Significance of 14-3-3 Self-Dimerization for Phosphorylation-dependent Target Binding

Abstract

14-3-3 proteins via binding serine/threonine-phosphorylated proteins regulate diverse intracellular processes in all eukaryotic organisms. Here, we examine the role of 14-3-3 self-dimerization in target binding, and in the susceptibility of 14-3-3 to undergo phosphorylation. Using a phospho-specific antibody developed against a degenerated mode-1 14-3-3 binding motif (RSxpSxP), we demonstrate that most of the 14-3-3-associated proteins in COS-7 cells are phosphorylated on sites that react with this antibody. The binding of these phosphoproteins depends on 14-3-3 dimerization, inasmuch as proteins associated in vivo with a monomeric 14-3-3 form are not recognized by the phospho-specific antibody. The role of 14-3-3 dimerization in the phosphorylation-dependent target binding is further exemplified with two well-defined 14-3-3 targets, Raf and DAF-16. Raf and DAF-16 can bind both monomeric and dimeric 14-3-3; however, whereas phosphorylation of specific Raf and DAF-16 sites is required for binding to dimeric 14-3-3, binding to monomeric 14-3-3 forms is entirely independent of Raf and DAF-16 phosphorylation. We also find that dimerization diminishes 14-3-3 susceptibility to phosphorylation. These findings establish a significant role of 14-3-3 dimerization in its ability to bind targets in a phosphorylation-dependent manner and point to a mechanism in which 14-3-3 phosphorylation and dimerization counterregulate each other

Dimerization Is Essential for 14-3-3ζ Stability and Function in Vivo

“…Nevertheless, in Zhou et al (34) and other studies (25,26,49), monomeric species appear abundant enough to permit functional analyses. However, these results were obtained by transfecting cultured cells with constructs encoding the monomeric proteins (25,26,34,49). Expression from transfected constructs is relatively high and acute, approximating our own experiments with the Gal80 ts system (Fig.…”

“…In vitro and in cultured cells, certain mutant dimerizationimpaired 14-3-3s have been shown to bind clients (25,26), often with similar affinities as their dimeric counterparts, but with some exceptions, they appear unable to support normal target activity (21,25,27,28). In addition, phosphorylation of Ser 58 on vertebrate 14-3-3 renders it unable to dimerize in transfected cultured cells (29), suggesting that monomerization may regulate some 14-3-3 functions.…”

Class IIa histone deacetylases: regulating the regulators

“…In this context, 14-3-3-dependent nuclear export of class IIa HDACs may serve as a supporting mechanism to ensure maximal activation of their target genes . Interestingly, the fact that 14-3-3 proteins are also regulated by phosphorylation may provide an additional level of regulation of class IIa HDAC nucleocytoplasmic shuttling (Fu et al, 2000;Tzivion and Avruch, 2002;Ellis et al, 2003;Shen et al, 2003).…”

Identification of Novel In Vivo Raf-1 Phosphorylation Sites Mediating Positive Feedback Raf-1 Regulation by Extracellular Signal-regulated Kinase

“…The MEK inhibitor U0126 was from Promega (Madison, WI). Phosphospecific antibodies for Raf-1 pS289, pS296, and pS301 were produced as described previously (Shen et al, 2003a) and screened for specificity as described in the text. …”