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In vivo and in vitro Studies of Adaptor-clathrin Interaction
17:14

In vivo and in vitro Studies of Adaptor-clathrin Interaction

Published on: January 26, 2011

The ankyrin repeat-containing adaptor protein Tvl-1 is a novel substrate and regulator of Raf-1

J H Lin1, A Makris, C McMahon

  • 1Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.

The Journal of Biological Chemistry
|May 18, 1999
PubMed

Insights

Tvl-1, an ankyrin repeat protein, interacts with and regulates the serine-threonine kinase Raf-1. This protein acts as both a target and a potentiator of Raf-1 activation, impacting cellular signaling pathways.

Area of Science:

  • Molecular Biology
  • Cell Signaling
  • Protein Interactions

Background:

  • Tvl-1 is an ankyrin repeat protein identified through a yeast two-hybrid screen.
  • It was found to interact with the serine-threonine kinase Raf-1.
  • Tvl-1 is expressed in thymus, lung, and testes.

Purpose of the Study:

  • To characterize the interaction between Tvl-1 and Raf-1.
  • To investigate the functional role of Tvl-1 in Raf-1 signaling.
  • To determine the localization and regulatory properties of Tvl-1.

Main Methods:

  • Yeast two-hybrid screening
  • Co-immunoprecipitation assays (transient transfection and endogenous proteins)
  • Immunofluorescence microscopy
  • In vitro and in vivo phosphorylation assays
  • Functional assays in insect and mammalian cells

Main Results:

  • Tvl-1 directly interacts with Raf-1 via its ankyrin repeat domain.
  • Tvl-1 also undergoes homodimerization through the same domain.
  • Tvl-1 is phosphorylated by activated Raf-1.
  • Tvl-1 potentiates Raf-1 activation by Src, Ras, and EGF.
  • Tvl-1 is localized in both the cytoplasm and nucleus.

Conclusions:

  • Tvl-1 functions as both a target and a regulator of Raf-1.
  • The ankyrin repeat domain is crucial for Tvl-1's interaction with Raf-1 and homodimerization.
  • Tvl-1 plays a significant role in modulating Raf-1 signaling pathways.
  • The human homologue of Tvl-1 is located on chromosome 19p12.

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