1. NF-κB1/p105 Regulates Lipopolysaccharide-Stimulated MAP Kinase Signaling by Governing the Stability and Function of the Tpl2 Kinase 
Michael R. Waterfield, Minying Zhang, Lourdes P. Norman, Shao-Cong Sun 
Molecular Cell 
Volume 11, Issue 3, Pages (March 2003)
DOI: /S (03)
Copyright © 2003 Cell Press Terms and Conditions

2. Figure 1
nfκb1 Deficiency Interrupts LPS-Stimulated ERK Activation and Expression of Its Target Gene EGR-1
(A) IB analysis of p105 and the control tubulin in WT and nfκb1−/− bone marrow-derived macrophages (BMDM) using anti-p105 and anti-tubulin antibodies.
(B) Impaired ERK activation by LPS in nfκb1−/− macrophages. WT and nfκb1−/− macrophages were stimulated for the indicated times. Activated (upper panel) and total (lower panel) ERK proteins were detected by IB using anti-phospho ERK (P-ERK) and anti-ERK, respectively.
(C–E) Normal activation of JNK and p38 and degradation of IκBα in nfκb1−/− cells. The same cell lysates used in (B) were subjected to IB for analyzing the activation of JNK (C) and p38 (D) using corresponding anti-phospho (upper panels) or pan antibodies (lower panels) or degradation of IκBα using anti-IκBα (E).
(F) ERK1/2 activation in WT and nfκb1−/− macrophages following prolonged LPS stimulation. Activated ERK1 and ERK2 were detected as described in (B).
(G) Normal ERK activation by the mitogen PMA in nfκb1−/− cells. WT and nfκb1−/− macrophages were stimulated with PMA followed by IB analysis of activated (upper panel) and total (lower panel) ERKs as described in (B).
(H) Impaired induction of EGR-1 protein expression by LPS in nfκb1−/− macrophages. WT and nfκb1−/− macrophages were either nontreated (NT) or stimulated for 2 hr with the indicated inducers. Expression of EGR-1 and the control protein tubulin were detected by IB using anti-EGR-1 and anti-tubulin.
(I) Impaired induction of EGR-1 mRNA and normal induction of IκBα mRNA in nfκb1−/− cells. The WT and nfκb1−/− macrophages were stimulated for 1 hr as indicated, and total RNA was isolated and subjected to RT-PCR analysis to determine the level of EGR-1, IκBα, and GAPDH mRNAs.
Molecular Cell  , DOI: ( /S (03) )
Copyright © 2003 Cell Press Terms and Conditions

3. Figure 2
NF-κB1 Is Essential for LPS-Stimulated Activation of MEK1 and Steady Expression of Its Upstream Regulator Tpl2
(A) Impaired MEK1 activation in nfκb1−/− macrophages stimulated by LPS, but not by PMA. WT and nfκb1−/− macrophages were stimulated with LPS or PMA followed by IB analysis of the activated (upper panel) and total (lower panel) MEK1 using anti-phospho MEK1 and anti-MEK1.
(B and C) Deficiency in activation and expression of Tpl2, but not Raf1, in nfκb1−/− macrophages. Tpl2 was immunoprecipitated with anti-Tpl2 antibody, and its catalytic activity measured by immunecomplex kinase assay (KA) using GST-MEK1 as substrate ([B], top panel). The expression of Tpl2 ([B], middle panel), IKKα ([B], bottom panel), the Tpl2-related upstream kinase Raf1 ([C], upper panel), and tubulin ([C], lower panel) was monitored by IB using anti-Tpl2, anti-IKKα, anti-Raf1, and anti-tubulin, respectively. The two isoforms of Tpl2 are known to result from alternative translational initiation (Aoki et al., 1991; Miyoshi et al., 1991).
(D) Restoration of Tpl2 expression in nfκb1−/− macrophages reconstituted with p105. nfκb1−/− macrophages (BMDM) were infected with retroviruses encoding either GFP or p105 and then subjected to IB to detect p105/p50 and Tpl2. As positive control, a cell lysate from wild-type (WT) macrophages was included in the Tpl2 IB. The presence of a background band (ns) in the infected cells (lanes 3 and 4) was due to the loading of higher amounts of cell lysates (24 μg) than the positive control (lane 5, 12 μg).
(E) Restoration of ERK signaling in nfκb1−/− macrophages reconstituted with p105. The retrovirus-infected nfκb1−/− macrophages were either not treated (NT) or stimulated with LPS for 30 min and then collected for IB to analyze activated and total ERK1/2 proteins.
(F) Normal Tpl2 expression and MEK/ERK activation in nfκb2−/− cells. WT and nfκb2−/− macrophages were stimulated with LPS, and the cell lysates analyzed by IB for detection of Tpl2 (top panel), activated MEK1 (phospho-MEK1, middle panel), and activated ERK1/2 (phospho-ERK1/2, bottom panel).
Molecular Cell  , DOI: ( /S (03) )
Copyright © 2003 Cell Press Terms and Conditions

