phosphothreonine and Carcinoma--Squamous-Cell

We previously found that the pro-apoptotic DNA damaging agent, cisplatin, mediated the proteasome-dependent degradation of Delta Np63 alpha associated with its increased phosphorylated status. Since Delta Np63 alpha usually plays an opposite role to p53 and TAp63 in human cancers, we tested the notion that phosphorylation events induced by DNA damage would affect the protein degradation of Delta Np63 alpha in HNSCC cells upon cisplatin exposure. We found that Delta Np63 alpha is phosphorylated in the time-dependent fashion at the following positions: S385, T397 and S466, which were surrounded by recognition motifs for ATM, CDK2 and p70s6K kinases, respectively. We showed that chemical agents or siRNA inhibiting the activity of ATM, CDK2 and p70s6K kinases blocked degradation of Delta Np63 alpha in HNSCC cells after cisplatin exposure. Site-specific mutagenesis of Delta Np63 alpha residues targeted for phosphorylation by ATM, CDK2 or p70s6k led to dramatic modulation of Delta Np63 alpha degradation. Finally, we demonstrated that the Delta Np63 alpha protein is a target for direct in vitro phosphorylation by ATM, CDK2 or p70s6K. Our results implicate specific kinases, and target phosphorylation sites in the degradation of Delta Np63 alpha following DNA damage.

Topics: Amino Acid Motifs; Amino Acid Sequence; Ataxia Telangiectasia Mutated Proteins; Carcinoma, Squamous Cell; Cell Cycle Proteins; Cell Line, Tumor; Cisplatin; Cyclin-Dependent Kinase 2; DNA Damage; DNA-Binding Proteins; Head and Neck Neoplasms; Humans; Molecular Sequence Data; Mutation; Phosphorylation; Phosphoserine; Phosphothreonine; Protein Kinase Inhibitors; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Ribosomal Protein S6 Kinases, 70-kDa; RNA, Small Interfering; Time Factors; Trans-Activators; Transcription Factors; Tumor Suppressor Proteins

GM3 ganglioside (II3NeuAcLacCer) inhibits epidermal growth factor (EGF)-dependent receptor autophosphorylation and cell growth (Bremer, E.G., Schlessinger, J., and Hakomori, S. (1986) J. Biol. Chem. 261, 2434-2440), whereas de-N-acetyl-GM3 (deNAcGM3; II3NeuNH2Lac-Cer) promotes these processes (Hanai, N., Dohi, T., Nores, G. A., and Hakomori, S. (1988) J. Biol. Chem. 263, 6296-6301). Receptor-receptor interaction has been proposed as an essential initial mechanism for EGF-dependent activation of EGF receptor kinase (EGF-RK) (Schlessinger, J. (1988) Trends Biochem. Sci. 13, 443-447). We studied the effects of GM3 and deNAcGM3 on EGF-RK function and EGF-R dimerization, and observed that (i) EGF-dependent in vitro and in vivo (in situ) phosphorylation of A431 cells at both monomeric and dimeric forms of EGF-R was inhibited in a dose-dependent manner by GM3, but unaffected by GM1. (ii) Quantities of both forms of EGF-R remained constant regardless of addition of various quantities of GM3 or GM1, as revealed by blotting with antibodies directed to the C-terminal region of EGF-R, or by cell surface 125I-labeling followed by immunoprecipitation. (iii) DeNacGM3 in the absence as well as in the presence of a minimal quantity of detergent significantly enhanced EGF-R phosphorylation, particularly Ser phosphorylation. (iv) DeNAcGM3 was detected in a large variety of actively growing tumor cells. Findings i and ii above indicate that GM3 directly inhibits EGF-dependent Tyr phosphorylation but does not affect receptor-receptor interaction. Findings iii and iv suggest that deNAcGM3 strongly promotes serine phosphorylation (in addition to Tyr phosphorylation) of EGF-R and may function as a second messenger in the process of cell growth stimulation.

Topics: Animals; Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Dogs; ErbB Receptors; Erythrocytes; G(M3) Ganglioside; Humans; Kinetics; Macromolecular Substances; Phosphorylation; Phosphoserine; Phosphothreonine; Phosphotyrosine; Serine; Tumor Cells, Cultured; Tyrosine

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