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Focal adhesion protein-tyrosine kinase phosphorylated in response to cell attachment to fibronectin
S K Hanks1, M B Calalb, M C Harper
1Department of Cell Biology, Vanderbilt University School of Medicine, Nashville, TN 37232.
Abstract:
A homology-based cDNA cloning approach was used to identify a widely expressed protein-tyrosine kinase designated as "focal adhesion kinase" (FadK). The entire mouse FadK amino acid sequence was deduced from cDNA clones, revealing a large (119-kDa) non-membrane-spanning protein-tyrosine kinase that lacks Src-homology SH2 and SH3 domains. Immunostaining of BALB/c 3T3 fibroblasts revealed that FadK is concentrated in focal adhesions. FadK is phosphorylated on tyrosine in growing cultures of BALB/c 3T3 cells but contains little or no phosphotyrosine in cells detached by trypsinization. The tyrosine-phosphorylated state is regained within minutes when the cells are replated onto fibronectin. Activation of FadK may be an important early step in intracellular signal transduction pathways triggered in response to cell interactions with the extracellular matrix.
Insights
Focal adhesion kinase (FadK), a protein-tyrosine kinase, is concentrated in cell focal adhesions. Its tyrosine phosphorylation is regulated by cell adhesion to the extracellular matrix, suggesting a role in cell signaling.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Protein-tyrosine kinases play crucial roles in cellular signaling pathways.
- Focal adhesions are key sites for cell-extracellular matrix interactions.
- Understanding the regulation of these kinases is vital for deciphering cell behavior.
Purpose of the Study:
- To identify and characterize a novel protein-tyrosine kinase involved in cell adhesion.
- To investigate the localization and regulation of this kinase in response to extracellular matrix interactions.
Main Methods:
- Homology-based cDNA cloning was employed to isolate the gene for the novel kinase.
- Amino acid sequencing was performed using deduced cDNA clones.
- Immunostaining was used to determine the subcellular localization of the protein.
- Analysis of protein phosphorylation status was conducted on cells under different adhesion conditions.
Main Results:
- A widely expressed protein-tyrosine kinase, named focal adhesion kinase (FadK), was identified.
- FadK is a large (119-kDa) non-membrane-spanning kinase lacking SH2 and SH3 domains.
- FadK localizes to focal adhesions in BALB/c 3T3 fibroblasts.
- FadK tyrosine phosphorylation increases in adherent cells and decreases upon detachment, rapidly recovering upon re-adhesion to fibronectin.
Conclusions:
- FadK is a novel protein-tyrosine kinase localized to focal adhesions.
- Its phosphorylation state is dynamically regulated by cell adhesion to the extracellular matrix.
- FadK activation may represent an early event in signal transduction initiated by cell-matrix interactions.