title: Hierarchical phosphorylation of the TNF-alpha receptor, TNF-R1, by p42Mapk/Erk at basic Pro-directed kinase sites.
abstract: Phosphorylation of the TNF-alpha receptor TNF-R1 has been shown to differentially regulate receptor signaling and function and promote changes in its subcellular localization. Previous studies have shown that p42(mapk/erk2) phosphorylates Ser and Thr residues (T236, S240, S244, and S270) in the membrane proximal region of TNF-R1 and that mutation of these residues to Glu and Asp residues (TNF-R1.4D/E) mimics the effect of phosphorylation on receptor signaling and localization. In the present study, we investigated whether the initial phosphorylation of these residues by p42(mapk/erk2) promotes hierarchical phosphorylation of additional sites within the cytoplasmic domain of TNF-R1. This question was addressed by investigating the ability of the TNF-R1.4D/E mutant receptor to be phosphorylated in in vitro kinase assays using GST-mutant cytoplasmic domain fusion proteins as substrates and in intact cells following mutant receptor expression. In addition, we determined the location of the additional phosphorylation sites. Incubation of Sepharose bead-bound GST-TNF-R1(207)(-)(425).4D/E fusion protein with lysates containing activated p42(mapk/erk2) led to the phosphorylation of Ser and Thr residues in addition to the previously defined sites at T236, S240, S244, and S270. Deletional mutagenesis localized these residues to a stretch of 14 amino acids that encompasses three basic Pro-directed ([S/T]P) kinase consensus sequences located between residues S256 and T267. Point mutagenesis of T257, S262, and T267 to Ala residues indicated that these sites are targets of phosphorylation by p42(mapk/)(erk2). These findings support the conclusion that p42(mapk/erk2) promotes extensive phosphorylation of the membrane proximal region in a hierarchical fashion at both consensus and nonconsensus ERK-phosphorylation sites.
authors.affiliation: Program in Cell Biology, Department of Pediatrics, National Jewish Medical and Research Center, Denver, Colorado 80206, USA.
authors.forename: Annemie A
authors.initials: AA
authors.lastname: Van Linden
authors.forename: Vincent
authors.initials: V
authors.lastname: Cottin
authors.forename: Stephen K
authors.initials: SK
authors.lastname: Frankel
authors.forename: David W H
authors.initials: DW
authors.lastname: Riches
mesh_terms: D000818:Animals; D001216:Asparagine / Q000378:metabolism; D019556:COS Cells; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D016384:Consensus Sequence; D003593:Cytoplasm / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008809:Mice, Inbred C3H; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011392:Proline / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000235:genetics; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion / Q000235:genetics; D012694:Serine / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
chemical_list: D047888:Receptors, Tumor Necrosis Factor, Type I; D014409:Tumor Necrosis Factor-alpha; D012694:Serine; D001216:Asparagine; D011392:Proline; D017346:Protein Serine-Threonine Kinases; D019950:Mitogen-Activated Protein Kinase 1