okadaic-acid and Lymphoma

Mitogens activate the mammalian target-of-rapamycin (mTOR) pathway through phosphatidylinositol 3-kinase (PI3K). The activated mTOR kinase phosphorylates/ activates ribosomal protein S6 kinase (p70S6K) and phosphorylates/inactivates eukaryotic initiation factor 4E-binding protein-1 (4E-BP1), resulting in the initiation of translation and cell-cycle progression. The prolactin receptor signaling cascade has been implicated in crosstalk with the mTOR pathway, but whether prolactin (PRL) directly activates mTOR is not known. This study showed that PRL stimulated the phosphorylation of mTOR, p70S6K, Akt, and Jak2 kinases in a dose- and time-dependent manner in PRL-dependent rat Nb2 lymphoma cells. PRL-stimulated phosphorylation of mTOR was detected as early as 10 min, closely following the phosphorylation of Akt (upstream of mTOR), but preceding that of the downstream p70S6K. PRL activation of mTOR was inhibited by rapamycin (mTOR inhibitor), LY249002, and wortmannin (P13K inhibitors), but not by AG490 (Jak2 inhibitor), indicating that it was mediated by the P13K/Akt, but not Jak2, pathway. PRL also stimulated phosphorylation of 4E-BP1 in Nb2 cells. PRL-induced phosphorylation of p70S6K and 4E-BP1 was inhibited by rapamycin, but not by okadaic acid (inhibitor of protein phosphatase, PP2A). PRL induced a transient interaction between p70S6K and the catalytic subunit of PP2A (PP2Ac) in 1 and 2 h, whereas a PP2Ac-4E-BP1 complex was constitutively present in quiescent and PRL-treated Nb2 cells. These results suggested that p70S6K and 4E-BP1 were substrates of PP2A and the inhibition of mTOR promoted their dephosphorylation by PP2A. In summary, PRL-stimulated phosphorylation of mTOR is mediated by PI3K. PRL-activated mTOR may phosphorylate p70S6K and 4E-BP1 by restraining PP2A.

Topics: Androstadienes; Animals; Blotting, Western; Carrier Proteins; Cell Line, Tumor; Chromones; Enzyme Inhibitors; Immunoprecipitation; Intracellular Signaling Peptides and Proteins; Lymphoma; Morpholines; Okadaic Acid; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphoproteins; Prolactin; Protein Kinases; Rats; Ribosomal Protein S6 Kinases, 70-kDa; Sirolimus; Stimulation, Chemical; TOR Serine-Threonine Kinases; Wortmannin

We previously reported that prolactin-mediated macromolecular synthesis and mitogenesis are coupled to the activation of mitogen-activated protein kinase (MAPK) and p70 S6-kinase (p70S6K). Full activation of MAPK requires tyrosine and threonine phosphorylation whereas that of p70S6K requires serine phosphorylation. In the present study, okadaic acid, which inhibits serine/threonine protein phosphatase activity, was used to explore the linkage of MAPK and p70S6K activation to down-stream effects of prolactin in Nb2 cells. The results show that 1 nM okadaic acid augmented prolactin-stimulated mitogenesis and synthesis of protein and DNA 250%, 42%, and 70%, respectively. Addition of okadaic acid alone a) stimulated and sustained p70S6K activity (5- to 8-fold) and MAPK (3.5- to 5-fold); and b) increased protein synthesis with the maximum effect being about equal to that of prolactin (2.1-fold with 1 nMokadaic acid versus 2.3-fold with 0.2 nMprolactin). However, okadaic acid did not affect DNA synthesis or mitogenesis. These results indicate that the activation of MAPK and p70S6K is necessary for stimulation of protein synthesis but not sufficient for prolactin-driven mitogenesis.

