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Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay
12:26

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Published on: May 3, 2018

Phosphorylation-dependent ubiquitination of cyclin D1 by the SCF(FBX4-alphaB crystallin) complex

Douglas I Lin1, Olena Barbash, K G Suresh Kumar

  • 1The Leonard and Madlyn Abramson Family Cancer Research Institute and Cancer Center, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

Molecular Cell
|November 4, 2006
PubMed

Insights

The Skp1-Cul1-F box (SCF) ubiquitin ligase, involving FBX4 and alphaB crystallin, targets phosphorylated cyclin D1 for degradation. Reduced expression of these proteins in cancers correlates with cyclin D1 overexpression and faster cell-cycle progression.

Area of Science:

  • Cell Biology
  • Molecular Oncology
  • Protein Degradation

Background:

  • Cyclin D1 proto-oncogene accumulation is regulated by phosphorylation-dependent proteolysis.
  • Threonine 286 phosphorylation triggers cyclin D1 ubiquitination by an E3 ligase, but its identity remained unknown.

Purpose of the Study:

  • To identify the E3 ubiquitin ligase responsible for degrading phosphorylated cyclin D1.
  • To investigate the role of FBX4 and alphaB crystallin in cyclin D1 regulation and cancer.

Main Methods:

  • Demonstrated recognition of Thr286-phosphorylated cyclin D1 by SCF(FBX4-alphaB crystallin) ubiquitin ligase.
  • Assessed the impact of FBX4 and alphaB crystallin overexpression and functional impairment on cyclin D1 ubiquitination and turnover.
  • Performed in vitro ubiquitination assays using purified SCF(FBX4-alphaB crystallin).
  • Analyzed FBX4 and alphaB crystallin expression in tumor cell lines and human cancers.

Main Results:

  • FBX4 and alphaB crystallin govern substrate specificity for Thr286-phosphorylated cyclin D1 recognition by the SCF ubiquitin ligase.
  • Overexpression of FBX4 and alphaB crystallin enhanced cyclin D1 ubiquitination and turnover.
  • Impaired SCF(FBX4-alphaB crystallin) function led to cyclin D1 overexpression and accelerated cell-cycle progression.
  • Reduced FBX4 and alphaB crystallin expression was observed in cancer cell lines and primary human cancers with high cyclin D1 levels.

Conclusions:

  • SCF(FBX4-alphaB crystallin) functions as an E3 ubiquitin ligase promoting the degradation of Thr286-phosphorylated cyclin D1.
  • The SCF(FBX4-alphaB crystallin) complex plays a critical role in regulating cyclin D1 levels and cell-cycle progression.
  • Downregulation of FBX4 and alphaB crystallin may contribute to cyclin D1 overexpression in tumorigenesis.

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