phosphothreonine and Adenocarcinoma

We have recently identified tumor necrosis factor (TNF)-α-induced phosphorylation of epidermal growth factor receptor (EGFR) at Thr-669 and Ser-1046/1047 via ERK and p38 pathways, respectively. In the present study, we investigated the roles of ligand-induced phosphorylation of serine and threonine residues in EGFR-overexpressing MDA-MB-468 breast cancer cells. Epidermal growth factor and heregulin, an ErbB3 ligand, induced the phosphorylation of Thr-669 and Ser-1046/1047. Inversely, constitutive tyrosine phosphorylation of the C-terminal domain, including Tyr-1068, was significantly downregulated on ligand stimulation. Inhibition of the ERK pathway by U0126 blocked ligand-induced Thr-669 phosphorylation as well as Tyr-1068 dephosphorylation. Downregulation of constitutive tyrosine phosphorylation of EGFR in HEK293 cells stably expressing the wild type was abolished by substitution of Thr-669 for Ala. In an asymmetric EGFR homodimer structure, one Thr-669 in the receiver kinase of the dimer was involved in downregulation. Similarly, Thr-669 in an EGFR-ErbB3 heterodimer also participated in tyrosine dephosphorylation. These results indicate that ERK-mediated Thr-669 phosphorylation suppresses constitutive tyrosine phosphosphorylation in the homo- and heterodimer asymmetric conformations of the EGFR.

Topics: Adenocarcinoma; Breast Neoplasms; Butadienes; Cell Line, Tumor; Dimerization; Epidermal Growth Factor; ErbB Receptors; Feedback, Physiological; Female; Humans; Ligands; MAP Kinase Signaling System; Neoplasm Proteins; Neuregulin-1; Nitriles; Phosphorylation; Phosphothreonine; Protein Conformation; Protein Kinase Inhibitors; Protein Processing, Post-Translational; Recombinant Proteins; Tumor Necrosis Factor-alpha

Serine 7 of centromere protein A (CENP-A) is a very important mitosis-specific phosphorylation site. In this study, we demonstrate the subcellular distribution of Ser7 phosphorylated CENP-A during mitosis in MCF-7 cells. The Ser7 phosphorylation of CENP-A was observed beginning at prophase at centromeres. Upon progression of mitosis, the fluorescence signals emerged in the central region of the metaphase plate and were maintained until anaphase at centromeres. At late anaphase, the fluorescence signals moved to the midzone gradually and transferred from the centromere to the midbody completely at telophase. They were compacted into the centre of the midbody in a thin cylinder consisting of a sandglass-like "mitotic machine" with microtubules and condensed chromosome. We also found that Ser10 phosphorylated H3 and Thr11 phosphorylated H3 were co-localized at the midbody in two bell-like symmetrical bodies with Ser7 phosphorylated CENP-A during the terminal stage of cytokinesis. Midbody isolation and immunoblotting experiments also indicated that Ser7 phosphorylated CENP-A are components of the midbody. These findings suggest that Ser7 phosphorylated CENP-A acts as a chromosomal passenger protein and may play an important role in cytokinesis.

Topics: Adenocarcinoma; Autoantigens; Biological Transport; Breast Neoplasms; Centromere; Centromere Protein A; Chromosomal Proteins, Non-Histone; Cytokinesis; Female; Histones; Humans; MCF-7 Cells; Microscopy, Confocal; Microscopy, Fluorescence; Mitosis; Neoplasm Proteins; Phosphorylation; Phosphoserine; Phosphothreonine; Pregnancy; Protein Processing, Post-Translational; Spindle Apparatus

Endoglin, a transmembrane glycoprotein that acts as a transforming growth factor-beta (TGF-beta) coreceptor, is downregulated in PC3-M metastatic prostate cancer cells. When restored, endoglin expression in PC3-M cells inhibits cell migration in vitro and attenuates the tumorigenicity of PC3-M cells in SCID mice, though the mechanism of endoglin regulation of migration in prostate cancer cells is not known. The current study indicates that endoglin is phosphorylated on cytosolic domain threonine residues by the TGF-beta type I receptors ALK2 and ALK5 in prostate cancer cells. Importantly, in the presence of constitutively active ALK2, endoglin did not inhibit cell migration, suggesting that endoglin phosphorylation regulated PC3-M cell migration. Therefore, our results suggest that endoglin phosphorylation is a mechanism with relevant functional consequences in prostate cancer cells. These data demonstrate for the first time that TGF-beta receptor-mediated phosphorylation of endoglin is a Smad-independent mechanism involved in the regulation of prostate cancer cell migration.

