okadaic-acid and Leukemia--Erythroblastic--Acute

We have investigated by electrophoretic mobility shift assay (EMSA) the level of GATA-1 DNA-binding activity in nuclear extracts prepared from the human erythroleukaemic cell line, K562, after erythroid induction by hemin, sodium butyrate (NaB) or Trichostatin A or treatment with N -acetylcysteine (NAC). Relative to extract from untreated cells, GATA-1 binding activity increased markedly in all cases. However, immunoblot analysis revealed unchanged levels of GATA-1 protein after induction. Incubation of induced but not uninduced K562 extracts with phosphatase prior to EMSA weakened the binding activity, suggesting that the increase in GATA-1 binding following induction of K562 cells was a consequence of phosphorylation. When the mouse erythroleukaemic cell line MEL was induced with dimethylsulphoxide (DMSO), NaB or NAC, GATA-1 binding activity fell with DMSO, rose significantly with NaB and remained at about the same level in NAC-induced cells. In this case immunoblotting revealed that GATA-1 protein levels were in accord with the EMSA data. The DNA-binding activities of induced and uninduced MEL cell nuclear extracts were decreased by incubation with phosphatase, showing that phosphoryl-ation and DNA binding of GATA-1 are already optimalin these cells. The DNA-binding activity of affinity-purified GATA-1 from MEL cells was also reduced by phosphatase treatment, showing that phosphorylation/dephosphorylation is directly affecting the factor. Furthermore, when a comparison was made by EMSA of nuclear extracts prepared from K562 and MEL cells untreated or incubated with okadaic acid, a phosphatase inhibitor, GATA-1 binding was seen to increase with K562 cells, whereas with MEL cells there was no change in GATA-1 binding. Overall the results suggest that the level of GATA-1 phosphorylation increases after the induction of K562, but not MEL cells, where GATA-1 is already highly phosphorylated. Furthermore, phosphorylation increases the binding affinity of GATA-1 for a canonical binding site.

Topics: Animals; Cell Differentiation; DNA; DNA-Binding Proteins; Enzyme Inhibitors; Erythroid-Specific DNA-Binding Factors; GATA1 Transcription Factor; Humans; K562 Cells; Leukemia, Erythroblastic, Acute; Mice; Nuclear Proteins; Okadaic Acid; Phosphoprotein Phosphatases; Phosphorylation; Transcription Factors

In the present study we investigated the possible involvement of the mitogen-activated protein kinase family members extracellular-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK) in mediating IL-6 gene expression in human monocytes, in particular their role in enhancing NF-kappa B activity. Freshly isolated monocytes treated with the protein phosphatase inhibitor okadaic acid secreted high levels of IL-6 protein, which coincided with enhanced binding activity of NF-kappa B as well as with phosphorylation and activation of the ERK1/2 and JNK proteins. The ERK pathway-specific inhibitor PD98059 inhibited IL-6 secretion from monocytes. Transient overexpression of inactive mutants of either Raf-1 or JNK1 showed that both pathways were involved in kappa B-dependent IL-6 promoter activity. By using PD98059, we demonstrated that the Raf1/MEK1/ERK1/2 pathway did not affect the DNA binding of NF-kappa B but, rather, acted at the level of transcriptional activity of NF-kappa B. Interestingly, it was shown that NF-kappa B-mediated gene transcription, both in the context of the IL-6 promoter as well as on its own, was dependent on both serine kinase activity and interaction with c-Jun protein. We conclude that okadaic acid-induced IL-6 gene expression is at least partly mediated through the ERK1/2 and JNK pathway-dependent activation of NF-kappa B transcriptional capacity. Our results suggest that the JNK pathway may regulate NF-kappa B-mediated gene transcription through its phosphorylation and activation of c-Jun.

Topics: Calcium-Calmodulin-Dependent Protein Kinases; Cells, Cultured; Down-Regulation; Enzyme Activation; Humans; Interleukin-6; JNK Mitogen-Activated Protein Kinases; Leukemia, Erythroblastic, Acute; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Monocytes; Mutagenesis, Site-Directed; NF-kappa B; Okadaic Acid; Phosphorylation; Promoter Regions, Genetic; Protein Binding; Proto-Oncogene Proteins c-jun; Proto-Oncogene Proteins c-raf; Transcription, Genetic; Tumor Cells, Cultured

Pre-mRNA splicing is catalyzed by a multitude of proteins including serine/arginine-rich (SR) proteins, which are thought to play a crucial role in the formation of spliceosomes and in the regulation of alternative splicing. SR proteins are highly phosphorylated, and their kinases are believed to regulate the recruitment of SR proteins from nuclear storage compartments known as speckles. Recently, a family of autophosphorylating kinases termed CLK (CDC2/CDC28-like kinases) was shown to phosphorylate SR proteins and to influence alternative splicing in overexpression systems. Here we used endogenous CLK2 protein to demonstrate that it displays different biochemical characteristics compared with its overexpressed protein and that it is differentially phosphorylated in vivo. Furthermore, CLK2 changed its nuclear localization upon treatment with the kinase inhibitor 5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole. We have also identified a CLK2 autophosphorylation site, which is highly conserved among all CLK proteins, and we show by site-directed mutagenesis that its phosphorylation influences the subnuclear localization of CLK2. Our data suggest that CLK2 localization and possibly activity are influenced by a balance of CLK2 autophosphorylation and the regulation by CLK2 kinases and phosphatases.