Topics: Animals; Calcium-Calmodulin-Dependent Protein Kinases; DNA, Neoplasm; Enzyme Inhibitors; Lymphoma; Mitosis; Molecular Mimicry; Okadaic Acid; Phosphoprotein Phosphatases; Prolactin; Protein Biosynthesis; Ribosomal Protein S6 Kinases; Signal Transduction; Tumor Cells, Cultured

Full-length cytosolic phospholipase A2 (cPLA2) was cloned from U937 cells and polymorphonuclear leukocytes (PMNLs) while a naturally occurring variant of cPLA2, which lacks residues Val473-Ala749 but has a C-terminal extension of ILMNLSEYMLWMSKVKRFM (DcPLA2) was cloned from PMNLs and mononuclear leukocytes. We were unable to clone DcPLA2 from U937 cells. When cPLA2 and DcPLA2 were expressed in insect cells, both proteins were detected in cell lysates by SDS/PAGE as single bands of apparent molecular masses 100 kDa and 57 kDa, respectively. Full-length cPLA2 was phosphorylated stoichiometrically by p42 mitogen-activated protein (MAP) kinase in vitro at a similar rate to other physiological substrates of this protein kinase and the major site of phosphorylation was identified by amino acid sequencing as Ser505. [32P]Ser(P)505 in cPLA2 was only dephosphorylated at a slow rate by mammalian tissue homogenates. Protein phosphatases 2A, 2B and 2C all contributed significantly to the overall dephosphorylation of cPLA2. The phosphorylation of cPLA2 by p42 MAP kinase correlated with an approximately 1.5-fold increase in specific enzyme activity which was reversed by dephosphorylation.

Topics: Amino Acid Sequence; Animals; Base Sequence; Calcium-Calmodulin-Dependent Protein Kinases; Cells, Cultured; Cloning, Molecular; Cytosol; Ethers, Cyclic; Humans; Insecta; Leukocytes, Mononuclear; Lymphoma; Mitogen-Activated Protein Kinase 1; Molecular Sequence Data; Neutrophils; Okadaic Acid; Phospholipases A; Phospholipases A2; Phosphoprotein Phosphatases; Phosphorylases; Phosphorylation; Protein-Tyrosine Kinases; Rats; Recombinant Proteins; Tissue Extracts; Tumor Cells, Cultured

Topics: Cell Line; Cytotoxicity, Immunologic; Enzyme Inhibitors; Ethers, Cyclic; Homeostasis; Humans; Killer Cells, Natural; Kinetics; Lymphoma; Marine Toxins; Okadaic Acid; Oxazoles; Protein Serine-Threonine Kinases; Protein Tyrosine Phosphatases; Thymus Neoplasms; Tumor Cells, Cultured

To address the role of protein phosphatases in regulating hormonal responses in mammalian cells, we investigated the effects of okadaic acid, a potent inhibitor of protein phosphatases 1 and 2A, on epinephrine and prostaglandin E1 stimulation of cAMP accumulation and adenylyl cyclase in S49 WT and kin- lymphoma cells. Depending on the dose and time of okadaic acid pretreatment of both cell lines, there were two distinguishable effects on cAMP accumulation, an augmentation and an inhibition. The augmentation occurred rapidly (t1/2 < 1 min), was maximal with 3 microM okadaic acid, and was observed with concentrations of okadaic acid as low as 0.3 microM. Prolonged (t1/2 of 5-15 min) pretreatment of cells with okadaic acid caused an inhibition of epinephrine-stimulated cAMP accumulation, which was characterized by a 2-3-fold increase in the EC50 for the response to epinephrine. The EC50 for the okadaic acid-mediated inhibition was similar to that for the augmentation. In assays of adenylyl cyclase in membrane fractions prepared from okadaic acid-pretreated cells the inhibitory, but not the stimulatory, effects of okadaic acid pretreatment were observed. The data demonstrate that protein phosphatases play an important role in regulating adenylyl cyclase and suggest that cAMP-dependent protein kinase is not involved in either of its actions.

Topics: Adenylyl Cyclases; Alprostadil; Animals; Colforsin; Cyclic AMP; Dose-Response Relationship, Drug; Epinephrine; Ethers, Cyclic; Kinetics; Lymphoma; Mice; Okadaic Acid; Phosphoprotein Phosphatases; Time Factors; Tumor Cells, Cultured