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Updated: Mar 10, 2026

Characterize Disease-related Mutants of RAF Family Kinases by Using a Set of Practical and Feasible Methods
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Cell Density-Dependent Increase in Tyrosine-Monophosphorylated ERK2 in MDCK Cells Expressing Active Ras or Raf

Noriyuki Kawabata1, Michiyuki Matsuda1

  • 1Department of Pathology and Biology of Diseases, Graduate School of Medicine, Kyoto University, Kyoto, Japan.

Plos One
|December 10, 2016
PubMed

Insights

Cell density impacts extracellular signal-regulated kinase (ERK) activity. High cell density suppresses ERK, even with active Ras or BRaf, suggesting phosphatases regulate this pathway in cancer.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Oncology

Background:

  • Extracellular signal-regulated kinase (ERK) is crucial for transmitting growth signals.
  • The role of active Ras and BRaf in increasing ERK activity in cancer is debated.
  • Cell density is a potential factor influencing ERK pathway activation.

Purpose of the Study:

  • To investigate the impact of cell density on ERK activity in cells expressing active Ras or BRaf.
  • To elucidate the mechanisms underlying cell density-dependent regulation of ERK signaling.

Main Methods:

  • Utilized Madin-Darby canine kidney (MDCK) cells expressing active HRas, NRas, KRas, or BRaf.
  • Employed a Förster resonance energy transfer (FRET)-based ERK biosensor to measure ERK activity.
  • Performed Western blotting with Phos-tag gel to analyze ERK phosphorylation states.
  • Investigated the effect of calyculin A, a protein phosphatase inhibitor.

Main Results:

  • Active Ras or BRaf expression increased ERK activity at low cell densities.
  • ERK activity was significantly suppressed at high cell densities, regardless of Ras/BRaf expression.
  • High cell density led to decreased active ERK and increased inactive ERK, as shown by Western blotting.
  • Calyculin A treatment reduced tyrosine-monophosphorylated ERK, indicating phosphatase involvement.

Conclusions:

  • Cell density-dependent regulation of ERK activity is evident in cells with oncogenic Ras/BRaf.
  • PPP-subfamily protein phosphatases likely mediate ERK dephosphorylation at high cell densities.
  • This finding offers insights into the complex regulation of the ERK pathway in cancer.

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