Topics: Alternative Splicing; Animals; Cell Nucleus; Cloning, Molecular; Kinetics; Leukemia, Erythroblastic, Acute; Mice; Okadaic Acid; Phosphorylation; Phosphoserine; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Recombinant Proteins; Saccharomyces cerevisiae; Serine; Tumor Cells, Cultured

After treatment of HD3 cells with erythroid-inducing agents (hemin and butyric acid) at 42 degrees C, the profile of phosphotyrosine-containing proteins was altered. Upon induction the overall level of phosphotyrosine-containing proteins increased. To examine the role of protein phosphorylation in HD3 cells differentiation, the cells were treated with specific inhibitors. In the presence of okadaic acid, cell proliferation was arrested and accompanied by a marked increase in haemoglobin synthesis, a differentiation marker of erythroid cells. Okadaic acid caused decrease of the phosphotyrosine-containing proteins, presumably to maintain a balance between phosphorylation/dephosphorylation processes in the cells. Addition of 3-isobutyl-1-methyl-xanthine, an activator of phosphatases, caused a decrease or disappearance of almost all phosphotyrosine-containing proteins and, at the same time, prevented the erythroid differentiation of HD3 cells. Sodium orthovanadate, a specific inhibitor of phosphotyrosine phosphatase, increased the level of phosphotyrosine proteins and induced differentiation of HD3 cells. These results indicate that phosphorylation of cellular proteins is coupled with a reaction(s) which is responsible for triggering the differentiation of HD3 cells. The phosphorylation/dephosphorylation processes are associated with an early event(s) during the differentiation of HD3 cells and may not be connected to tyrosine residues.

Topics: 1-Methyl-3-isobutylxanthine; Animals; Butyric Acid; Cell Differentiation; Chickens; Enzyme Inhibitors; Erythroblasts; Hemin; Hemoglobins; Leukemia, Erythroblastic, Acute; Neoplasm Proteins; Okadaic Acid; Phosphoprotein Phosphatases; Phosphoproteins; Phosphorylation; Phosphotyrosine; Protein Processing, Post-Translational; Protein Tyrosine Phosphatases; Protein-Tyrosine Kinases; Vanadates

Treatment of the human myeloid leukemia K562 cells with the protein phosphatase inhibitors okadaic acid or calyculin A resulted in down-regulation of both c-myc and max genes at the mRNA and protein levels. The extent of the down-regulation was similar for both genes and was dependent on the dose and on the treatment time. Interestingly, c-myc and max down-regulation was concomitant with apoptosis induced by okadaic acid and calyculin A in K562 cells. The expression of c-myc and max returned to control levels after the removal of okadaic acid from the media, although apoptosis was irreversible. These effects were observed at okadaic acid concentrations (15 nM) that inhibited the activity of protein phosphatase type 2A but not of phosphatase type 1. We conclude that the inhibition of protein phosphatase 2A is associated to decreased levels of c-Myc/Max heterodimers in K562 cells.

Topics: Apoptosis; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Basic-Leucine Zipper Transcription Factors; Blotting, Northern; Cell Line; DNA-Binding Proteins; Dose-Response Relationship, Drug; Enzyme Inhibitors; Ethers, Cyclic; Gene Expression Regulation, Neoplastic; Genes, myc; Humans; Kinetics; Leukemia, Erythroblastic, Acute; Marine Toxins; Okadaic Acid; Oxazoles; Protein Biosynthesis; Protein Phosphatase 2; Protein Tyrosine Phosphatase, Non-Receptor Type 1; Protein Tyrosine Phosphatases; Proto-Oncogene Proteins c-myc; RNA, Messenger; Time Factors; Transcription Factors; Transcription, Genetic; Tumor Cells, Cultured

Hexamethylenebisacetamide-induced terminal differentiation of Friend virus-transformed murine erythroleukemia (MEL) cells can be inhibited by okadaic acid, an inhibitor of type 1 and type 2A protein phosphatases. The inhibition is shown to be correlated with prevention of dephosphorylation of retinoblastoma protein (pRB) in cells and bypass of G1 prolongation in the cell cycle. These results suggest that pRB-mediated G1 prolongation is necessary for MEL cells to commit to terminal differentiation. However, further experiments demonstrate that the simple cell cycle exit is not sufficient for commitment to terminal differentiation. Induction of dephosphorylation of pRB and subsequent G1 prolongation by forskolin does not lead MEL cells to differentiate. Additional pRB has been expressed in MEL cells by transfection with a neo-resistant plasmid containing RB cDNA under the control of a cytomegalovirus promoter. Exogenously expressed pRB is hyperphosphorylated in logarithmically growing MEL cells without any noticeable change in growth rate between the transfected cell line and the parental cell line. This result suggests that pRB in MEL cells is regulated by protein kinases and protein phosphatases and not by transcription.

Topics: Acetamides; Animals; Antineoplastic Agents; Cell Cycle; Cell Differentiation; Cell Line; Cell Line, Transformed; Ethers, Cyclic; Friend murine leukemia virus; G1 Phase; Gene Expression; Kinetics; Leukemia, Erythroblastic, Acute; Mice; Okadaic Acid; Phosphoprotein Phosphatases; Retinoblastoma Protein; Tumor Cells, Cultured