JNK3 Enzyme Binding to Arrestin-3 Differentially Affects the Recruitment of Upstream Mitogen-activated Protein (MAP) Kinase Kinases

Abstract

Non‐visual arrestins‐2 and‐3 interact with numerous GPCRs and dozens of non‐receptors partners, such as MAP kinases. Arrestin‐3 promotes JNK3 activation, scaffolding ASK1‐MKK4‐JNK3 cascade. Full activation of JNKs, unlike other MAP kinases, requires two upstream kinases, MKK4 and MKK7, and each preferentially phosphorylating a distinct site, tyrosine (MKK4) and theronine (MKK7). It remains unclear whether arrestin‐3 can promote the activation of JNK3 by MKK7. Using phospho‐Tyr and phospho‐Thr antibodies we found that arrestin‐3 promotes the phosphorylation of both Tyr and Thr within the T‐P‐Y motif in COS‐7 cells. We also found that arrestin‐3 directly binds MKK7 comparably to MKK4. The binding of JNK3 enhanced the association of arrestin‐3 with MKK4, while reduced the binding of MKK7. With pure proteins we show that arrestin‐3 scaffolds MKK7‐JNK3 module at higher concentration than MKK4‐JNK3 module. Arresint‐3 also facilitates JNK3 activation in COS‐7 cells over‐expressing either MKK4 or MKK7. This is the first evidence that arrestin‐3 promotes JNK3 phosphorylation by MKK7. The data suggest that JNK3 binding has opposite effect on arrestin‐3 interactions with MKK4 and MKK7. Support: NIH GM081756, GM077561 and EY011500 (VVG) and GM059802 and Welch Foundation Grant F‐1390 (KND).

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.…”