Analyzing the degradation of PERIOD protein by the ubiquitin-proteasome pathway in cultured Drosophila cells

Hyuk Wan Ko, Isaac Edery

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

Time-of-day specific changes in the levels of key clock proteins are critical for the normal progression of circadian pacemakers. Evidence indicates a major role for the ubiquitin-proteasome pathway (UPP) in the temporal control of clock protein stability. A conserved feature of animal clocks is that PERIOD (PER) proteins undergo daily rhythms in abundance. The stability of PER proteins is regulated by differential phosphorylation, whereby hyperphosphorylated isoforms are selectively degraded by the UPP. The use of transformed stable cell lines has been instrumental in advancing our understanding of the mechanisms underlying the intersection of the UPP and clock protein metabolism. This article describes several standard methodologies used to analyze the UPP-mediated degradation of Drosophila PER (dPER) expressed in cultured Drosophila cells (Ko et al., 2002). Although this article focuses on dPER as a case study, general issues are discussed that should have broad application to other cell culture-based systems and clock proteins. For example, we discuss (i) advantages/disadvantages of cultured cells, (ii) types of expression vectors and "peptide tags" for recombinant protein production and surveillance, and (iii) standard approaches to determine whether a protein of interest is modified by ubiquitin and degraded by the proteasome. Prior to the discussion on methodologies, the article provides a brief overview of diverse strategies by which clock proteins in a variety of systems are regulated by the UPP.

Original languageEnglish (US)
Article number18
Pages (from-to)394-408
Number of pages15
JournalMethods in enzymology
Volume393
DOIs
StatePublished - Jan 1 2005

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Proteasome Endopeptidase Complex
Ubiquitin
Proteolysis
Drosophila
Cultured Cells
Clocks
Degradation
Proteins
Protein Stability
Transformed Cell Line
Cells
Recombinant Proteins
Pacemakers
Phosphorylation
Protein Isoforms
Cell Culture Techniques
Cell culture
Metabolism
Peptides
Animals

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology

Cite this

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Analyzing the degradation of PERIOD protein by the ubiquitin-proteasome pathway in cultured Drosophila cells. / Ko, Hyuk Wan; Edery, Isaac.

In: Methods in enzymology, Vol. 393, 18, 01.01.2005, p. 394-408.

Research output: Contribution to journalArticle

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