IκB kinase-induced interaction of TPL-2 kinase with 14-3-3 is essential for Toll-like receptor activation of ERK-1 and -2 MAP kinases

Abstract

The MEK-1/2 kinase TPL-2 is critical for Toll-like receptor activation of the ERK-1/2 MAP kinase pathway during inflammatory responses, but it can transform cells following C-terminal truncation. IκB kinase (IKK) complex phosphorylation of the TPL-2 C terminus regulates full-length TPL-2 activation of ERK-1/2 by a mechanism that has remained obscure. Here, we show that TPL-2 Ser-400 phosphorylation by IKK and TPL-2 Ser-443 autophosphorylation cooperated to trigger TPL-2 association with 14-3-3. Recruitment of 14-3-3 to the phosphorylated C terminus stimulated TPL-2 MEK-1 kinase activity, which was essential for TPL-2 activation of ERK-1/2. The binding of 14-3-3 to TPL-2 was also indispensible for lipopolysaccharide-induced production of tumor necrosis factor by macrophages, which is regulated by TPL-2 independently of ERK-1/2 activation. Our data identify a key step in the activation of TPL-2 signaling and provide a mechanistic insight into how C-terminal deletion triggers the oncogenic potential of TPL-2 by rendering its kinase activity independent of 14-3-3 binding.

Keywords: MAP3K8; NF-κB; inflammation.

Fig. 1.

Map3k8^S400A mutation blocks LPS activation of ERK-1/2 in macrophages. (A) WT and Map3k8^S400A/S400A BMDMs were stimulated with LPS for the indicated times. Lysates were immunoblotted. (B) WT and Map3k8^S400A/S400A mice were injected intraperitoneally (i.p.) with LPS or PBS control. After 10 min, peritoneal cells were aspirated and stained for surface F4/80 and intracellular phospho–ERK-1/2 (P-ERK). Anti–P-ERK mean fluorescence intensity (MFI) of F4/80⁺ cells was determined by flow cytometry (mean ± SEM; n = 6 mice per genotype). (C) Triplicate cultures of BMDMs were stimulated with LPS for the indicated times, and TNF levels in supernatants were assayed. (D) WT and Map3k8^S400A/S400A mice were injected i.p. with LPS. After 1 h, mice were bled, and serum TNF levels were quantified (mean ± SEM; n = 8 mice per genotype). **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 2.

Map3k8^S400A mutation does not affect LPS-induced release of TPL-2 from NF-κB1 p105 or TPL-2 intrinsic kinase activity. WT and Map3k8^S400A/S400A (Map3k8^S400A) BMDMs were stimulated with LPS. (A) Total and p105-depleted lysates were immunoblotted. (B) TPL-2 was immunoprecipitated and assayed for its ability to phosphorylate GST–MEK-1^K207A in vitro. MEK-1 phosphorylation and TPL-2 levels were assayed by immunoblotting.

Fig. 3.

S400 is required for TPL-2 interaction with 14-3-3. (A) Biolayer interferometry was used to measure the ability of TPL-2^393–407 peptide nonphosphorylated (□) and phosphorylated on S400 (●) to displace 14-3-3ζ protein associated with biotinylated phospho-S621–cRaf peptide bound to a Streptavidin sensor chip. A K_d value of 0.7 μM for the phospho-S400 peptide was calculated. (B) GST–14-3-3γ pulldowns from lysates of BMDMs with or without LPS (15 min) were immunoblotted. (C) IL-1R–293 cells were transiently transfected with expression constructs encoding WT or S400A Flag–TPL-2 or empty vector (EV). Anti-Flag immunoprecipitates and cell lysates were immunoblotted. (D) BMDMs were preincubated with BI605906 IKK2 inhibitor or vehicle control for 1 h, before stimulation with LPS for 15 min. GST–14-3-3γ pulldowns from cells lysates were immunoblotted. (E and F) BMDMs of the indicated genotypes were stimulated with LPS or left unstimulated. GST–14-3-3γ pulldowns and total cell lysates were immunoblotted.

Fig. 4.

