Dual engagement of 14-3-3 proteins controls signal relay from ASK2 to the ASK1 signalosome.

Faithful and efficient transmission of biological signals through mitogen-activated protein kinase (MAPK) pathways requires engagement of highly regulated cellular machinery in response to diverse environmental cues. Here, we report a novel mechanism controlling signal relay between two MAP3Ks, apoptosis signal-regulating kinase (ASK) 1 and ASK2. We show that ASK2 specifically interacts with 14-3-3 proteins through phosphorylated S964. Although a 14-3-3-binding defective mutant of ASK1 (S967A) has no effect on the ASK2/14-3-3 interaction, both overexpression of the analogous ASK2 (S964A) mutant and knockdown of ASK2 dramatically reduced the amount of ASK1 complexed with 14-3-3. These data suggest a dominant role of ASK2 in 14-3-3 control of ASK1 function. Indeed, ASK2 S964A-induced dissociation of 14-3-3 from ASK1 correlated with enhanced phosphorylation of ASK1 at T838 and increased c-Jun N-terminal kinase phosphorylation, the two biological readouts of ASK1 activation. Our results suggest a model in which upstream signals couple ASK2 S964 phosphorylation to the ASK1 signalosome through dual engagement of 14-3-3.

RGS14 is a bifunctional regulator of Galphai/o activity that exists in multiple populations in brain.

Members of the regulators of G protein signaling (RGS) family modulate Galpha-directed signals as a result of the GTPase-activating protein (GAP) activity of their conserved RGS domain. In addition to its RGS domain, RGS14 contains a Rap binding domain (RBD) and a GoLoco motif. To define the cellular and biochemical properties of RGS14 we utilized two different affinity purified antisera that specifically recognize recombinant and native RGS14. In brain, we observed two RGS14-like immunoreactive bands of distinct size (60 kDa and 55 kDa). Both forms are present in brain cytosol and in two, biochemically distinct, membrane subpopulations: one detergent-extractable and the other detergent-insensitive. Recombinant RGS14 binds specifically to activated Galphai/o, but not Galphaq/11, Galpha12/13, or Galphas in brain membranes. In reconstitution studies, we found that RGS14 is a non-selective GAP for Galphai1 and Galphao and that full-length RGS14 is an approximately 10-fold more potent stimulator of Galpha GTPase activity than the RGS domain alone. In contrast, neither full-length RGS14 nor the isolated RBD domain is a GAP for Rap1. RGS14 is also a highly selective guanine nucleotide dissociation inhibitor (GDI) for Galphai but not Galphao, and this activity is restricted to the C-terminus containing the GoLoco domain. These findings highlight previously unknown biochemical properties of RGS14 in brain, and provide one of the first examples of an RGS protein that is a bifunctional regulator of Galpha actions.