascade. Here, we show that SV40 small t can substitute for tumor necrosis factor‐α (TNF‐α) or serum and stimulate atypical protein kinase C ζ (PKC ζ) activity, resulting in MEK activation, cell proliferation and NF‐κB‐dependent gene transcriptional activation in CV‐1 and NIH 3T3 cells. These effects were abrogated by co‐expression of kinase‐deficient PKC ζ and inhibition of phosphatidylinositol 3‐kinase p85α‐p110 by wortmannin, LY294002 and a dominant‐negative mutant of p85α. In contrast, expression of kinase‐inactive ERK2 inhibited small t‐dependent cell growth but was unable to abolish small t‐induced NF‐κB transactivation. Our results provide the first in vivo evidence for a critical regulatory role of PP2A in bifunctional PKC ζ signaling pathways controlled by phosphatidylinositol 3‐kinase. Constitutive activation of PKC ζ and NF‐κB following inhibition of PP2A supports new mechanisms by which SV40 small t promotes cell growth and transformation. By establishing PP2A as a key player in the response of cells to growth factors and stress signals like TNF‐α, our findings could explain why PP2A is a primary target utilized during SV40 infection to alter cellular behavior.

Protein phosphatase 1α is a Ras‐activated Bad phosphatase that regulates interleukin‐2 deprivation‐induced apoptosis

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.

A human villin cDNA clone to investigate the differentiation of intestinal and kidney cells in vivo and in culture.

Villin, a Ca2+‐regulated actin‐binding protein is a major component of microvilli of intestinal epithelial cells and kidney proximal tubule cells. Villin expression during assembly of the brush border can be investigated using a human colon adenocarcinoma cell line HT29‐18. This cell line is able to differentiate under nutritional control and develops an enterocyte‐like phenotype. A cDNA library from a subclone HT29‐18‐C1 was constructed in an expression vector and a cDNA specific for human villin was isolated. This cDNA codes for the 110 carboxy‐terminal residues of villin. Within that region, the 76 carboxy‐terminal residues present 65% homology with the chicken villin ‘head piece’. We show that two mRNA species 4.0 kb and 3.2 kb long hybridize with this cDNA probe in humans, whereas in rat and chicken only one mRNA species can be detected. The two villin mRNA species are co‐expressed in normal human small and large intestinal mucosa and tumoral HT29‐18 cells as well as in normal kidney. No villin mRNAs were detected in other normal or malignant epithelial cell types. Finally, we observed an accumulation of the two mRNA species coding for villin when HT29‐18 cells become differentiated, suggesting that control of villin expression during terminal differentiation can occur at the transcription level or by RNA stabilization.

Bcl-2 Targets Protein Phosphatase 1α to Bad

The diverse forms of protein phosphatase 1 (PP1) in vivo result from the association of the catalytic subunit with different regulatory subunits. We recently have described that PP1α is a Ras-activated Bad phosphatase that regulates IL-2 deprivation-induced apoptosis. With the yeast two-hybrid system, GST fusion proteins, indirect immunofluorescence, and coimmunoprecipitation, we found that Bcl-2 interacts with PP1α and Bad. In contrast, Bad did not interact with 14-3-3 protein. Bcl-2 depletion decreased phosphatase activity and association of PP1α to Bad. Bcl-2 contains the RIVAF motif, analogous to the well characterized R/KXV/IXF consensus motif shared by most PP1-interacting proteins. This sequence is involved in the binding of Bcl-2 to PP1α. Disruption of Bcl-2/PP1α association strongly decreased Bcl-2 and Bad-associated phosphatase activity and formation of the trimolecular complex. These results suggest that Bcl-2 targets PP1α to Bad.

Constitutive PI3-K activity is essential for proliferation, but not survival, of Theileria parva-transformed B cells

Theileria 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‐κB, 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.

The anti‐apoptotic molecules Bcl‐xL and Bcl‐w target protein phosphatase 1α to Bad

Bcl‐xL and Bcl‐w specifically interact with PP1α and Bad. A phosphatase activity sensitive to okadaic acid was detected in Bcl‐xL, Bcl‐w and Bad immunoprecipitates. Serine phosphorylation of Bcl‐xL and Bcl‐w correlates with the number of trimolecular complexes formed. Depletion of Bcl‐xL and Bcl‐w decreases the remaining Bad‐associated phosphatase activity and association of protein phosphatase 1 (PP1)α to Bad. Bcl‐xL and Bcl‐w contain the R/K X V/I X F consensus motif shared by PP1 targeting subunits. This motif, in addition to F X X R X R motif, is involved in binding of Bcl‐xL and Bcl‐w to PP1α. Disruption of Bcl‐xL/PP1α or Bcl‐w/PP1α association strongly decreases Bad‐associated phosphataseactivity and stability of trimolecular complexes. These results suggest that Bcl‐xL and Bcl‐w are PP1α targeting subunits and this trimolecular complex may be involved in the control of apoptosis.

