Vanderbilt Institute of Chemical Biology



Discovery at the VICB







Amplifying the Signal with Scaffolding


By: Carol A. Rouzer, VICB Communications
Published:  January 9, 2019


Studies of arrestin-3 interactions with JNK3 signaling cascade proteins reveal how arrestin-3-mediated scaffolding promotes JNK3 activation.


Scaffolding proteins play an important role in many signaling pathways. These proteins serve as a site of binding and organization for the multiple components involved in signal transduction, increasing the efficiency of their interaction and promoting signal propagation. However, if the interactions between signaling pathway components and the scaffold are too tight, the pathway may suffer from poor ability to amplify the signal and rapidly respond to changing conditions. The precise mechanisms that regulate the interactions between scaffolding proteins and signaling pathway components are poorly understood, leading Vanderbilt Institute of Chemical Biology members Vsevolod Gurevich, Carlos Lopez, and Tina Iverson, along with their labs to investigate how arrestin-3 acts as a scaffold in the activation of the JNK3 (c-Jun N-terminal kinase-3) signaling pathway [N. A. Perry et al., (2018) Proc. Natl. Acad. Sci. U.S.A., published December 27, DOI: 10.1073/pnas.1819230116].

The arrestins are proteins initially recognized for their role in modulating signal transduction mediated by G protein-couple receptors (GPCRs). However, growing evidence indicates that arrestins also play a role in cell signaling via mechanisms that are independent of GPCRs. Prior work had shown that arrestin-3 (also known as β-arrestin2 and hTHY-ARRX) binds to and promotes the activation of JNK3, a mitogen-activated protein kinase (MAPK) that modulates the apoptotic pathway in neuronal tissue. MAPK activation occurs through a three-step process in which a MAPK kinase kinase (MAP3K) phosphorylates and activates a MAPK kinase (MAP2K), which then phosphorylates and activates the MAPK (Figure 1). In the case of JNK3, the MAP3K is ASK1 (apoptosis signal-related kinase 1), and the role of MAP2K is carried out by two enzymes, MKK4 and MKK7, which phosphorylate JNK3 at Tyr-223 and Thr-221, respectively. Full JNK3 activation requires phosphorylation at both sites.



FIGURE 1. Outline of the MAPK activation pathway. Reproduced with permission from N. A. Perry et al., (2018) Proc. Natl. Acad. Sci. U.S.A., published December 27, DOI: 10.1073/pnas.1819230116. Copyright 2018, N. A. Perry, et al.




Arrestins are highly flexible proteins that adopt both active and inactive conformations. The active conformation is stabilized by interaction with GPCRs and other modulatory signaling molecules, including inositol hexakisphosphate (IP6) (Figure 2A). Alternatively, selected mutations of the protein promote adoption of the active conformation. Among these is deletion of portions of the C-terminal tail (Figure 2B), a structural element that stabilizes the inactive conformation. To simplify their studies, the investigators used a C-terminal deletion mutant of the protein [arrestin-3(1-393)], so that they did not have to include GPCRs or other modulators to ensure the active state. They confirmed that use of the deletion mutant did not lead to spurious results by repeating some of their work using full-length arrestin-3 activated by IP6.




FIGURE 2. (A) Diagrammatic representation of the crystal structure of arrestin-3 complexed with IP6, which results in an active conformation. The protein has formed a trimer, with each monomer shown in a different color (blue, gold, and gray). The IP6 molecules are shown in stick format. The circle highlights the finger loops believed to play a role in the activation process. (B) Diagrammatic representation of the N-domain of basal arrestin-3 (gray) overlaid on the N-domain of arrestin-3 as seen in the complex with IP6 (blue). The C-terminal tail of basal arrestin-3 is shown in pink. Figure reproduced under the Creative Commons Attribution 4.0 International License from Q. Chen et al. Nat. Commun., 2017, 8:1426 DOI: 10.1038/s41467-017-01218-8).



The research began with the expression of full-length arrestin-3 or the arrestin-3 T1A peptide (comprising amino acids 1-25) fused to maltose-binding protein (MBP). The fusion to MBP enabled the investigators to capture the proteins, along with any binding partners, using amylose resin. They employed this approach to show that both arrestin-3 and T1A bind to the unphosphorylated (inactive) forms of MKK4, MKK7, and JNK3 in vitro. For all three proteins, binding was stronger in the presence of ATP. MKK4 bound equally well to T1A and full-length arrestin-3, whereas MKK7 and JNK3 exhibited moderate and substantial reduction, respectively, in binding to T1A as compared to the intact protein.

The researchers next used microscale thermophoresis to directly measure the affinity of the phosphorylated and unphosphorylated proteins for arrestin-3(1-393) in the presence of physiological concentrations of MgCl2 and ATP. The results revealed that MKK7 bound to arrestin-3(1-393) with similar affinity regardless of its state of phosphorylation. In contrast, phosphorylation reduced the binding affinity of MKK4 by approximately 10-fold. Phosphorylation of JNK3 at a single site similarly reduced binding affinity for arrestin-3(1-393) by approximately 10-fold, whereas the doubly phosphorylated protein exhibited a much greater reduction in affinity.

