C-terminal Recognition by 14-3-3 Proteins for Surface Expression of Membrane Receptors

Abstract

Diverse functions of 14-3-3 proteins are directly coupled to their ability to interact with targeted peptide substrates. RSX(pS/pT)XP and RX⌽X(pS/pT)XP are two canonical consensus binding motifs for 14-3-3 proteins representing the two common binding modes, modes I and II, between 14-3-3 and internal peptides. Using a genetic selection, we have screened a random peptide library and identified a group of C-terminal motifs, termed SWTY, capable of overriding an endoplasmic reticulum localization signal and redirecting membrane proteins to cell surface. Here we report that the C-terminal SWTY motif, although different from mode I and II consensus, binds tightly to 14-3-3 proteins with a dissociation constant (K D ) of 0.17 M, comparable with that of internal canonical binding peptides. We show that all residues but proline in -SWTX-COOH are compatible for the interaction and surface expression. Because SWTY-like sequences have been found in native proteins, these results support a broad significance of 14-3-3 interaction with protein C termini. The C-terminal binding consensus, mode III, represents an expansion of the repertoire of 14-3-3-targeted sequences.The 14-3-3 proteins were initially identified from brain for their abundance and acidic properties (1). They have been found in all eukaryotes and recognized for their diverse functional roles in biological processes including metabolism, cell signaling, intracellular trafficking, stress responses, cell cycle progression, and malignant transformation (see reviews in Refs. 2-4). Multiple isoforms of 14-3-3 are commonly found in a given species. Saccharomyces cerevisiae has two 14-3-3 isoforms, encoded by BMH1 and BMH2. Deletion of either does not cause growth defects, but the double deletion of both is lethal in most genetic backgrounds (5-7). Essential to 14-3-3 function, in most cases, is the ability to bind a short peptide substrate only upon phosphorylation. As the first protein module identified with preferential affinity to phosphorylated Ser/Thr substrates, 14-3-3 binding provides an inducible mechanism of translating enzymatic activity to protein-protein interaction, a molecular feature with broad significance in regulation.Structural studies and examination of the interactive sequences in human proto-oncogene Raf-1 and from random phosphopeptide libraries have established two binding consensuses, RSX(pS/pT)XP and RX⌽X(pS/pT)XP, which are also known as mode I and II recognition (8, 9). The two binding modes have provided molecular insights into the recognition of 14-3-3 binding to a large number of interacting proteins. The vast majority of binding sites identified so far lies internally and matches either mode I or II binding consensus. In addition to phosphorylation-mediated induction of 14-3-3 binding, there is evidence for additional mechanisms of interaction. For example, a tight association between 14-3-3 and multiple regions of plant plasma membrane H ϩ -ATPase is induced by additional binding of a fungal toxin, fusicoccin, which leads to the forma...

Membrane Proteins as 14-3-3 Clients in Functional Regulation and Intracellular Transport

“…The construct consisted of a truncated Kir2.1 channel, an RXR motif (from Kir6.2) inserted at the COOH terminus of Kir2.1, and an artificial COOH terminus generated by using a peptide library. With the use of this approach, mode III 14-3-3 binding motifs (located at the extreme COOH terminus) were shown to override an RXR motif located upstream (FIGURE 1B) (12,80). In agreement with other work (55,56,68), the serine or threonine residue at the penultimate position was found to be absolutely required for efficient surface expression (80).…”

“…It should be noted that the proline (P) located at position ϩ2 of the phosphorylation site occurs in only about one-half of known 14-3-3 binding motifs (35). In addition to these two canonical binding motifs, 14-3-3 can bind to the extreme COOH terminus of several proteins, recently defined as a mode III binding site (12,80). The motif is R-X-XpS/pT-X-COOH, with serine or threonine at position Ϫ2 being absolutely required, whereas an arginine (R) residue at position Ϫ5 increases binding affinity (68,80).…”

Phosphorylation-dependent C-terminal Binding of 14-3-3 Proteins Promotes Cell Surface Expression of HIV Co-receptor GPR15

“…4C). This was consistent with our earlier finding that the minimal component of C-terminal mode III 14-3-3 binding motif is RXX(S/T)X-COOH (22 seem to be the most critical components of 14-3-3 binding activity within the C-terminal 9 amino acids of GPR15. However, the higher surface expression of S356A compared with that of S359A in spite that both mutants completely lack 14-3-3 binding suggests that Arg 356 may be also a component of the ER localization signal.…”

An Obligatory Heterodimer of 14-3-3β and 14-3-3ϵ Is Required for Aldosterone Regulation of the Epithelial Sodium Channel

“…Initial studies to determine 14-3-3 binding sites revealed that their optimal target sequences could be characterized as either mode-1 (RSXpSXP; where pS represents phosphoserine) or mode-2 (RXXXpSXP) (19,37). Using a genetic screen, Coblitz et al (38) identified the C-terminal sequence SWpTX as a "mode-3" 14-3-3 binding motif (38). This collection of target sites is described as canonical, but variations on these motifs have also been documented to mediate 14-3-3 protein interactions with substrates (39).…”