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Updated: Aug 8, 2026

Identification of Kinase-substrate Pairs Using High Throughput Screening
11:13

Identification of Kinase-substrate Pairs Using High Throughput Screening

Published on: August 29, 2015

Novel tyrosine kinase substrates from Rous sarcoma virus-transformed cells are present in the membrane skeleton

J R Glenney1, L Zokas

  • 1Department of Biochemistry, Markey Cancer Center, University of Kentucky College of Medicine, Lexington 40536-0084.

The Journal of Cell Biology
|June 1, 1989
PubMed

Insights

Researchers developed new monoclonal antibodies to identify phosphotyrosine (PY)-containing proteins. These antibodies revealed altered protein distributions in Rous sarcoma virus-transformed cells, highlighting changes in the cytoskeleton and cell adhesion upon tyrosine kinase activation.

Area of Science:

  • Cell Biology
  • Biochemistry
  • Molecular Oncology

Background:

  • Phosphotyrosine (PY) modification is crucial, regulated by oncogene products and growth factor receptors.
  • Previous work established monoclonal antibodies against phosphotyrosine.

Purpose of the Study:

  • To generate monoclonal antibodies targeting specific phosphotyrosine-containing proteins.
  • To analyze the subcellular localization and cytoskeletal association of these proteins in normal and Rous sarcoma virus-transformed chick embryo fibroblasts (RSV-CEFs).

Main Methods:

  • Affinity chromatography using an anti-phosphotyrosine antibody (py20) to isolate PY-proteins.
  • Immunization of mice with isolated PY-proteins to generate monoclonal antibodies against specific substrates.
  • Immunofluorescence microscopy and detergent extraction to determine subcellular localization and cytoskeletal association.

Main Results:

  • Fifteen monoclonal antibodies were generated against phosphotyrosine-containing proteins of 215, 76, 60, and 22 kD.
  • Antibodies to 215- and 76-kD proteins stained focal contacts; anti-22-kD antibodies showed punctate marginal staining.
  • Transformed cells exhibited altered staining patterns for these proteins.
  • Significant fractions of 76-kD (50%) and 22-kD ( >90%) proteins were associated with the cytoskeleton in detergent-extracted cells.

Conclusions:

  • This study presents a novel method for identifying membrane skeletal proteins in fibroblasts.
  • The findings demonstrate significant alterations in protein localization and cytoskeletal association upon transformation by activated tyrosine kinases.

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