Binding and Transcriptional Regulation by 14-3-3 (Bmh) Proteins Requires Residues Outside of the Canonical Motif

Abstract

Evolutionarily conserved 14-3-3 proteins have important functions as dimers in numerous cellular signaling processes, including regulation of transcription. Yeast 14-3-3 proteins, known as Bmh, inhibit a post-DNA binding step in transcription activation by Adr1, a glucose-regulated transcription factor, by binding to its regulatory domain, residues 226 to 240. The domain was originally defined by regulatory mutations, ADR1 c alleles that alter activator-dependent gene expression. Here, we report that ADR1 c alleles and other mutations in the regulatory domain impair Bmh binding and abolish Bmh-dependent regulation both directly and indirectly. The indirect effect is caused by mutations that inhibit phosphorylation of Ser230 and thus inhibit Bmh binding, which requires phosphorylated Ser230. However, several mutations inhibit Bmh binding without inhibiting phosphorylation and thus define residues that provide important interaction sites between Adr1 and Bmh. Our proposed model of the Adr1 regulatory domain bound to Bmh suggests that residues Ser238 and Tyr239 could provide cross-dimer contacts to stabilize the complex and that this might explain the failure of a dimerization-deficient Bmh mutant to bind Adr1 and to inhibit its activity. A bioinformatics analysis of Bmh-interacting proteins suggests that residues outside the canonical 14-3-3 motif might be a general property of Bmh target proteins and might help explain the ability of 14-3-3 to distinguish target and nontarget proteins. Bmh binding to the Adr1 regulatory domain, and its failure to bind when mutations are present, explains at a molecular level the transcriptional phenotype of ADR1 c mutants.

General Amino Acid Control and 14-3-3 Proteins Bmh1/2 Are Required for Nitrogen Catabolite Repression-Sensitive Regulation of Gln3 and Gat1 Localization

“…The Bmh1/2 proteins are involved in a wide variety of control pathways by virtue of binding to and positively or negatively regulating phosphorylated regulatory molecules ranging from Adr1, at the level of transcription (Parua et al 2010;Parua and Young 2014), to their participation in Mks1-dependent negative regulation of nuclear Rtg1/3 localization and retrograde transcription (Liu et al 2003(Liu et al , 2005.…”

Snf1-Dependent Transcription Confers Glucose-Induced Decay upon the mRNA Product

“…The chimeric activator was expressed under the control of the constitutive ADH1 promoter. We hypothesized that the chimeric activator would be glucose and SNF1 independent because it lacks the major regulatory region of Adr1, the 14-3-3 (also known as Bmh in yeast) binding domain (40,65), and is highly expressed from the ADH1 promoter. To test the glucose and SNF1 dependence of gene activation by Adr1-EV, we measured the expression of three ADR1-dependent genes (ADH2, ACS1, and POX1) in a strain lacking ADR1 and SNF1 (CKY27) and grown under either repressing or derepressing conditions.…”

Arbuscular Mycorrhizal Fungal 14-3-3 Proteins Are Involved in Arbuscule Formation and Responses to Abiotic Stresses During AM Symbiosis

“…Moreover, 14-3-3 proteins form homo- or hetero-dimers by two subunits harboring the independent ligand-binding channels. Until now, it is extensively studied that these proteins generally serve as adapters, chaperones, activators, or repressors in the regulation of signal transduction pathways by reorganization of specific phosphoserine/phosphothreonine-inclusive binding motifs phosphorylated by protein kinase A ( Smith et al, 1998 ; van Heusden, 2009 ; Smith et al, 2011 ; Parua and Young, 2014 ). Additionally, 14-3-3 proteins also play important roles in the pseudohyphal growth of Saccharomyces cerevisiae and the pathogenic fungal infection, such as Ustilago maydis ( Gancedo, 2001 ; Rispail et al, 2009 ; Ballou et al, 2013 ; Liu et al, 2015 ).…”