Chk1-mediated phosphorylation of receptor-associated late transducer at serine 250 increases its stability by stimulating its interaction with 14-3-3.
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.
Mesh Terms:
14-3-3 Proteins, Amino Acid Motifs, Animals, COS Cells, Cercopithecus aethiops, Checkpoint Kinase 1, Epidermal Growth Factor, ErbB Receptors, Humans, Intracellular Signaling Peptides and Proteins, Mutant Proteins, Mutation, Phosphorylation, Phosphoserine, Protein Binding, Protein Isoforms, Protein Kinases, Protein Stability, Protein Transport, Rats, Substrate Specificity
14-3-3 Proteins, Amino Acid Motifs, Animals, COS Cells, Cercopithecus aethiops, Checkpoint Kinase 1, Epidermal Growth Factor, ErbB Receptors, Humans, Intracellular Signaling Peptides and Proteins, Mutant Proteins, Mutation, Phosphorylation, Phosphoserine, Protein Binding, Protein Isoforms, Protein Kinases, Protein Stability, Protein Transport, Rats, Substrate Specificity
Genes Cells
Date: May. 01, 2013
PubMed ID: 23432726
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