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Identification of Protein Interacting Partners Using Tandem Affinity Purification
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Published on: February 25, 2012

PTPIP51 in protein interactions: regulation and in situ interacting partners

Alexander Brobeil1, Manuel Bobrich, Claudia Tag

  • 1Institute of Anatomy and Cell Biology, Justus-Liebig-University, 35392, Giessen, Germany. alexander.brobeil@anatomie.med.uni-giessen.de

Cell Biochemistry and Biophysics
|May 1, 2012
PubMed

Insights

Tyrosine phosphorylation of PTPIP51 by c-Src regulates its interaction with 14-3-3β and Raf-1. This phosphorylation impacts interactions with MAPK pathway partners, but does not trigger apoptosis.

Area of Science:

  • Cellular signaling
  • Protein-protein interactions
  • Kinase regulation

Background:

  • PTPIP51 is a protein involved in cellular signaling pathways.
  • The interaction of PTPIP51 with binding partners is crucial for its function.
  • The role of PTPIP51 tyrosine phosphorylation in regulating these interactions is largely unknown.

Purpose of the Study:

  • To investigate how tyrosine phosphorylation of PTPIP51 affects its binding to 14-3-3β and other partners.
  • To elucidate the role of c-Src in phosphorylating PTPIP51 at tyrosine 176.
  • To determine the impact of PTPIP51 phosphorylation on its interactions with mitogen-activated protein kinase (MAPK) pathway modulators.

Main Methods:

  • PTPIP51 phosphorylation was modulated in human keratinocytes by inhibiting phosphatase activity and using PP2 to block Src kinases.
  • Interactions of PTPIP51 with 14-3-3β, Raf-1, PTP1B, and c-Src were quantified.
  • Interactions with protein kinase A (PKA) and diacylglycerol kinase alpha (DAGKα) were also assessed.
  • Immunostaining was used to investigate the functional implications in apoptotic processes.

Main Results:

  • Increased PTPIP51 tyrosine phosphorylation significantly reduced interactions with 14-3-3β and Raf-1.
  • PTPIP51 interactions with PKA and DAGKα were also found to be regulated by tyrosine phosphorylation.
  • Enhanced phosphorylation did not lead to increased apoptosis in treated HaCaT cells.

Conclusions:

  • PTPIP51 interaction with 14-3-3β and Raf-1 is dependent on tyrosine phosphorylation in vivo.
  • PTPIP51 exhibits tyrosine-dependent interactions with DAGKα and PKA.
  • The disruption of PTPIP51-14-3-3 interaction is insufficient to induce apoptosis.

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