4. Figure 3
NF-κB1 Is Not Required for Tpl2 Gene Expression, but Controls the Stability of the Tpl2 Protein
(A) WT and nfκb1−/− BMDM, peritoneal macrophages (PTM), and splenocytes were subjected to IB analysis for expression of Tpl2, p105, and the loading control tubulin.
(B) Normal expression of Tpl2 mRNA in nfκb1−/− cells. Total RNA was isolated from WT and nfκb1−/− BMDM and splenocytes and then subjected to RT-PCR assays to detect the level of Tpl2 mRNA (upper panel). GAPDH and β-actin were included as controls (lower panel).
(C) Rapid degradation of Tpl2 in nfκb1−/− macrophages. WT, nfκb1−/−, and nfκb2−/− macrophages were pulse labeled with 35S-methionine/cysteine for 1.5 hr followed by the indicated times of chase. The radiolabeled Tpl2 was isolated by IP, separated by SDS-PAGE, and visualized by autoradiography.
Molecular Cell  , DOI: ( /S (03) )
Copyright © 2003 Cell Press Terms and Conditions

5. Figure 4
The NF-κB1 Precursor Protein p105 Stabilizes Tpl2 by Forming a p105/Tpl2 Complex
(A) 293 cells were transfected by DEAE-dextran with HA-tagged Tpl2 (0.5 μg), either alone or together with the indicated expression vectors (0.5 μg). The transfected cells were subjected to pulse-chase labeling followed by IP using anti-HA. Since HA is tagged to the N terminus of the protein, the shorter isoform of Tpl2 (lacking the N-terminal region) shown in Figure 3C was not detected. To monitor the expression of the cotransfected NF-κB proteins, a small aliquot of the cell lysates used in the IP (top panel) was subjected to IB (lower panel) using the indicated antibodies.
(B) Enhancement of Tpl2 steady expression by p cells were transfected with HA-tagged Tpl2 (0.5 μg) together with either an empty vector or the indicated expression vectors (0.5 μg). Cell lysates were analyzed by IB using HRP-conjugated anti-HA (3F10).
(C) p105 and p105ΔN, but not p100 or p50, physically interact with Tpl cells were transfected as described in (B) except that more Tpl2 expression vector was transfected in lanes 1, 3, 4, and 5 to obtain similar level of Tpl2 expression. The cell lysates were subjected to IP using the antibodies indicated above the figure, followed by detecting the coprecipitated HA-Tpl2 by IB using HRP-conjugated anti-HA (top panel). The expression level of the transfected proteins was analyzed by IB using anti-HA (middle panel) or anti-p105N (bottom panel, lanes 1-3), anti-p100 (lane 4), or anti-p105C (lanes 5 and 6). The transfected p50 contains some additional amino acid sequences and thus is larger than the natural p105 processing product.
Molecular Cell  , DOI: ( /S (03) )
Copyright © 2003 Cell Press Terms and Conditions

