Chk1-mediated phosphorylation of receptor-associated late transducer at serine 250 increases its stability by stimulating its interaction with 14-3-3

Genes Cells. 2013 May;18(5):369-86. doi: 10.1111/gtc.12043. Epub 2013 Feb 22.


Receptor-associated late transducer (RALT) acts as a negative feedback inhibitor of ErbB receptor signaling via physical interaction with ErbB. Although RALT contains a 14-3-3 binding motif (247-RSHSGP-252), little is known about the molecular basis and significance of binding to 14-3-3. Here, we report that 14-3-3 interacts with RALT in H9c2 and COS-7 cells in a Ser-250 phosphorylation-dependent manner. An in vitro kinase assay showed that RALT is a substrate for checkpoint kinase 1 (Chk1). Interaction between ectopically expressed RALT and endogenous 14-3-3 was partially suppressed by pretreatment with the Chk1 inhibitor, UCN-01. In addition, expression of constitutively active Chk1 (Chk11-365 ) resulted in increased phosphorylation of the RALT 14-3-3 binding motif and enhanced the interaction between RALT and 14-3-3θ. Furthermore, fluorescence microscopy revealed that rapid trafficking of RALT to endosome-like vesicle structures was decelerated by coexpression of Chk11-365 , whereas this coexpression had no significant impact on trafficking of the RALT S250A mutant. Finally, a cycloheximide chase assay indicated that coexpression of Chk11-365 decelerated the degradation of ectopically expressed RALT, but not that of the S250A mutant. Collectively, these results suggest that Chk1 plays a role in regulating RALT protein stability by facilitating the interaction between 14-3-3 and RALT.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 14-3-3 Proteins / metabolism*
  • Amino Acid Motifs
  • Animals
  • COS Cells
  • Checkpoint Kinase 1
  • Chlorocebus aethiops
  • Epidermal Growth Factor / pharmacology
  • ErbB Receptors / metabolism
  • Humans
  • Intracellular Signaling Peptides and Proteins / metabolism*
  • Mutant Proteins / metabolism
  • Mutation / genetics
  • Phosphorylation / drug effects
  • Phosphoserine / metabolism*
  • Protein Binding / drug effects
  • Protein Isoforms / metabolism
  • Protein Kinases / metabolism*
  • Protein Stability / drug effects
  • Protein Transport / drug effects
  • Rats
  • Substrate Specificity / drug effects


  • 14-3-3 Proteins
  • Intracellular Signaling Peptides and Proteins
  • Mutant Proteins
  • Protein Isoforms
  • Phosphoserine
  • Epidermal Growth Factor
  • Protein Kinases
  • ErbB Receptors
  • CHEK1 protein, human
  • Checkpoint Kinase 1
  • Chek1 protein, rat