Hierarchical Phosphorylation of the TNF-α Receptor, TNF-R1, by p42mapk/erk at Basic Pro-Directed Kinase Sites

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.