IκB kinase 2 regulates TPL-2 activation of extracellular signal-regulated kinases 1 and 2 by direct phosphorylation of TPL-2 serine 400

Abstract

Tumor progression locus 2 (TPL-2) functions as a MEK-1/2 kinase, which is essential for Toll-like receptor 4 (TLR4) activation of extracellular signal-regulated kinase 1 and 2 (ERK-1/2) mitogen-activated protein (MAP) kinases in lipopolysaccharide (LPS)-stimulated macrophages and for inducing the production of the proinflammatory cytokines tumor necrosis factor and interleukin-1β. In unstimulated cells, association of TPL-2 with NF-κB1 p105 prevents TPL-2 phosphorylation of MEK-1/2. LPS stimulation of TPL-2 MEK-1/2 kinase activity requires TPL-2 release from p105. This is triggered by IκB kinase 2 (IKK-2) phosphorylation of the p105 PEST region, which promotes p105 ubiquitination and degradation by the proteasome. LPS activation of ERK-1/2 additionally requires transphosphorylation of TPL-2 on serine 400 in its C terminus, which controls TPL-2 signaling to ERK-1/2 independently of p105. However, the identity of the protein kinase responsible for TPL-2 serine 400 phosphorylation remained unknown. In the present study, we show that TPL-2 serine 400 phosphorylation is mediated by IKK2. The IKK complex therefore regulates two of the key regulatory steps required for TPL-2 activation of ERK-1/2, underlining the close linkage of ERK-1/2 MAP kinase activation to upregulation of NF-κB-dependent transcription.

Fig 1

IKK2 regulates LPS-induced phosphorylation of TPL-2. Cell lysates were immunoblotted for the antigens shown. (A, D, and E) BMDM of the indicated genotypes were pretreated with BI605906 or vehicle control (DMSO) and then stimulated with LPS (10 ng/ml) or left unstimulated. WT cells were used in panel A. In panels D and E, no ERK-1/2 phosphorylation was detected after LPS stimulation with or without BI605906 (data not shown). (B) QBI-293A cells were pretreated with BI605906 or vehicle control (DMSO) and then stimulated with TNF for 15 min or left unstimulated. (C) BMDM of the indicated genotypes were stimulated with LPS or left unstimulated.

Fig 2

Phosphorylation of TPL-2 by IKK2 in vitro. (A) Coomassie blue-stained SDS-PAGE gel of purified TPL-2^D270A/NF-κB1 p105/ABIN-2 complex. (B) TPL-2^D270A/NF-κB1 p105/ABIN-2 complex was incubated with recombinant IKK2 plus [³²P]ATP for a kinase assay (KA). Labeled proteins were revealed by autoradiography after SDS-PAGE. Cell lysates were immunoblotted (IB) for the indicated antigens. (C) Extracted ion chromatograms of phosphorylated S62, S62/S66, and S400 peptides identified by mass spectrometry of TPL-2^D270A complex without (left) and with (right) IKK2 phosphorylation. Absolute intensities and retention times are shown (Table 1).

Fig 3

TPL-2 S62 and S66 are not required for TPL-2 activation of ERK. Map3k8^−/− BMDM were transduced with recombinant retroviruses (RV) encoding TPL-2 (WT), TPL-2^S62A, TPL-2^S66A, and TPL-2^S400A. Cells were stimulated with LPS (100 ng/ml), and lysates were immunoblotted. P-ERK, phospho-ERK-1/2; P-p38, phospho-p38.

Fig 4

Phosphorylation of TPL-2 S400 by IKK2 in cells. (A) FLAG–TPL-2/HA-p105/ABIN-2-StrepII complex was isolated by anti-FLAG immunoprecipitation (IP) from transiently transfected QBI-293A cells coexpressing HA-IKK2 (+) or empty vector (−). Isolated proteins were resolved by SDS-PAGE and immunoblotted. WCL, whole-cell lysate. (B) C6 cells were transiently transfected with a vector encoding Myc-TPL-2 together with either a vector encoding IKK2 or empty vector (EV). After 48 h, cells were stimulated with IL-1β (20 ng/ml) for 20 min, or left unstimulated. Myc-TPL-2 was immunoprecipitated from cell lysates with Myc MAb and then immunoblotted. (C) C6 cells were transfected with a vector encoding FLAG–TPL-2 or EV. After 48 h, cells were pretreated with BI605906 (+) or vehicle control (−) and then stimulated with IL-1β for 20 min or left unstimulated. FLAG immunoprecipitates were immunoblotted. (D) Nfkb1^−/− BMDM, transduced with retrovirus expressing wild-type TPL-2, were pretreated with BI605906 (+) or vehicle control (−) and then stimulated with LPS (100 ng/ml) or left unstimulated. Cell lysates were immunoblotted. P-ERK, phospho-ERK-1/2; T-ERK, total ERK-1/2.

Fig 5

Regulation of TPL-2 activation by IKK. In unstimulated cells, TPL-2 is complexed with NF-κB1 p105. Agonist stimulation induces IKK2, a catalytic subunit of the IKK complex, to phosphorylate p105 on serines 927 and 932 (human p105 numbering) in its PEST region, which triggers p105 K48-linked polyubiquitination and degradation by the proteasome. This releases TPL-2 from p105-mediated inhibition, allowing TPL-2 access to its substrates MEK-1/2. TPL-2 activation of MEK-1/2 also requires phosphorylation of the TPL-2 C terminus on S400, which is shown here to be mediated by IKK2. The IKK complex therefore regulates two critical steps in the activation of TPL-2 MEK-1/2 kinase activity.