Functional analysis of C-TAK1 substrate binding and identification of PKP2 as a new C-TAK1 substrate

EMBO J. 2003 Sep 1;22(17):4431-42. doi: 10.1093/emboj/cdg426.

Abstract

Cdc25C-associated kinase 1 (C-TAK1) has been implicated in cell cycle regulation and Ras signaling through its interactions with two putative substrates, the Cdc25C phosphatase and the MAPK scaffold KSR1. Here, we identify sequence motifs required for stable C-TAK1 association and substrate phosphorylation. Using a mutational approach to disrupt binding of C-TAK1 to KSR1 and Cdc25C, we demonstrate that C-TAK1 contributes to the regulation of these proteins in vivo through the generation of 14-3-3-binding sites. KSR1 proteins defective in C-TAK1 binding had severely reduced phosphorylation at the 14-3-3-binding site in vivo, were constitutively localized to the plasma membrane and had increased biological activity. Disruption of the Cdc25C-C-TAK1 interaction resulted in reduced 14-3-3-binding site phosphorylation and nuclear accumulation of Cdc25C in interphase cells. Finally, utilizing the acquired C-TAK1 binding and substrate phosphorylation data, we identify plakophilin 2 (PKP2) as a novel C-TAK1 substrate. Phosphorylation of PKP2 by C-TAK1 also generates a 14-3-3-binding site that influences PKP2 localization. These findings underscore the importance of C-TAK1 as a regulator of 14-3-3 binding and protein localization.

MeSH terms

  • 14-3-3 Proteins
  • 3T3 Cells
  • Amino Acid Sequence
  • Animals
  • Binding Sites / genetics
  • COS Cells
  • Cell Cycle Proteins / genetics
  • Cell Cycle Proteins / metabolism
  • Female
  • Humans
  • In Vitro Techniques
  • Mice
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Oocytes / metabolism
  • Phosphorylation
  • Plakophilins
  • Protein Kinases / genetics
  • Protein Kinases / metabolism
  • Protein-Serine-Threonine Kinases / genetics
  • Protein-Serine-Threonine Kinases / metabolism*
  • Proteins / genetics
  • Proteins / metabolism*
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Substrate Specificity
  • Transfection
  • Tyrosine 3-Monooxygenase / metabolism
  • Xenopus
  • cdc25 Phosphatases / genetics
  • cdc25 Phosphatases / metabolism

Substances

  • 14-3-3 Proteins
  • Cell Cycle Proteins
  • PKP2 protein, human
  • Pkp2 protein, mouse
  • Plakophilins
  • Proteins
  • Recombinant Proteins
  • Tyrosine 3-Monooxygenase
  • Protein Kinases
  • KSR-1 protein kinase
  • MARK3 protein, human
  • Protein-Serine-Threonine Kinases
  • CDC25C protein, human
  • Cdc25c protein, mouse
  • cdc25 Phosphatases