To further explore the binding sites of JNK3 and its activating kinases to arrestin-3, the researchers examined the abilities of the unphosphorylated and phosphorylated proteins to interact with an array of peptides derived from the T1A sequence. The results showed that ASK1, MKK4, and MKK7, all bind to residues 1-15 of arrestin-3, with subtle differences in binding site resulting from phosphorylation. Phosphorylated JNK3 bound preferentially to residues 1-15, whereas the unphosphorylated protein interacted with moderate affinity with peptides across the broader T1A sequence. These findings strongly suggested that MKK4 and MKK7 share a common binding site on arrestin-3, although all components interact with varying affinities with the N-terminal T1A region.

Through an in vitro kinase assay, the researchers characterized the time course of JNK3 phosphorylation in the presence or absence of arrestin-3 by phosphorylated MKK4 and MKK7. They combined these data, along with the results from their binding assay and literature values to create and calibrate JARMv1.0, a reaction model of the interactions of MKK4, MKK7, and JNK3 with arrestin-3 (Figure 3). Bayesian parameter inference enabled them to obtain probability distributions for model parameters that were not available experimentally. The modeling results revealed 2.2-fold more phosphorylation of Thr-221 than Tyr-223 and an overall 1.2- to 1.8-fold increase in dual phosphorylation of JNK3 when arrestin-3 is present as compared to when it is absent. At early time points, arrestin-3 increased the formation of doubly phosphoryated JNK3 ~3-fold. An interesting finding was that the kinetic constant for phosphorylation of Thr-221 by MKK7 is two orders of magnitude higher than the corresponding constant for phosphorylation of Tyr-223 by MKK4. Thus, the step catalyzed by MKK4 is rate-limiting, and JNK3 phosphorylated at Thr-221 tends to accumulate early in the process.




FIGURE 3. Schematic diagram of all of the interactions included in the JARMv1.0 reaction model for JNK3 activation by MKK4 and MKK7. Reproduced with permission from N. A. Perry et al., (2018) Proc. Natl. Acad. Sci. U.S.A., published December 27, DOI: 10.1073/pnas.1819230116. Copyright 2018, N. A. Perry, et al.



A key question arises regarding the role of protein dissociation from the scaffold during signal transduction events. To explore this question, the investigators expressed either arrestin-3 or arrestin-3 fused to JNK3 in HEK293 cells in which the genes for both arrestin-2 and arrestin-3 had been knocked out. The investigators found that the JNK3 fused to arrestin-3 in cells expressing the fused protein became phosphorylated, but phosphorylation of free JNK3 was substantially reduced in comparison to JNK3 phosphorylation observed in cells expressing arrestin-3. These results suggest that the inability of active JNK3 to dissociate from arrestin-3 impedes phosphorylation and activation of the kinase.

In summary, the researchers combined their data to propose a "conveyor belt" model for arrestin-3-facilitated activation of JNK3 (Figure 4). They hypothesized that ASK1 binds initially, followed by either MKK4 or MKK7. As the two latter proteins are thought to share a binding site, they likely associate with arrestin-3 individually. This association leads to their phosphorylation by ASK1, resulting in activation. Next, inactive JNK3 binds and is phosphorylated by either MKK4 or MKK7, whichever is present. Then a second phosphorylation event by the other MKK enzyme occurs, and JNK3, now doubly phosphorylated, loses its affinity for arrestin-3 and is released. Thus, the complex assembled by the arrestin-3 scaffold can sequentially activate several JNK3 molecules, thereby resulting in signal amplification. Clearly, further work is needed to test the hypotheses underlying this model and to better define the timing of binding and exchange of MKK4 and MKK7. However, the findings lay important groundwork for a better understanding of how arrestin-3-mediated scaffolding leads to activation of JNK3, and the mechanism might be applicable to the function of other scaffolds facilitating MAPK signaling in all eukaryotic cells.




FIGURE 4. Proposed conveyor belt model for arrestin-3-mediated JNK3 activation. (A) ASK1 binds to arrestin-3. (B) Either MKK4 or MKK7 binds to the ASK1-arrestin-3 complex. (C) Bound MKK4/MKK7 is phosphorylated and activated by ASK1, and JNK3 binds. (D) Activated MKK4 and MKK7 phosphorylate JNK3, which dissociates, allowing a new, unphosphorylated molecule to bind. Reproduced with permission from N. A. Perry et al., (2018) Proc. Natl. Acad. Sci. U.S.A., published December 27, DOI: 10.1073/pnas.1819230116. Copyright 2018, N. A. Perry, et al.


View Proc. Natl. Acad. Sci. U.S.A., article: Arrestin-3 scaffolding of the JNK3 cascade suggests a mechanism for signal amplification







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