PerspectiveSignal Transduction

An Arresting Start for MAPK

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Science  24 Nov 2000:
Vol. 290, Issue 5496, pp. 1515-1518
DOI: 10.1126/science.290.5496.1515

Protein kinases are ubiquitous enzymes that are able to modulate the activities of other proteins by adding phosphate groups to their tyrosine, serine, or threonine amino acids (phosphorylation). Mitogen-activated protein kinases (MAPKs), which are activated by many different signals, belong to a large family of serine/threonine protein kinases that are conserved in organisms as diverse as yeast and humans. MAPKs deliver extracellular signals from activated receptors to various cellular compartments, notably the nucleus, where they direct the execution of appropriate genetic programs. A unique feature of MAPKs is that they themselves can be activated by addition of phosphate groups to both their tyrosine and threonine amino acids (dual phosphorylation) after stimulation of a receptor by growth factors, mitogens, hormones, cytokines, or environmental stresses. MAPKs operate in modules composed of three protein kinases that phosphorylate and activate each other sequentially: MAP kinase kinase kinase (MKKK) activates MAP kinase kinase (MKK), which then activates MAP kinase. These kinase modules have been duplicated with slight variations, allowing cells to instigate multiple biological responses through a set of MAP kinase-wiring networks.

The coexistence of conserved protein kinase modules within the same cellular compartment, however, poses an enormous challenge for the cell because protein kinases are rather promiscuous enzymes. How does the cell solve the crucial problems of substrate specificity and the prevention of inappropriate cross talk between signaling pathways? How are these highly conserved signaling circuits “insulated” from nonspecific interactions with other molecules? Cells seem to have adopted two solutions. The specificity and fidelity of kinases is ensured by the existence of specific docking sites on kinases and their protein substrates (1, 2). Insulation and signal efficiency are provided by scaffold proteins that assemble the components of a given MAPK module into a single signaling complex (3). Attempts to identify scaffold proteins have been frustrated by the fact that they are not enzymes and seem to have emerged as nonconserved evolutionary “bricolage.” The first MAPK scaffold protein identified was Ste5 in budding yeast. An astonishing and intriguing new example of a mammalian scaffold protein is presented by McDonald et al. (4) on page 1574 of this issue. The investigators provide evidence that this new scaffold protein, β-arrestin 2, brings together components of the MAPK module, resulting in activation of c-Jun amino-terminal kinase-3 (JNK3) in response to activation of G protein-coupled receptors (GPCRs).

The β-arrestin 2 scaffold protein is not homologous to c-Jun amino-terminal kinase interacting protein (JIP), a member of the mammalian JNK scaffold protein family (3). This discovery is intriguing because for years it has been known that arrestins stop signals from growth factors, hormones, or environmental stressors by uncoupling the activated GPCR from its G protein signaling molecule (5, 6). But how can arrestins be both “Stop” molecules and signal activators? What is the evidence that arrestins are scaffold proteins for the MAPK module that activates JNK3 through the activation of ASK1 and MKK4?

With a yeast two-hybrid screening assay, McDonald et al. identified a direct interaction between β-arrestin 2 and the carboxyl-terminal portion of JNK MAPK family members, in particular with the p54 splice variants of JNK. Immunoprecipitation of β-arrestin 1 and 2 from extracts of cultured cells expressing epitope-tagged JNK1, JNK2, and JNK3 isoforms established that only β-arrestin 2 coimmunoprecipitated with the JNK3 isoform. The authors then explored the functional consequence of this interaction on JNK3 activation by measuring phosphorylation of c-Jun, a typical transcription factor targeted by the JNK signaling pathway. JNK3 phosphorylated c-Jun only when it was coexpressed in cultured cells with both β-arrestin 2 and ASK1 (an upstream MKKK activator of the pathway). Because ASK1 cannot directly activate JNK3, this result suggested that MKK4 or MKK7 is present in the complex and that β-arrestin 2 may serve as a scaffold protein, holding together all of the components of the JNK cascade. ASK1 binds directly to the amino-terminal region of β-arrestin 2 at a site distinct from JNK3 binding. Whereas MKK4 or MKK7 displays a very weak interaction with β-arrestin 2, expression of ASK1 or JNK3 is sufficient to promote MKK4 recruitment to the complex. This finding suggests that MKK4 interacts indirectly with β-arrestin 2 through ASK1 or JNK3.

According to this evidence, the designation of β-arrestin 2 as a specific scaffold protein (2) is convincing. But still unanswered is the question of whether this β-arrestin 2-JNK3 complex is involved in GPCR-mediated activation of JNK3. The investigators addressed this question with cultured cells that expressed both JNK3 and a GPCR called angiotensin II receptor type IA. When the ligand for this receptor, angiotensin II, was added to the cells, there was a very weak and slow activation of JNK3. Remarkably, after introduction of β-arrestin 2 into these cells, angiotensin II initiated a time-dependent and robust activation of JNK3. Because no ASK1 was coexpressed in these cells, this finding implied that an endogenous MKKK was co-opted into the complex. This was nicely confirmed by the finding that the three components of the MAPK module—ASK1, MKK4, and JNK3—were found in β-arrestin 2 immunoprecipitates; however, the amounts detected were not affected by addition of angiotensin II. This result suggests that the β-arrestin 2-JNK module is preformed in unstimulated cells and raises the question of how GPCR is involved in JNK3 activation.

