Nonvisual Arrestins Function as Simple Scaffolds Assembling the MKK4–JNK3α2 Signaling Complex

Abstract

Arrestins are a small family of proteins with four mammalian members that play key roles in the regulation of multiple GPCR-dependent and -independent signaling pathways. Although arrestins were reported to serve as scaffolds for MAP kinase cascades, promoting the activation of JNK3, ERK1/2, and p38, the molecular mechanisms involved were not elucidated and even the direct binding of arrestins with MAP kinases were never demonstrated. Here using purified proteins we show that both non-visual arrestins directly bind JNK3α2 and its upstream activator MKK4, and that the affinity of arrestin-3 for these kinases is higher than that of arrestin-2. Reconstitution of the MKK4-JNK3α2 signaling module from pure proteins in the presence of different arrestin-3 concentrations showed that arrestin-3 acts as a “true” scaffold, facilitating JNK3α2 phosphorylation by bringing the two kinases together. Both JNK3α2 phosphorylation by MKK4 and JNK3α2 activity towards its substrate ATF2 increase at low and then decrease at high arrestin-3 levels, yielding bell-shaped concentration dependence expected with true scaffolds that do not activate the upstream kinase or its substrate. Thus, direct binding of both kinases and true scaffolding is the molecular mechanism of arrestin-3 action on the MKK4-JNK3α2 signaling module.

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

“…At optimal concentrations arrestin-3 increases the phosphorylation of JNK1␣1 and JNK2␣2 by both MKK4 and MKK7, similar to the effect of purified arrestin-3 on JNK3␣2 phosphorylation by these upstream kinases (10,12). Endogenous arrestin-3 co-immunoprecipitates with endogenous JNK1/2.…”

“…Cell culture reagents and media were from Mediatech (Manassas, VA) or Invitrogen. All other chemicals were from sources recently described (10,12).…”

“…JNK phosphorylation was assayed by Western blotting using an antibody specific for phosphorylated JNKs to detect phospho (active) JNKs (10,12). Whole cell lysates were boiled for 5 min and centrifuged at 10,000 ϫ g for 10 min, and the supernatants were used for Western blotting.…”

“…The ability of overexpressed arrestin-3 5 to promote the activation of exogenous c-Jun N-terminal kinase 3 (JNK3) in cells was demonstrated more than a decade ago (6). Numerous subsequent studies showed that this function does not depend on arrestin-3 association with GPCRs (7)(8)(9)(10)(11)(12). In all these studies JNK3, which is predominantly expressed in neurons, heart, and testes (13), was either exogenously expressed in cultured cells that normally do not contain this isoform (6 -9, 11, 12) or used in purified form for in vitro reconstitution experiments (10,12).…”

“…Numerous subsequent studies showed that this function does not depend on arrestin-3 association with GPCRs (7)(8)(9)(10)(11)(12). In all these studies JNK3, which is predominantly expressed in neurons, heart, and testes (13), was either exogenously expressed in cultured cells that normally do not contain this isoform (6 -9, 11, 12) or used in purified form for in vitro reconstitution experiments (10,12). Although arrestin-3 is expressed in virtually every cell in the body (3,14,15), its ability to affect the activation of equally ubiquitous JNK1 and JNK2 isoforms (16 -18) has never been reported.…”