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Activation of pp60c-src depending on cell density in PC12h cells
S Kobayashi1, N Okumura, T Nakamoto
1Division of Protein Metabolism, Institute for Protein Research, Osaka University, 3-2 Yamada-Oka, Suita, Osaka 565, Japan.
The Journal of Biological Chemistry
|June 27, 1997
Summary
Cell density increases Src family tyrosine kinase (Src) activity, impacting cell interactions. This activation occurs without altering Src
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Src family tyrosine kinases regulate cell-cell and cell-matrix interactions.
- p130(cas) is a known substrate for Src family kinases.
- pp60(c-src) kinase activity is modulated by phosphorylation at its C-terminal negative regulatory site.
Purpose of the Study:
- To investigate the effect of cell density on Src family tyrosine kinase activity.
- To explore the mechanism by which cell density influences pp60(c-src) activity.
Main Methods:
- Measurement of tyrosine phosphorylation levels of intracellular proteins, including p130(cas).
- Assay of pp60(c-src) kinase activity in cell cultures at varying densities.
- Subcellular fractionation to determine the localization of pp60(c-src).
Main Results:
- Increased cell density enhanced tyrosine phosphorylation of p130(cas) and elevated pp60(c-src) activity across multiple cell lines (PC12h, PC12, Balb/c 3T3, Swiss 3T3, Hela).
- The tyrosine phosphorylation level of the pp60(c-src) negative regulatory site remained unchanged with increasing cell density.
- In high-density cultures, pp60(c-src) translocated from detergent-soluble to detergent-insoluble cellular fractions.
Conclusions:
- Cell-cell interactions appear to induce pp60(c-src) activation.
- Activation of pp60(c-src) by cell density occurs independently of changes in its regulatory site phosphorylation.
- Translocation of pp60(c-src) to detergent-insoluble fractions may be a key mechanism in its activation by cell-cell contact.