Identification of arrestin‐3‐specific residues necessary for JNK3 activation

Arrestins regulate GPCR signaling and scaffold mitogen‐activated protein kinase (MAPK) cascades. Both arrestin‐2 and ‐3 bind JNK3 and upstream kinases MKK4 and ASK1, but only arrestin‐3 promotes JNK3 activation. To identify the residues responsible, we constructed a series of arrestin‐2/3 chimeras and arrestin‐3 mutants with selected residues substituted with their arrestin‐2 homologues. The chimera with the N‐domain of arrestin‐2 and the C‐domain of arrestin‐3, but not arrestin‐2‐based chimeras with smaller arrestin‐3 elements, facilitated JNK3 phosphorylation, suggesting that several elements are necessary. Arrestin‐3 residues Ser264, Leu278, Ser280, Val343, His350, Asp 351, His352, and Ile353 participate JNK3 activation, and that elimination of Val343 was sufficient to block it. However, introduction of arrestin‐3‐specific residues into arrestin‐2 did not yield arrestin‐3‐like ability to activate JNK3. JNK3 co‐immunoprecipitation with the mutants did not correlate with their ability to activate it. Thus, the strength of JNK3 binding is not the decisive factor. Multiple residues in different elements of the C‐domain of arrestin‐3 are necessary for productive scaffolding of ASK1‐MKK4‐JNK3 cascade. JNK3 activation via arrestin‐3 likely requires correct arrangement of MAP kinases, rather than a single docking motif for JNK3. NIH grants GM77561 and GM81756 (VVG)