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Oligopeptide Competition Assay for Phosphorylation Site Determination
09:16

Oligopeptide Competition Assay for Phosphorylation Site Determination

Published on: May 18, 2017

Dynamic changes in C-Raf phosphorylation and 14-3-3 protein binding in response to growth factor stimulation:

Mirko Hekman1, Stefan Wiese, Renate Metz

  • 1Institute for Medical Radiation and Cell Research, University of Wuerzburg, 97078 Wuerzburg.

The Journal of Biological Chemistry
|December 23, 2003
PubMed

Insights

Researchers developed phosphospecific antibodies to study Raf protein phosphorylation, revealing distinct 14-3-3 binding affinities and the sequence of C-Raf activation events following nerve growth factor stimulation.

Area of Science:

  • Cellular signaling pathways
  • Protein phosphorylation
  • Signal transduction

Background:

  • Phosphorylation is critical for Raf kinase activation and 14-3-3 protein association.
  • Specific serine residues (S259/S621 in C-Raf, S364/S728 in B-Raf) are implicated in 14-3-3 binding.

Purpose of the Study:

  • To develop and characterize phosphospecific antibodies for studying Raf phosphorylation at 14-3-3 binding sites.
  • To investigate the functional consequences of Raf phosphorylation on 14-3-3 binding and kinase activity.
  • To elucidate the temporal sequence of C-Raf phosphorylation events during activation.

Main Methods:

  • Development of phosphospecific monoclonal (6B4 for pS621) and polyclonal antibodies.
  • Western blot analysis of C-Raf and B-Raf.
  • Kinase-dead mutant analysis.
  • Direct protein binding and competition assays.
  • Time-course analysis of endogenous C-Raf phosphorylation in response to nerve growth factor (NGF) using phosphospecific antibodies.

Main Results:

  • The 6B4 antibody specifically recognizes native C-Raf, not B-Raf.
  • Serine 259 phosphorylation is interdependent with Serine 621 phosphorylation in C-Raf.
  • The pS621 epitope represents a high-affinity 14-3-3 binding site, while pS259 mediates lower affinity binding.
  • NGF stimulation induced rapid Ser-621 phosphorylation preceding Ser-259 phosphorylation and kinase activity.
  • Phosphorylation of Tyr-340/341 occurred after Ser-621 phosphorylation and coincided with kinase activation.

Conclusions:

  • Substantial differences exist between C-Raf 14-3-3 binding epitopes pS259 and pS621.
  • The study provides the first visualization of the sequential phosphorylation events essential for C-Raf activation in mammalian cells upon growth factor stimulation.

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