Related Experiment Videos
DNA synthesis induced by some but not all growth factors requires Src family protein tyrosine kinases
1Differentiation Programme, European Molecular Biology Laboratory, Heidelberg, Germany.
Abstract:
The Src family of protein tyrosine kinases have been implicated in the response of cells to several ligands. These include platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and colony stimulating factor type 1 (CSF-1, in macrophages and in fibroblasts engineered to express the receptor). We recently described a microinjection approach which we used to demonstrate that Src family kinases are required for PDGF-induced S phase entry of fibroblasts. We now use this approach to ask whether other ligands also require Src kinases to stimulate cells to replicate DNA. An antibody specific for the carboxy terminus of Src, Fyn, and Yes (anti-cst.1) inhibited Src kinase activity in vitro and caused morphological reversion of Src transformed cells in vivo. Microinjection of this antibody was used to demonstrate that Src kinases were required for both CSF-1 and EGF to drive cells into the S phase. Expression of a kinase-inactive form of Src family kinases also prevented EGF- and CSF-1-stimulated DNA synthesis. However, even though the Src family kinases were necessary for both PDGF- and EGF-induced DNA synthesis in Swiss 3T3 cells, the responses to two other potent growth factors for these cells, lysophosphatidic acid and bombesin, were unaffected by the neutralizing antibodies. Therefore, some but not all growth factors required functional Src family kinases to transmit mitogenic responses.
Insights
Src family kinases are essential for some growth factor-stimulated cell proliferation, including platelet-derived growth factor (PDGF) and epidermal growth factor (EGF). However, these kinases are not required for responses to lysophosphatidic acid or bombesin.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Src family kinases are key signaling molecules involved in cellular responses to various growth factors.
- Previous studies indicated a role for Src kinases in PDGF-induced fibroblast proliferation.
Purpose of the Study:
- To investigate whether other ligands, such as EGF and CSF-1, also require Src kinases for mitogenic signaling.
- To determine the specificity of Src kinase involvement in growth factor-induced DNA synthesis.
Main Methods:
- Microinjection of a Src-specific antibody (anti-cst.1) to inhibit Src kinase activity.
- Expression of kinase-inactive Src family kinases.
- Assessment of DNA synthesis (S phase entry) in response to various growth factors.
Main Results:
- Microinjected anti-cst.1 antibody inhibited Src kinase activity and blocked EGF- and CSF-1-stimulated DNA synthesis.
- Expression of kinase-inactive Src also prevented EGF- and CSF-1-induced DNA synthesis.
- Responses to lysophosphatidic acid and bombesin were not affected by Src inhibition, despite their mitogenic effects.
Conclusions:
- Src family kinases are necessary for PDGF, EGF, and CSF-1 to stimulate DNA synthesis in Swiss 3T3 cells.
- The requirement for Src kinases in mitogenesis is ligand-specific, as responses to other growth factors remain unaffected.