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Activated R-ras, Rac1, PI 3-kinase and PKCepsilon can each restore cell spreading inhibited by isolated integrin
A L Berrier1, A M Mastrangelo, J Downward
1Center for Cell Biology and Cancer Research, Albany Medical College, Albany, New York 12208, USA.
The Journal of Cell Biology
|January 3, 2001
Summary
Cell spreading, crucial for cell adhesion and migration, requires integrin beta cytoplasmic domains. Signaling proteins R-Ras, PI 3-kinase, Rac1, and PKCepsilon all depend on these domains to regulate cell spreading.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Cell attachment to extracellular matrix proteins initiates cell spreading, enhancing adhesion and enabling migration, survival, and proliferation.
- Integrins, particularly their beta cytoplasmic domains, are essential for cell spreading by linking to the actin cytoskeleton and activating signaling pathways.
- While R-Ras, PI 3-kinase, PKCepsilon, and Rac1 are known regulators of cell spreading, their dependence on integrin beta cytoplasmic domains remains unclear.
Purpose of the Study:
- To investigate the role of integrin beta cytoplasmic domains in regulating cell spreading.
- To determine if activated signaling proteins (R-Ras, PI 3-kinase, PKCepsilon, Rac1) can restore cell spreading inhibited by dominant-negative integrin mutants.
- To elucidate the signaling pathways and dependencies involved in integrin-mediated cell spreading.
Main Methods:
- Inhibition of cell spreading using a dominant-negative integrin inhibitor (tac-beta1).
- Restoration of cell spreading by co-expressing constitutively active forms of signaling proteins: V38R-Ras, p110alpha-CAAX, myr-PKCepsilon, and L61Rac1.
- Assessing cell spreading by measuring cell area and utilizing GTP-dependent assays, kinase domain requirements, and dominant-negative Rac1 mutants.
Main Results:
- Activated R-Ras, PI 3-kinase, PKCepsilon, and Rac1 restored cell spreading inhibited by tac-beta1.
- Restoration by R-Ras and Rac1 was GTP-dependent; PKCepsilon required an intact kinase domain.
- All tested signaling proteins required intact integrin beta cytoplasmic domains for rescue, with R-Ras rescue dependent on PI 3-kinase activity.
- Rac1 was found to be downstream of PI 3-kinase and PKCepsilon in this integrin-dependent pathway.
Conclusions:
- Integrin beta cytoplasmic domains are essential for R-Ras, PI 3-kinase, Rac1, and PKCepsilon to regulate cell spreading.
- Rac1 acts downstream of PI 3-kinase and PKCepsilon, highlighting a specific signaling cascade within integrin-mediated cell spreading.
- These findings clarify the molecular mechanisms by which integrins control cell spreading and adhesion-dependent cellular processes.