A key feature of arrestins is their capacity to translocate from the cytosol to the vicinity of the phosphorylated GPCR in the plasma membrane, after activation of the receptor by an agonist (a molecule that resembles the receptor's natural ligand). Addition of angiotensin II to cells initiated a redistribution of intracellular β-arrestin 2 and JNK3 pools. A pool of β-arrestin 2 was found associated with endosomal vesicles, and part of the JNK3 pool, previously sequestered in the cytoplasm, moved to the nucleus. This nuclear JNK3 pool was, however, inactive. This intriguing finding hints that activation of the JNK3 module proceeds at the plasma membrane by recruitment of the β-arrestin 2-JNK3 complex to the activated GPCR (see the figure). Activated JNK3 is predicted to rapidly dissociate from the scaffolding (“activating”) complex and to phosphorylate cytoplasmic and nuclear protein substrates. The inactive nuclear pool of JNK3 is reminiscent of the stimulation of p42/p44 MAPKs (also called extracellular signal-regulated kinases, or ERKs) by growth factors. In response to such stimulation, ERKs rapidly dissociate from the cytoplasmic activating complex. They then translocate to the nucleus where they accumulate and become progressively inactivated by nuclear MAPK phosphatases (which remove their phosphate groups).

β-arrestins court MAP kinases.

Binding of a G protein-coupled receptor (GPCR) to its ligand activates a heterotrimeric G protein (Gαbg). The activated Gα subunit interacts with an effector (for example, adenylyl cyclase, phosphodiesterase, or an ion channel) to deliver signal 1. The ligand-occupied receptor is then phosphorylated (P) by one of the GPCR-kinases, providing a binding site for a β-arrestin scaffolding protein. β-arrestin then moves from the cytosol to the phosphorylated GPCR in the plasma membrane, thereby leading to G protein uncoupling and attenuation of signal 1. The complex between β-arrestin 2 and the JNK MAPK module (ASK1 activates MKK4, which activates JNK3) is constitutively preformed in the cytoplasm, and this entire complex translocates to the membrane-bound GPCR after ligand binding. β-arrestin promotes internalization of the entire complex through clathrin-coated pits (9), leading to activation of the MAPK module and generation of signal 2. JNK3 is released from the β-arrestin 2 scaffold, translocates to the nucleus, and switches on transcription of target genes. JNK3 is then inactivated in turn by the nuclear MAPK phosphatases (MKPs) and is recycled back to the “activating” scaffold complex in the cytoplasm.

The model that emerges from the McDonald study is one in which β-arrestin 2 is a dual signaling switch. Its translocation to the GPCR in the plasma membrane initiates the JNK3 signaling pathway, an action that simultaneously stops the G protein-mediated signal (see the figure). This appealing model immediately raises several questions: How is the arrestin-JNK module complex activated? Is the tethering of arrestin to the phosphorylated GPCR the initiating event? Is this multicomponent complex consisting of GPCR, β-arrestin 2, and the MAPK module formed in cells in vivo? Can we mimic JNK3 activation simply by inducing nonspecifically the translocation of β-arrestin 2 to the plasma membrane? How general is this signaling mechanism, and how important is it likely to be in cells that have not been artificially transfected with the components of this signaling pathway? Do all arrestin isoforms primarily serve as scaffold molecules for a subset of MAPK modules?

An emerging body of evidence lends strong support to the generality of this model. Several GPCRs, when activated by their appropriate ligands and engaged by β-arrestin 1 during endocytosis, have been found to be associated with the p42/p44 MAPK (ERK) module. The proteinase-activated receptor 2 (PAR2) complex contains the internalized receptor, β-arrestin 1, Raf-1, and p42/p44 MAPK (7). When activated under these conditions, p42/p44 MAPK does not translocate to the nucleus and, as expected, PAR2 does not transmit mitogenic signals inducing the cell to divide (8). Engineering mutations in PAR2 that interfere with its interaction with β-arrestin prevents this receptor from becoming internalized by the cell and instead triggers activation of p42/p44 MAPK through a separate route, resulting in translocation of p42/p44 MAPK to the nucleus and mitogenic stimulation of the cell (7).

Several findings now converge to enlarge the part played by arrestins in signal termination. Receptor signaling and desensitization of the receptor to the activating signal are in reality two intimately coupled processes. Molecules viewed as “signal terminators” in one pathway may in fact be “activators” in another. With the McDonald et al. work, we are witnessing an important new beginning for arrestins as activators of MAPK signaling.

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