Arrestin-3-Dependent Activation of c-Jun N-Terminal Kinases (JNKs)

Abstract

Only 1 out of 4 mammalian arrestin subtypes, arrestin-3, facilitates the activation of c-Jun N-terminal kinase (JNK) family kinases. Here, we describe two different sets of protocols used for elucidating the mechanisms involved. One is based on reconstitution of signaling modules from the following purified proteins: arrestin-3, MKK4, MKK7, JNK1, JNK2, and JNK3. The main advantage of this method is that it unambiguously establishes which effects are direct because only intended purified proteins are present in these assays. The key drawback is that the upstream-most kinases of these cascades, ASK1 or other MAP3Ks, are not available in purified form, limiting reconstitution to incomplete two-kinase modules. The other approach is used for analyzing the effects of arrestin-3 on JNK activation in intact cells. In this case, signaling modules include ASK1 and/or other MAP3Ks. However, as every cell expresses thousands of different proteins, their possible effects on the readout cannot be excluded. Nonetheless, the combination of in vitro reconstitution from purified proteins and cell-based assays makes it possible to elucidate the mechanisms of arrestin-3-dependent activation of JNK family kinases. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Construction of arrestin-3-scaffolded MKK4/7-JNK1/2/3 signaling modules in vitro using purified proteins Alternate Protocol 1: Characterization of arrestin-3-mediated JNK1/2 activation by MKK4/7 by measurement of JNK1/2 phosphorylation using immunoblotting with anti-phospho-JNK antibody Support Protocol 1: Expression, purification, and activation of GST-MKK4 Support Protocol 2: Expression, purification, and activation of GST-MKK7-His₆ Support Protocol 3: Expression, purification, and activation of tagless JNK1Α1 Support Protocol 4: Expression, purification, and activation of tagless JNK2Α2 Basic Protocol 2: Analysis of the role of arrestin-3 in ASK1/MKK4/MKK7-induced JNK activation in intact cells Alternate Protocol 2: Analysis of the role of arrestin-3 in MKK4-induced JNK activation in intact cells Basic Protocol 3: Characterization of the biphasic effect of arrestin-3 on ASK1/MKK7-stimulated JNK phosphorylation in cells.

Keywords: activation; arrestin; biphasic dependence; c-Jun N-terminal kinase (JNK); scaffold.

FIGURE 2.12.1

Arrestin-3-mediated JNK3α2 activation by MKK4/7. (A) A three-state model showing the scaffolding mechanism of the two-kinase signaling module. A, J, and M designate arrestin-3, JNK3α2, and upstream kinases MKK4/7, respectively. Kinases can exist in three states: (a) interacting in solution, (b) bound to the scaffold to form incomplete complexes containing a single kinase, and (c) simultaneously assembled by arrestin-3 to form a complete signaling complex. (B) Six affinity constants (k1 through k6) describe the indicated binding equilibria. Calculated concentrations of JM (dotted line, left y axis) and JAM (solid line, right y axis) complexes at different arrestin-3 concentrations (KinTek Explorer 3.0; all six K_d values were set at 5 μM). (C) Representative autoradiograms showing JNK3α2 phosphorylated by MKK4 (upper panel) or MKK7 (lower panel) at the indicated concentration of arrestin-3 (10-sec incubation). The optimal arrestin-3 concentrations are indicated (*, for MKK4; **, for MKK7). (D) The effect of arrestin-3 concentration on JNK3α2 phosphorylation by both MKK4 and MKK7 is biphasic. The bands from the gels were excised and the radioactivity measured in a Tri-Carb liquid scintillation counter to quantify the incorporation of [³²P]phosphate from [γ-³²P]ATP into JNK3α2. Data from (Zhan et al., 2013b).

Figure 2.12.2

Full activation of JNKs requires dual phosphorylation. For full activity, JNK family kinases must be phosphorylated at Thr (by MKK7) and Tyr (by MKK4). Reprinted with permission from (Zhan et al., 2013a).

Figure 2.12.3

Arrestin-3-mediated JNK1/2 activation by MKK4/7. In vitro phosphorylation of JNK1α1 and JNK2α2 in the presence of MKK4 (A) or MKK7 (B) yielded bell-shaped curves as functions of arrestin-3 concentration. Means ± SD of three independent experiments are shown. ANOVA analysis with arrestin-3 as main factor demonstrated significance of arrestin-3 concentration in the presence of MKK4 and MKK7 for both JNK1α1 and JNK2α2 (p < 0.001). * –p <0.001, ** –p <0.01, * –p <0.05 to maximal values (at 5 or 10 μM of arrestin-3, respectively) according to Bonferroni/Dunn post-hoc test with correction for multiple comparisons. Data from Kook et al. (2014).

Figure 2.12.4

Arrestin-3 promotes JNK1/2 activation induced by the expression of ASK1 in intact cells. (A) Representative immunoblot showing phosphorylation of endogenous JNK1/2 isoforms with or without arrestin-3 in COS-7 cells expressing varying amounts of ASK1. Upper p-JNK blot is the same as lower blot exposed for longer time to visualize p54 isoforms. (B) Quantification of phosphorylation of JNK p46H and p54 isoforms with or without arrestin-3. ANCOVA with arrestin-3 as factor and ASK1 concentration as co-variate showed significant effect of ASK1 concentration on the level of p46H and p54 phosphorylation (p < 0.0001). * –p < 0.05, ** –p < 0.01 to - Arr3, Student’s t-test for individual points. Data from Kook et al. (2014).

Figure 2.12.5

Arrestin-3 enhances MKK7-dependent phosphorylation of endogenous JNK1/2 in intact cells. (A) Representative immunoblot 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. ANCOVA with arrestin-3 as factor and MKK7 concentration as co-variate showed significant effect of MKK7 concentration of the level of p46H and p54 phosphorylation (p <0.0001). The presence of arrestin-3 significantly affected the level of p46H, but not p54, phosphorylation across MKK7 concentrations (F(1,38) = 8.592 p = 0.0057). * –p < 0.05 to -Arr3, Student’s t-test for individual points. Data from Kook et al. (2014).

Figure 2.12.6

Arrestin-3 enhances ASK1- and MKK7-dependent phosphorylation of endogenous JNK1/2 in intact cells. (A,C) Representative immunoblots 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,D) Quantification of the levels of p46H JNK isoform phosphorylation. One-way ANOVA analysis with arrestin-3 concentration as factor yielded significant effect of arrestin-3 on JNK phosphorylation both in the presence of ASK1 and MKK7 (p <0.0001). ** –p < 0.01, * –p <0.05 to the value at 0 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 Bonferroni/Dunn post-hoc test. Data from Kook et al. (2014).