Tpl2 (tumor progression locus 2) phosphorylation at Thr 290 is induced by lipopolysaccharide via an Iκ-B kinase-β-dependent pathway and is required for Tpl2 activation by external signals

Abstract

The serine-threonine protein kinase encoded by the tumor progression locus 2 (Tpl2) proto-oncogene transduces Toll-like receptor and death receptor signals in a variety of cell types. Here we show that Tpl2 undergoes phosphorylation at Thr 290 both in cells overexpressing Tpl2 and in cells stimulated with lipopolysaccharide (LPS) or tumor necrosis factor-α and that phosphorylation on this site parallels Tpl2 activation. Reconstitution of Tpl2 -/- macrophages with wild type Tpl2 or Tpl2 T290D restored ERK activation by LPS, whereas reconstitution of the same cells with Tpl2 T290A did not, suggesting that phosphorylation at Thr 290 is required for the physiological activation of Tpl2 by external signals. Both the wild type Tpl2 and the kinase-inactive mutant Tpl2 K167M undergo Thr 290 phosphorylation, suggesting that Thr 290 may be a site of trans-phosphorylation rather than auto-phosphorylation. Pretreatment of 293 cells and primary macrophages with the κ-B kinase-β (IKKβ) inhibitor PS-1145 blocked Tpl2 phosphorylation at Thr 290, suggesting that phosphorylation depends on IKKβ, an obligatory positive regulator of Tpl2. We conclude that Tpl2 phosphorylation at Thr 290 is induced by LPS, depends on IKKβ, and is required for the physiological activation of Tpl2 by external signals. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.