Arrestin-3 binds c-Jun N-terminal kinase 1 (JNK1) and JNK2 and facilitates the activation of these ubiquitous JNK isoforms in cells via scaffolding

Abstract

Non-visual arrestins scaffold mitogen-activated protein kinase (MAPK) cascades. The c-Jun N-terminal kinases (JNKs) are members of MAPK family. Arrestin-3 has been shown to enhance the activation of JNK3, which is expressed mainly in neurons, heart, and testes, in contrast to ubiquitous JNK1 and JNK2. Although all JNKs are activated by MKK4 and MKK7, both of which bind arrestin-3, the ability of arrestin-3 to facilitate the activation of JNK1 and JNK2 has never been reported. Using purified proteins we found that arrestin-3 directly binds JNK1α1 and JNK2α2, interacting with the latter comparably to JNK3α2. Phosphorylation of purified JNK1α1 and JNK2α2 by MKK4 or MKK7 is increased by arrestin-3. Endogenous arrestin-3 interacted with endogenous JNK1/2 in different cell types. Arrestin-3 also enhanced phosphorylation of endogenous JNK1/2 in intact cells upon expression of upstream kinases ASK1, MKK4, or MKK7. We observed a biphasic effect of arrestin-3 concentrations on phosphorylation of JNK1α1 and JNK2α2 both in vitro and in vivo. Thus, arrestin-3 acts as a scaffold, facilitating JNK1α1 and JNK2α2 phosphorylation by MKK4 and MKK7 via bringing JNKs and their activators together. The data suggest that arrestin-3 modulates the activity of ubiquitous JNK1 and JNK2 in non-neuronal cells, impacting the signaling pathway that regulates their proliferation and survival.

Keywords: Arrestin; Jun N-terminal Kinase (JNK); MAP Kinases (MAPKs); Protein Phosphorylation; Scaffold Proteins; Signal Transduction.

FIGURE 1.

Arrestin-3 binds JNK1α1 and JNK2α2 isoforms in vitro and in cells. A, Coomassie Blue-stained gel of the eluted bait proteins (JNK1α1, JNK2α2, and JNK3α2). B, the amount of arrestin-3 retained was analyzed by Western blot (IB). Representative gels are shown. Bar graph, quantification of the Western blot data. The intensity of the arrestin-3 bands specifically bound to indicated JNK isoforms in three independent experiments was quantified and statistically analyzed. The means ± S.E. are shown. All JNK isoforms showed significant specific binding (p < 0.001 as compared with control without JNK, repeated measure ANOVA); JNK isoforms showed significantly different efficacy in arrestin-3 binding (one-way ANOVA F(2,6) = 9.975 p = 0.0124); *, −p < 0.05 compared to JNK1 according to Bonferroni/Dunn post hoc test with correction for multiple comparisons. C, results of a representative immunoprecipitation (IP) experiment in COS-7 cells expressing arrestin-3 without (−) or with FLAG-JNK1α1, FLAG-JNK2α2, or FLAG-JNK3α2 using anti-FLAG antibody. Arrows point to arrestin-3 bands.

FIGURE 2.

Endogenous arrestin-3 interacts with endogenous JNK1/2 in non-neuronal and neuronal cells. A, immunoprecipitation of endogenous arrestin-3 from arrestin-2 knock-out MEFs using rabbit polyclonal arrestin antibody (ab) F431 (upper panels) or mouse monoclonal arrestin-3 antibody (lower panels). Respective rabbit or mouse IgGs were used as controls. Co-immunoprecipitated JNK isoforms were visualized using mouse or rabbit JNK antibodies. Black arrows point to arrestin-3 bands, black arrowheads point to JNK bands, and white arrows point to IgG bands. IB, immunoblot. B, immunoprecipitation experiments performed in Neuro2a cells. All conditions and labels are the same as in panel A. Rabbit arrestin antibody immunoprecipitated arrestin-3 much more efficiently than mouse arrestin-3 antibody, as seen in the images in the left in A and in B. Note that the amount of arrestin-3 standard loaded on each gel was the same (Arr3 Std), but the exposure times differed. Thus, the amount of co-precipitated JNK1/2 was also higher when rabbit arrestin antibody was used.

FIGURE 3.