Segregation of Bad from lipid rafts is implicated in the induction of apoptosis

Many molecules relocate subcellularly in cells undergoing apoptosis. Using coimmunoprecipitation experiments we demonstrate that Bad is not associated to 14-3-3 protein, suggesting a new mechanism for the control of the proapoptotic role of Bad. Here we show, by confocal microscopy and cellular fractionation, that Bad is attached to lipid rafts in IL-4-stimulated cells and thymocytes while associated with mitochondria in IL-4-deprived cells. Disruption of lipid rafts by methyl-β-cyclodextrin treatment induces segregation of Bad from rafts, which correlates with apoptosis. Our results suggest that the interaction of Bad with rafts is a dynamic process regulated by IL-4 and involved in the control of apoptosis.

A novel role for protein phosphatase 2A in the dopaminergic regulation of Na,K-ATPase.

Stimulation of dopaminergic type 1 (D1) receptors increases lung edema clearance by regulating Na,K‐ATPase function in the alveolar epithelium. We studied the role of serine/threonine protein phosphatases in the Na,K‐ATPase regulation by D1 agonists in A549 cells. We found that low doses of the type 1/2A protein phosphatase inhibitor okadaic acid as well as SV40 small t antigen transiently transfected into A549 cells prevented the D1 agonist‐induced increase in Na,K‐ATPase activity and translocation from intracellular pools to the plasma membrane. This was associated with a rapid and transient increase in protein phosphatase 2A activity. We conclude that D1 stimulation regulates Na,K‐ATPase activity by promoting recruitment of Na,K‐ATPases from intracellular pools into the basolateral membranes of A549 cells via a type 2A protein phosphatase.

High-affinity and Intermediate-affinity Forms of the Human Interleukin-2 Receptor, Expressed in An Interleukin-9-dependent Murine T-cell Line, Deliver Proliferative Signals Via Differences in Their Transduction Pathways

Although several reports have claimed that the p70 IL-2R per se transduces a growth signal in lymphoid and non-lymphoid systems, there is no convincing evidence for this in lymphoid T cell lines. In order to investigate the mechanism of IL-2R-dependent signal transduction pathways via the p70 IL-2R in lymphoid T cells, we have established two IL-9-dependent murine (TS1) cell lines stably expressing the human p70 IL-2R subunit or the human p55-p70 IL-2R complex. Whereas the parental T cell line, TS1, proliferated in response to IL-9 or IL-4 but not IL-2, cell lines stably expressing human p70 IL-2R or p55-p70 IL-2R complex proliferated in response to IL-2. This implies that the murine T cell, TS1, contains all the intracellular components necessary for directing IL-2 signalling. In human p55-p70 IL-2R-transfected cells, the expression of a functional murine p55 IL-2R seemed to be induced and regulated by IL-2. In human p70 IL-2R-transfected cells, in the presence of IL-2 an interaction requiring only intermediate-affinity was sufficient for transduction of a proliferative signal. In addition human p70 IL-2R per se, is biologically functional via a transduction pathway not requiring induction of murine p55 IL-2R and consecutive high-affinity complex reconstitution. Thus, although both transfected cell lines can transduce a proliferative signal in the presence of IL-2, a difference in their transduction pathway is probably involved for the induction of p55 IL-2R.

The Bcl-2 gene is differentially regulated by IL-2 and IL-4: role of the transcription factor NF-AT.

The murine TS1αβ T cell line expresses the anti-apoptotic protein Bcl-2 upon IL-2 stimulation, whereas IL-4-mediated growth of this cell line proceeds in the absence of Bcl-2 expression. In addition, IL-4 stimulation inhibits Bcl-2 expression and modulates its mRNA level. IL-2-induced DNA binding activity for these transcription factors is sensitive to phosphatidylinositol 3 kinase inhibitor wortmannin and to Rho inhibitor Clostridium difficile toxin B, which inhibit IL-2-induced Bcl-2 expression. NF-AT transcription factor appears to be the most important in the control Bcl-2 expression, since inhibition of the calcium-calmodulin-dependent phosphatase calcineurin, which regulates NF-AT activity, downregulates Bcl-2 expression in IL-2-stimulated cells. Constitutive expression of this phosphatase also upregulates Bcl-2 expression in IL-4-stimulated cells. In addition, a dominant negative NF-AT expression vector downregulates Bcl-2 expression in IL-2-stimulated cells. These results suggest that IL-2 induction of Bcl-2 expression may be directly or indirectly mediated by NF-AT.