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Mechanical Strain on Osteoblasts Activates Autophosphorylation of Focal Adhesion Kinase and Proline-rich Tyrosine Kinase 2 Tyrosine Sites Involved in ERK Activation
- Source :
- Journal of Biological Chemistry. 279:30588-30599
- Publication Year :
- 2004
- Publisher :
- Elsevier BV, 2004.
-
Abstract
- The mechanisms involved in the mechanical loading-induced increase in bone formation remain unclear. In this study, we showed that cyclic strain (CS) (10 min, 1% stretch at 0.25 Hz) stimulated the proliferation of overnight serum-starved ROS 17/2.8 osteoblast-like cells plated on type I collagen-coated silicone membranes. This increase was blocked by MEK inhibitor PD-98059. Signaling events were then assessed 0 min, 30 min, and 4 h after one CS period with Western blotting and coimmunoprecipitation. CS rapidly and time-dependently promoted phosphorylation of both ERK2 at Tyr-187 and focal adhesion kinase (FAK) at Tyr-397 and Tyr-925, leading to the activation of the Ras/Raf/MEK pathway. Cell transfection with FAK mutated at Tyr-397 completely blocked ERK2 Tyr-187 phosphorylation. Quantitative immunofluorescence analysis of phosphotyrosine residues showed an increase in focal adhesion plaque number and size in strained cells. CS also induced both Src-Tyr-418 phosphorylation and Src to FAK association. Treatment with the selective Src family kinase inhibitor pyrazolopyrimidine 2 did not prevent CS-induced FAK-Tyr-397 phosphorylation suggesting a Src-independent activation of FAK. CS also activated proline-rich tyrosine kinase 2 (PYK2), a tyrosine kinase highly homologous to FAK, at the 402 phosphorylation site and promoted its association to FAK in a time-dependent manner. Mutation of PYK2 at the Tyr-402 site prevented the ERK2 phosphorylation only at 4 h. Intra and extracellular calcium chelators prevented PYK2 activation only at 4 h. In summary, our data showed that osteoblast response to mitogenic CS was mediated by MEK pathway activation. The latter was induced by ERK2 phosphorylation under the control of FAK and PYK2 phosphorylation orchestrated in a time-dependent manner.
- Subjects :
- Male
Time Factors
Blotting, Western
PTK2
Biology
Transfection
Models, Biological
Biochemistry
Cell Line
Mice
Cell Adhesion
Animals
Enzyme Inhibitors
Phosphorylation
Rats, Wistar
Molecular Biology
Chelating Agents
Flavonoids
Binding Sites
Osteoblasts
PTK2B
MAP kinase kinase kinase
Autophosphorylation
Cell Biology
Protein-Tyrosine Kinases
Focal Adhesion Kinase 2
Precipitin Tests
Molecular biology
Rats
Enzyme Activation
Pyrimidines
Microscopy, Fluorescence
Focal Adhesion Kinase 1
Focal Adhesion Protein-Tyrosine Kinases
Tyrosine
Calcium
Cyclin-dependent kinase 9
Stress, Mechanical
Mitogen-Activated Protein Kinases
Cell Division
Plasmids
Signal Transduction
Proto-oncogene tyrosine-protein kinase Src
Subjects
Details
- ISSN :
- 00219258
- Volume :
- 279
- Database :
- OpenAIRE
- Journal :
- Journal of Biological Chemistry
- Accession number :
- edsair.doi.dedup.....68612d8b4ef48a8117c974edf0f54723