Arrestin-3 promotes JNK1/2 activation induced by the expression of ASK1. A, representative Western blot (IB) showing phosphorylation of endogenous JNK1/2 isoforms with or without arrestin-3 in COS-7 cells expressing varying amounts of ASK1. The upper p-JNK blot is the same as the lower blot exposed for a longer time to visualize p54 isoforms. B, quantification of phosphorylation of JNK p46H and p54 isoforms with or without arrestin-3. Analysis of covariance with Arr3 as factor and ASK1 concentration as co-variate showed a significant effect of ASK1 concentration on the level of p46H and p54 phosphorylation (p < 0.0001). Significant Arr3 × ASK1 interaction (p < 0.0001) indicated a difference in slopes due to co-expression of arrestin-3. *, −p < 0.05; **, −p < 0.01 to −Arr3; Student's t test for individual points.

FIGURE 4.

Arrestin-3 enhances MKK7-dependent phosphorylation of endogenous JNK1/2 in intact cells. A, a representative Western blot (IB) showing phosphorylation of endogenous JNK1/2 isoforms with or without arrestin-3 in COS-7 cells expressing varying amounts of MKK7. B, quantification of phosphorylation of JNK p46H and p54 isoforms with or without arrestin-3. Analysis of covariance with Arr3 as factor and MKK7 concentration as co-variate showed a significant effect of MKK7 concentration on the level of p46H and p54 phosphorylation (p < 0.0001). The presence of Arr3 significantly affected the level of p46H, but not p54, phosphorylation across MKK7 concentrations (F(1,38) = 8.592; p = 0.0057). Significant Arr3 × MKK7 interaction (p = 0.0335 for p46H and p < 0.0001 for p54) indicated a difference in slopes due to co-expression of arrestin-3. *, −p < 0.05 to −Arr3; Student's t test for individual points.

FIGURE 5.

Arrestin-3 enhances MKK4-dependent phosphorylation of endogenous JNK1/2 in intact cells. A, representative Western blot (IB) showing phosphorylation of endogenous JNK1/2 isoforms with or without arrestin-3 in COS-7 cells expressing varying amounts of MKK4. Note that there is no progression in the level of p46H phosphorylation with increased MKK4 concentration without arrestin-3 co-expression. P54 isoforms were poorly activated by the expression of MKK4, in contrast to ASK1 and MKK7 (Figs. 3 and 4), although some increase in the presence of arrestin-3 is visible. B, quantification of phosphorylation of JNK p46H isoforms with or without arrestin-3. Analysis of covariance with Arr3 as factor and MKK4 concentration as co-variate showed significant Arr3 X MKK4 interaction (p < 0.0001), indicating a difference in slopes due to co-expression of arrestin-3. *, −p < 0.05; **, −p < 0.01; *, −p < 0.001 −arrestin-3; Student's t test for individual points.

FIGURE 6.

Arrestin-3 facilitates phosphorylation of purified JNK1α1 and JNK2α2 by MKK4 and MKK7. Representative blots show JNK1α1 (A and D) or JNK2α2 (B and E) phosphorylation in the presence of MKK4 (A and B) or MKK7 (D and E) and increasing amounts of arrestin-3. Quantification of phosphorylation data for JNK1α1 and JNK2α2 in the presence of MKK4 (C) or MKK7 (F) yielded bell-shaped curves as functions of arrestin-3 concentration. Means ± S.D. of three independent experiments are shown. IB, immunoblot. ANOVA analysis with arrestin-3 as the main factor demonstrated significance of arrestin-3 concentration in the presence of MKK4 for both JNK1α1 and JNK2α2 (p < 0.001) and in the presence of MKK7 for both JNK isoforms (p < 0.001). *, −p < 0.001; **, −p < 0.01; *, −p < 0.05 to maximal values (at 5 or 10 μm arrestin-3, respectively) according to Bonferroni/Dunn post-hoc test with correction for multiple comparisons.

FIGURE 7.

Arrestin-3 enhances ASK1- and MKK7-dependent phosphorylation of endogenous JNK1/2 in intact cells. A and C, representative Western blots (IB) showing phosphorylation of endogenous JNK1/2 isoforms in cells expressing ASK1 (A) or MKK7 (C) in the presence of increasing concentrations of arrestin-3. B and D, quantification of the levels of p46H JNK isoform phosphorylation. One-way ANOVA analysis with arrestin-3 concentration as factor yielded a significant effect of arrestin-3 on JNK phosphorylation both in the presence of ASK1 and MKK7 (p < 0.0001). *, −p < 0.001; **, −p < 0.01; *, −p < 0.05 to the value at 0 μg of arrestin-3; b, −p < 0.01; c, −p < 0.001 to the maximal value (at 1 or 4 μg of arrestin-3 DNA) according to the Bonferroni/Dunn post-hoc test.