14-3-3γ binds regulator of G protein signaling 14 (RGS14) at distinct sites to inhibit the RGS14:Gαi–AlF4− signaling complex and RGS14 nuclear localization

Abstract

Regulator of G protein signaling 14 (RGS14) is a multifunctional brain scaffolding protein that integrates G protein and Ras/ERK signaling pathways. It is also a nucleocytoplasmic shuttling protein. RGS14 binds active Gα via its RGS domain, Raf and active H-Ras-GTP via its R1 Ras-binding domain (RBD), and inactive Gα via its G protein regulatory (GPR) domain. RGS14 suppresses long-term potentiation (LTP) in the CA2 region of the hippocampus, thereby regulating hippocampally based learning and memory. The 14-3-3 family of proteins is necessary for hippocampal LTP and associative learning and memory. Here, we show direct interaction between RGS14 and 14-3-3γ at two distinct sties, one phosphorylation-independent and the other phosphorylation-dependent at Ser-218 that is markedly potentiated by signaling downstream of active H-Ras. Using bioluminescence resonance energy transfer (BRET), we show that the pSer-218-dependent RGS14/14-3-3γ interaction inhibits active Gα-AlF binding to the RGS domain of RGS14 but has no effect on active H-Ras and inactive Gα-GDP binding to RGS14. By contrast, the phosphorylation-independent binding of 14-3-3 has no effect on RGS14/Gα interactions but, instead, inhibits (directly or indirectly) RGS14 nuclear import and nucleocytoplasmic shuttling. Together, our findings describe a novel mechanism of negative regulation of RGS14 functions, specifically interactions with active Gα and nuclear import, while leaving the function of other RGS14 domains intact. Ongoing studies will further elucidate the physiological function of this interaction between RGS14 and 14-3-3γ, providing insight into the functions of both RGS14 and 14-3-3 in their roles in modulating synaptic plasticity in the hippocampus.

Human genetic variants disrupt RGS14 nuclear shuttling and regulation of LTP in hippocampal neurons

“…We found that cytoplasmic RGS14 translocated to the nucleus within 40 minutes and was maximally nuclear by two hours ( Figure 3B), suggesting that cytoplasmic pools of RGS14 are constantly targeted for nuclear import/export with an equilibrium favoring the cytosol. These results are consistent with our recently published report demonstrating that nuclear import of RGS14 is regulated by 14-3-3g in neurons (36).…”

“…We and others have reported that recombinant RGS14 shuttles into and out of the nucleus in cultured cell lines (25)(26)(27) and neurons (36). As noted, RGS14 contains a functional nuclear localization sequence (NLS) between the RGS domain and the first Ras-binding domain (R1), and a functional NES embedded within the GPR motif of RGS14 (25,26) ( Figure 3A).…”

“…We identified the GPR motif as being critical for RGS14 functions in hippocampal neurons. Numerous reports have shown that RGS14 is a dynamically regulated protein which exhibits a nuclear-cytoplasm-membrane shuttling phenotype favoring cytoplasmic localization in resting cells (25)(26)(27)31,36). The C-terminus of RGS14 harbors the GPR motif with an embedded NES that governs the intracellular dynamics of RGS14, and dictates its availability to interact with signaling partners necessary to carry out its many functions (41)(42)(43)(44).…”

Aberrant localization of signaling proteins in skin cancer: Implications for treatment

“…79,80 In regard to 14-3-3 protein's effect on subcellular localization, they have been shown to promote the cytoplasmic retention of various proteins. [81][82][83] This was originally demonstrated when 14-3-3 was shown to cloister proapoptotic Bcl-2-associated death promoter (BAD) and Bcl-2-associated X protein (BAX) into the cytoplasm, leading to their inactivation and the evasion of apoptosis. 84,85 Accumulation of 14-3-3-BAD and 14-3-3-BAX in the cytoplasm and the ensuing inhibition of apoptosis is often utilized by cancer cells to avoid apoptotic cell death.…”

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“…Protein RGS14 is implicated in suppression of LTP in the CA2 region of the hippocampus, thereby regulating hippocampalbased learning and memory. These proteins were shown to directly interact in cells, and one of the two distinct interaction sties was shown to be phosphorylation independent while the other was phosphorylation-dependent, contributing to negative regulation of RGS14 functions [52].…”