Reconstitution of TPL-2 signaling activity with 14-3-3–binding R18 motif. (A) IL-1R–293 cells, transiently transfected with empty vector (EV) or plasmids encoding Myc-tagged TPL-2, TPL-2^S400A, and TPL-2^S400–R18, were stimulated with IL-1β or left unstimulated. (B–D) GST–14-3-3γ pulldowns and lysates were immunoblotted. BMDMs generated from Map3k8^−/− (B and C) or Nfkb1^−/− (D) mice were transduced with retroviruses encoding the indicated TPL-2 proteins. In B and D, cells were stimulated with LPS for 15 min or left unstimulated, and lysates were immunoblotted. In C, cells were stimulated with LPS for 9 h, and sTNF in culture supernatants was assayed (mean ± SEM; n = 3). ****P < 0.0001.

Fig. 5.

The 14-3-3 interaction with phospho-S443 is required for optimal TPL-2 signaling. (A) Binding between 14-3-3ζ and a synthetic phospho-S443–TPL-2^436–450 peptide as in Fig. 3A. A K_d value of 1.9 ± 0.4 μM was calculated. (B) IL-1R–293 cells, transiently transfected with plasmids encoding WT, S400A, or S443 AMyc–TPL-2, were stimulated with IL-1β or left unstimulated. Anti-Myc immunoprecipitates and cell lysates were immunoblotted. (C) IL-1R–293 cells expressing Myc–TPL-2 were pretreated with C34 (TPL-2 inhibitor), BI605906 (IKK2 inhibitor), or vehicle control and then stimulated with IL-1β or left unstimulated. Anti-Myc immunoprecipitates were immunoblotted. (D) IL-1R–293 cells expressing Myc–TPL-2 or kinase-inactive Myc–TPL-2^D270A were stimulated with IL-1β or left unstimulated. Anti-Myc immunoprecipitates were immunoblotted. (E) IL-1R–293 cells transiently expressing the indicated Myc–TPL-2 proteins were stimulated with IL-1β or left unstimulated. GST–14-3-3γ pulldowns and lysates were immunoblotted. (F) Map3k8^−/− BMDMs, transduced with retroviruses encoding the indicated TPL-2 proteins, were stimulated with LPS (15 min) or left unstimulated. Lysates were immunoblotted.

Fig. 6.

Optimal TPL-2 signaling requires 14-3-3 interaction with both phospho-S400 and -S443. (A) IL-1R–293 cells expressing the indicated Myc–TPL-2 constructs or empty vector (EV) were stimulated with IL-1β or left unstimulated. GST–14-3-3γ pulldowns and lysates were immunoblotted. (B and E) Map3k8^−/− BMDMs, transduced with retroviruses encoding the indicated TPL-2 proteins, were stimulated with LPS for 15 min or left unstimulated. Lysates were immunoblotted. (C and D) WT and mutant forms of Myc–TPL-2 were immunoprecipitated from lysates of IL-1R–293 cells with or without IL-1β stimulation, and immunoblotted. (F) IL-1R–293 cells expressing the indicated Myc–TPL-2 constructs were stimulated with IL-1β or left unstimulated. Lysates were immunoblotted.

Fig. 7.

TPL-2 MEK kinase activity is enhanced by 14-3-3. (A) IL-1R–293 cells, transiently coexpressing Flag–TPL-2 or Flag–TPL-2^S400A and HA–IKK2, were stimulated with IL-1β. Immunoprecipitated Flag–TPL-2 was assayed for its ability to phosphorylate GST–MEK-1^K207A (KA) with or without recombinant 14-3-3ζ protein. (B) Endogenous TPL-2 was immunoprecipitated from lysates of BMDMs with or without LPS (15 min) and assayed for its ability to phosphorylate GST–MEK-1^K207A (KA) with or without recombinant 14-3-3ζ protein. (C) IL-1R–293 cells were transfected with the indicated amounts of HA–MEK-1 plasmid together with a fixed quantity of Myc–TPL-2 plasmid. Lysates, prepared with or without IL-1β stimulation, were immunoblotted.