Topics: Activin Receptors, Type I; Adenocarcinoma; Animals; Antigens, CD; Bone Morphogenetic Protein 7; Cell Line, Tumor; Cell Movement; Endoglin; Female; Humans; Male; Mice; Mice, Inbred NOD; Mice, SCID; Neoplasm Invasiveness; Neoplasm Proteins; Phosphorylation; Phosphothreonine; Prostatic Neoplasms; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptors, Cell Surface; Receptors, Transforming Growth Factor beta; Recombinant Fusion Proteins; Sequence Deletion; Transforming Growth Factor beta; Transforming Growth Factor beta1; Transplantation, Heterologous

Several non-small cell lung carcinomas (squamous cell carcinomas and adenocarcinomas) were analyzed for protein kinase activity. Soluble protein extracts derived from these tumors and from the lung parenchyma adjacent to the tumors were resolved by Mono Q anion exchange chromatography, and the fractions were assayed for phosphotransferase activity towards in vitro substrates. Myelin basic protein, casein, and a ribosomal S6-1 COOH-terminus peptide were efficient substrates for protein kinases that exhibited elevated phosphotransferase activity in the tumor extracts when compared to extracts derived from the adjacent nonneoplastic lung or from the lung parenchyma from patients with nonneoplastic lung disorders. Casein phosphotransferase activity was resolved into two peaks that eluted at 0.44 M NaCl and 0.56 M NaCl. The second peak was identified as casein kinase 2, based upon immunoreactivity to casein kinase 2-specific antipeptide antibodies and its sensitivity to inhibition by heparin sulfate. Myelin basic protein phosphotransferase activity eluted at 0.44 M NaCl, but Western blot analysis revealed that this could not be ascribed to mitogen-activated protein (MAP) kinases. This tumor associated protein kinase, designated p40TAK, exhibited a molecular mass of approximately 40 kDa upon gel filtration. In addition to myelin basic protein, it phosphorylated S6 peptide analogues and histone H1 on seryl residues. Like casein kinase 2, p40TAK exhibited elevated basal phosphotransferase activity in squamous cell carcinomas and adenocarcinomas of the lung when compared to the nonneoplastic lung parenchyma adjacent to the tumor.

Topics: Adenocarcinoma; Amino Acid Sequence; Antibodies; Blotting, Western; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Casein Kinases; Caseins; Chromatography, Ion Exchange; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Humans; Kinetics; Lung; Lung Neoplasms; Molecular Sequence Data; Myelin Basic Protein; Peptides; Phosphoserine; Phosphothreonine; Phosphotyrosine; Protein Kinases; Protein Serine-Threonine Kinases; Ribosomal Protein S6; Ribosomal Proteins; Substrate Specificity; Tyrosine

Slices of human lung cancer tissue were incubated with [32P]-orthophosphoric acid, and the radiolabeled beta-glucuronidase was isolated by a procedure including immunoaffinity chromatography on anti-human liver beta-glucuronidase IgG Sepharose. Following removal of endo-beta-N-acetyl-glucosaminidase H-releasable carbohydrate portions of the enzyme, the protein moiety was acid-hydrolyzed. Two-dimensional separation of the hydrolysate identified phosphoserine and phosphothreonine. This is the first demonstration of protein phosphorylation in lysosomal beta-glucuronidase.

Topics: Adenocarcinoma; Chemical Phenomena; Chemistry; Electrophoresis, Polyacrylamide Gel; Glucuronidase; Humans; Hydrolysis; Immunodiffusion; Liver; Lung; Lung Neoplasms; Phosphorylation; Phosphoserine; Phosphothreonine; Serine; Threonine