title: Chk1-mediated phosphorylation of receptor-associated late transducer at serine 250 increases its stability by stimulating its interaction with 14-3-3.
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.
authors: Department of Cardiology, Kanazawa Medical University, 1-1 Daigaku, Uchinada, Ishikawa, 920-0293, Japan.
authors: Kenji
authors: K
authors: Takeda
authors: Takanobu
authors: T
authors: Takata
authors: Yasuyuki
authors: Y
authors: Kawai
authors: Yasuhito
authors: Y
authors: Ishigaki
authors: Kouji
authors: K
authors: Kajinami
mesh_terms: D048948:14-3-3 Proteins / Q000378:metabolism*; D020816:Amino Acid Motifs; D000818:Animals; D019556:COS Cells; D000071877:Checkpoint Kinase 1; D002522:Chlorocebus aethiops; D004815:Epidermal Growth Factor / Q000494:pharmacology; D066246:ErbB Receptors / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D010766:Phosphorylation / Q000187:drug effects; D010768:Phosphoserine / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D020033:Protein Isoforms / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism*; D055550:Protein Stability / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D051381:Rats; D013379:Substrate Specificity / Q000187:drug effects
chemical_list: D048948:14-3-3 Proteins; D047908:Intracellular Signaling Peptides and Proteins; D050505:Mutant Proteins; D020033:Protein Isoforms; D010768:Phosphoserine; D004815:Epidermal Growth Factor; D011494:Protein Kinases; D066246:ErbB Receptors; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; C000605871:Chek1 protein, rat