6. Figure 5
NF-κB1 Is Required for LPS-Stimulated Activation of Rsk1 and Msk1 and Their Target Transcription Factor CREB, as Well as Induction of the Downstream Gene cox-2
(A) LPS-mediated COX-2 induction requires NF-κB1. Cell lysates were prepared from LPS-stimulated WT (lanes 1–5) and nfκb1−/− (lanes 6–10) BMDM, as well as nfκb1−/− BMDM infected with retroviruses encoding GFP or p105 (lanes 11–14). The lysates were analyzed by IB using anti-Cox-2 (upper panel) or anti-ERK (lower panel) antibodies.
(B) Attenuated CREB activation in nfκb1−/− macrophages. Nuclear extracts were isolated from WT and nfκb1−/− BMDM, and equal amounts (5 μg) were subjected to EMSA using a 32P-radiolabeled CREB binding sequence derived from the cox-2 gene.
(C) Reduced CREB phosphorylation in nfκb1−/− macrophages. Cell lysates derived from LPS-stimulated WT and nfκb1−/− BMDM were subjected to IB using phospho-specific (upper panel) and regular (lower panel) anti-CREB antibodies. The phospho-specific antibody also reacts with the phosphorylated ATF1 (P-ATF1).
(D) Diminished actvation of Rsk1 and Msk1 in nfκb1−/− macrophages. Cell lysates derived LPS-stimulated WT, and nfκb1−/− BMDM were subjected to IB using phospho-specific or regular anti-Rsk1 and anti-Msk1 antibodies. ERK blots were used as controls.
Molecular Cell  , DOI: ( /S (03) )
Copyright © 2003 Cell Press Terms and Conditions

7. Figure 6
Tpl2 Activation Involves Its Dissociation from p105 and Rapid Degradation
(A) Degradation of Tpl2L by LPS. WT macrophages (BMDM) were stimulated with LPS followed by IB using anti-Tpl2 (top panel), anti-p105C (middle panel), and tubulin (bottom panel). The long (Tpl2L) and short (Tpl2S) isoforms of Tpl2 are indicated.
(B) Dissociation of Tpl2L from p105. Lysates of LPS-stimulated BMDM were subjected to IP using anti-p105C, and the p105-associated Tpl2 proteins were detected by IB using HRP-conjugated anti-Tpl2 (top panel). The amounts of Tpl2 and p105 in the cell lysates were monitored by IB (middle and bottom panels).
(C) p105-associated Tpl2 is inactive. Macrophages were stimulated with LPS for the indicated times and subjected to IP using either anti-p105C (lanes 1–5) or anti-Tpl2 (lanes 6–10) followed by kinase assays using GST-MEK1 as substrate (upper panel). After autoradiography, the kinase assay filter was subjected to IB using HRP-conjugated anti-Tpl2 (lower panel). The thick Tpl2S band is largely due to generation of multiple phosphorylated isoforms (migrate as smeary bands) in the in vitro kinase assays, which could be visualized with short exposure (data not shown).
(D) p105 inhibits the activity of Tpl2 in transfected cells. 293 cells were transfected with HA-tagged Tpl2 together with either an empty vector (V) or increasing amounts of p105 or p100. Tpl2 was isolated by IP using anti-HA, and its activity measured by kinase assay using GST-MEK1 as substrate (top panel). The expression levels of Tpl2 and the cotransfected NF-κB proteins were detected by IB using anti-HA (middle panel) or anti-p105N together with anti-p100 (bottom panel).
Molecular Cell  , DOI: ( /S (03) )
Copyright © 2003 Cell Press Terms and Conditions

8. Figure 7 Model for Tpl2 Regulation by p105
Newly synthesized Tpl2 proteins are rapidly captured by p105 to form a stable and latent kinase complex, with any free Tpl2 being degraded. In response to LPS stimulation, the long isoform of Tpl2 (Tpl2L) is liberated from p105, and this activated subset of Tpl2 mediates activation of the MEK/ERK signaling. This response is transient since the activated Tpl2L is rapidly degraded.
Molecular Cell  , DOI: ( /S (03) )
Copyright © 2003 Cell Press Terms and Conditions