Nogo-B is a new physiological substrate for MAPKAP-K2

Abstract

The neurite outgrowth inhibitor protein Nogo is one of 300 proteins that contain a reticulon homology domain, which is responsible for their association with the endoplasmic reticulum. Here we have found that the Nogo-B spliceform becomes phosphorylated at Ser107 in response to lipopolysaccharide in RAW264 macrophages or anisomycin in HeLa cells. The phosphorylation is prevented by SB 203580, an inhibitor of SAPK2a (stress-activated protein kinase 2a)/p38a and SAPK2b/p38b, and does not occur in embryonic fibroblasts generated from SAPK2a/p38a-deficient mice. Nogo-B is phosphorylated at Ser107 in vitro by MAPKAP-K2 [MAPK (mitogen-activated protein kinase)-activated protein kinase-2] or MAPKAP-K3, but not by other protein kinases that are known to be activated by SAPK2a/p38a. The anisomycin-induced phosphorylation of Ser107 in HeLa cells can be prevented by 'knockdown' of MAPKAP-K2 using siRNA (small interfering RNA). Taken together, our results identify Nogo-B as a new physiological substrate of MAPKAP-K2.

Figure 1. An unknown 43 kDa band is phosphorylated by the SAPK2/p38 pathway

(A) RAW cells were left untreated or pre-treated for 15 min with 10 μM SB 203580 (‘SB’) or 10 μM PD 184352 (‘PD’) and then stimulated for 45 min with 50 ng/ml LPS. Lysates were separated by SDS/PAGE, transferred to nitrocellulose and immunoblotted with the 5-LO phospho-specific antibody. (B) RAW cells were left untreated or incubated with 50 ng/ml LPS for the times indicated. Lysates were denatured in SDS, followed by SDS/PAGE and immunoblotting with the 5-LO phospho-specific antibody or an antibody that recognizes phosphorylated SAPK2a/p38α (pSAPK2a/p38α). (C) HeLa cells were left untreated or pretreated for 15 min with 10 μM SB 203580 (‘SB’) or 10 μM PD 184352 (‘PD’) and then stimulated for 15 min with 10 μg/ml anisomycin. Lysates were processed as described in (A). Similar results were obtained in at least six independent experiments.

Figure 2. Nogo-B is phosphorylated at Ser¹⁰⁷ by MAPKAP-K2

(A) Nogo-B (1 μM) was phosphorylated for 60 min with 10 units/ml active MAPKAP-K2, 10 mM MgCl₂ and 0.1 mM [γ-³²P]ATP and subjected to SDS/PAGE. The band corresponding to Nogo-B was excised, digested with trypsin and the resulting peptides separated by chromatography on a C₁₈ column equilibrated in 0.1% (v/v) trifluoroacetic acid. ³²P radioactivity is shown by the continuous line and the acetonitrile gradient by the broken line. (B) The peak T1 in (A) was shown by Edman sequencing to be a mixture of two peptides. The major peptide T1a started at residue 92 and the minor peptide T1b at residue 105. Solid-phase sequencing [40] identified Ser¹⁰⁷ as the site of phosphorylation in both peptides. (C) Bacterially expressed Nogo-B or Nogo-B[S107A] were left unphosphorylated (−) or phosphorylated for 60 min with 1 unit/ml MAPKAP-K2 or MAPKAP-K3 (+) and Mg-[γ³²-P]ATP. The proteins were denatured in SDS, subjected to SDS/PAGE and immunoblotted with the 5-LO phospho-specific antibody (‘p-Nogo-B’) as described in Figure 1 or with an antibody that recognizes all forms of Nogo-B (‘Nogo-B’). In contrast with the endogenous Nogo-B in cells, bacterially expressed Nogo-B migrates as two major bands, the lower generated by proteolysis near the C-terminus. (D) RAW cells were left untreated or incubated for 15 min with 5 μM SB 203580 or 10 μM PD 184352 and then stimulated for 45 min with 50 ng/ml LPS. Nogo-B was immunoprecipitated from the lysates, using 1 μg of anti-Nogo-B per mg of cell lysate, then denatured in SDS, followed by immunoblotting with the 5-LO phospho-specific antibody (‘p-Nogo-B’) as described in Figure 1 or with an antibody that recognizes all forms of Nogo-B (‘Nogo-B’). Similar results were obtained in two independent experiments.

Figure 3. Nogo-B is phosphorylated at Ser¹⁰⁷ in cells from wild-type mice, but not in mice deficient in SAPK2a/p38α

Wild-type (WT) and SAPK2a/p38α^−/− MEFs were left untreated (−) or preincubated for 15 min with 5 μM SB 203580 (‘SB’; +) and then incubated for 30 min without (−) or with (+) 10 μg/ml anisomycin. Nogo-B was immunoprecipitated from the lysates and denatured in SDS. After separation by SDS/PAGE and transfer to nitrocellulose, the membrane was probed with the 5-LO phospho-specific antibody (‘p-Nogo-B’) as described in Figure 1 or with an antibody that recognizes all forms of Nogo-B (‘Nogo-B’). Another aliquot of the cell lysate was immunoblotted (without immunoprecipitation) with a phospho-specific antibody that recognizes hnRNP A0 phosphorylated at Ser⁸⁴ (‘p-hnRNP A0’) or with an antibody that recognizes all forms of hnRNP A0 (‘hnRNP A0’). Similar results were obtained in two independent experiments.

Figure 4. Knock-down of MAPKAP-K2 and MAPKAP-K3 using siRNA prevents the phosphorylation of Nogo-B at Ser¹⁰⁷

HeLa cells were either mock-transfected (−) or transfected with siRNA against MAPKAP-K2, siRNA against MAPKAP-K3 or siRNA against both MAPKAP-K2 and MAPKAP-K3 (+) as indicated. (A) The cells were then left untreated or exposed for 30 min to 10 μg/ml anisomycin. MAPKAP-K2 (‘K2’) or MAPKAP-K3 (‘K3’) were immunoprecipitated from HeLa cell extracts as described [18] and assayed. Activity is shown in units/mg of cell extract. (B) The HeLa cell extracts from (A) (30 μg of protein) were immunoblotted with antibodies that recognize phosphorylated SAPK2a/p38α (‘pSAPK2a’), all forms of SAPK2a/p38α (‘SAPK2a’), HSP27 phosphorylated at Ser¹⁵ (‘pHSP27’), all forms of HSP27 (HSP27) or with the 5-LO phospho-specific antibody (‘p-Nogo-B’) and antibodies that recognize all forms of Nogo-B (‘Nogo-B’). Similar results were obtained in three independent experiments.

Figure 5. Nogo-B co-localizes with microtubules

HeLa cells were plated on Lab-Tek chamber slides (Nunc, Roskilde, Denmark) coated with fibronectin (5 μg/ml), left to adhere for 1 h and then incubated for 30 min without (A, B, G, H, I and J) or with 10 μM cytochalasin D (C and D) or 5 μM nocodazole (E and F). Nogo-B was visualized using the antibody that recognizes all forms of Nogo-B equally well coupled to anti-sheep IgG (A, C, E and G), whereas microtubules were visualized with an anti-(tyrosinated α-tubulin) antibody (YL-1/2) coupled to anti-rat IgG (B, D, F and H). The ER was visualized with an antibody that recognizes PDI and calreticulin coupled to anti-mouse IgG (I and J). The Figure shows that Nogo-B co-localizes with microtubules. Similar results were obtained in four (A and B) or two (C–J) independent experiments.