Related Experiment Videos
Distinct mechanisms mediate the initial and sustained phases of integrin-mediated activation of the
1Department of Pharmacology and The Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, North Carolina 27599-7365, USA.
Abstract:
Integrin-mediated adhesion to the extracellular matrix activates the canonical mitogen-activated protein kinase cascade, although the exact mechanism is not fully resolved. We show that integrin-mediated activation of Raf-1, an upstream regulator of mitogen-activated protein kinase, occurs in two phases. Efficient early activation of Raf required Raf-Ras interaction but was not affected by protein kinase C (PKC) inhibitors, while a lower, sustained level of activity was independent of Raf-Ras interaction but was reduced by PKC inhibitors. The combination of PKC inhibition and lack of Ras binding completely blocked integrin-mediated Raf activity. The activity of a membrane-bound Raf mutant that is deficient in Ras binding (Raf-R89L-CAAX) was also regulated by adhesion. Raf-R89L-CAAX activity was low in nonadherent cells, was rapidly stimulated to wild-type levels by cell adhesion, and remained at nearly maximal levels longer than wild-type activity. The activation of wild-type and mutant Raf proteins was ablated by cytochalasin D, demonstrating that cytoskeletal organization is required for activation of Raf, even when targeted to the membrane. These data suggest distinct initial and sustained phases of integrin-mediated Raf activation that require Raf membrane localization and possibly PKC activity, respectively, and that integrin-mediated adhesion may regulate a cytoskeleton-associated factor(s) responsible for Raf activation.
Insights
Integrin adhesion activates Raf-1 kinase in two distinct phases. Early activation needs Ras binding, while sustained activity involves protein kinase C (PKC) and cytoskeletal organization for full integrin-mediated signaling.
Area of Science:
- Cell Biology
- Molecular Signaling
- Extracellular Matrix Interactions
Background:
- Integrin-mediated cell adhesion is crucial for cellular processes.
- The canonical mitogen-activated protein kinase (MAPK) cascade is activated by integrins.
- The precise mechanism of integrin-mediated Raf-1 activation remains incompletely understood.
Purpose of the Study:
- To elucidate the distinct phases and molecular requirements of integrin-mediated Raf-1 activation.
- To investigate the roles of Ras interaction, protein kinase C (PKC), and cytoskeletal organization in Raf-1 activation.
Main Methods:
- Utilized integrin-mediated cell adhesion models.
- Employed biochemical assays to assess Raf-1 kinase activity.
- Investigated the impact of Ras binding inhibition and PKC inhibitors.
- Analyzed the function of a membrane-bound Raf mutant (Raf-R89L-CAAX).
- Assessed the effect of cytochalasin D on Raf activation.
Main Results:
- Integrin-mediated Raf-1 activation occurs in two phases: early (Ras-dependent) and sustained (PKC-dependent).
- A membrane-bound Raf mutant (Raf-R89L-CAAX) showed adhesion-regulated activity, independent of Ras binding.
- Cytoskeletal organization, disrupted by cytochalasin D, is essential for Raf activation, even for membrane-targeted Raf.
- Complete inhibition of integrin-mediated Raf activity required both PKC inhibition and blocked Ras binding.
Conclusions:
- Integrin-mediated Raf-1 activation involves distinct early and sustained phases.
- Sustained Raf activation appears to depend on protein kinase C (PKC) activity.
- Cytoskeletal organization is a critical component for integrin-induced Raf activation.
- Cell adhesion may regulate cytoskeleton-associated factors that control Raf activation.