14-3-3 proteins interact with a hybrid prenyl-phosphorylation motif to inhibit G proteins

Summary Signaling through G proteins normally involves conformational switching between GTP- and GDP-bound states. Several Rho GTPases are also regulated by RhoGDI binding and sequestering in the cytosol. Rnd proteins are atypical constitutively GTP-bound Rho proteins, whose regulation remains elusive. Here, we report a high-affinity 14-3-3-binding site at the C terminus of Rnd3 consisting of both the Cys241-farnesyl moiety and a Rho-associated coiled coil containing protein kinase (ROCK)-dependent Ser240 phosphorylation site. 14-3-3 binding to Rnd3 also involves phosphorylation of Ser218 by ROCK and/or Ser210 by protein kinase C (PKC). The crystal structure of a phosphorylated, farnesylated Rnd3 peptide with 14-3-3 reveals a hydrophobic groove in 14-3-3 proteins accommodating the farnesyl moiety. Functionally, 14-3-3 inhibits Rnd3-induced cell rounding by translocating it from the plasma membrane to the cytosol. Rnd1, Rnd2, and geranylgeranylated Rap1A interact similarly with 14-3-3. In contrast to the canonical GTP/GDP switch that regulates most Ras superfamily members, our results reveal an unprecedented mechanism for G protein inhibition by 14-3-3 proteins.
Graphical Abstract
Highlights • Rnd small G proteins bind to 14-3-3 via C-terminal phosphorylation and lipid groups • This interaction negatively regulates Rnd proteins by inducing membrane extraction • Structural analysis shows 14-3-3 binding to a hybrid lipid-phosphorylation motif • This motif identifies new 14-3-3-binding proteins, including Rap1A
14-3-3 inhibit Rnd proteins by extracting them from their site of action on membranes, which is regulated by Rnd phosphorylation.