RRC ID 28586
Author Takeda K, Takata T, Kawai Y, Ishigaki Y, Kajinami K.
Title Chk1-mediated phosphorylation of receptor-associated late transducer at serine 250 increases its stability by stimulating its interaction with 14-3-3.
Journal Genes Cells
Abstract 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.
Volume 18(5)
Pages 369-86
Published 2013-5-1
DOI 10.1111/gtc.12043
PMID 23432726
MeSH 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
IF 1.655
Times Cited 4
DNA material His-Xpress-RALT (RDB012225) Flag-Chk1 (1-365) (RDB012229) His-Xpress-RALT S250A mutant (RDB012226) SNAP-RALT (RDB012227) SNAP-RALT S250A mutant (RDB012228) Flag-Chk1 (1-365) K38M mutant (RDB012230)