Related Experiment Videos
Platelet-derived growth factor stimulates tyrosine-specific protein kinase activity in Swiss mouse 3T3 cell membranes
Abstract:
Platelet-derived growth factor (PDGF) stimulates the incorporation of 32P from [gamma-32P]ATP into a Mr approximately 170,000 protein by an endogenous tyrosine-specific protein kinase in membrane preparations of Swiss mouse 3T3 cells. Epidermal growth factor (EGF), but not fibroblast growth factor (FGF) or insulin, stimulates limited incorporation of 32P into a protein of similar molecular weight. The ligand concentration required for half-maximal activity (S0.5) for PDGF stimulation of phosphorylation is 50 ng/ml; saturation is achieved at 300 ng/ml. The S0.5 for ATP is 15 microM. Mg2+ or Mn2+ is required for protein kinase activity. Stimulation of PDGF results in the preferential phosphorylation of tyrosine residues in this Mr approximately 170,000 membrane protein. The Mr approximately 170,000 protein can be resolved into Mr approximately 180,000 and 160,000 components in 4% NaDodSO4 gels. PDGF stimulates 32P incorporation preferentially into the Mr approximately 180,000 and less extensively into the Mr approximately 160,000 protein. EGF stimulates 32P incorporation predominantly into a protein of Mr approximately 160,000. The similarity of PDGF and EGF in stimulating phosphotyrosine-specific protein kinase activity and the stimulation of a similar activity by viral transformation (src) genes suggest that a common mechanism may exist for the phenotypic expression of increased DNA synthesis and cell growth stimulated by these separate factors.
Insights
Platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) activate a tyrosine-specific protein kinase, phosphorylating a 170,000-dalton protein in Swiss mouse 3T3 cells. This suggests a common pathway for growth factor-stimulated cell proliferation.
Area of Science:
- Cell signaling
- Molecular biology
- Biochemistry
Background:
- Growth factors like PDGF and EGF are crucial for cell growth and proliferation.
- Protein phosphorylation, particularly on tyrosine residues, plays a key role in signal transduction pathways.
- Understanding the specific proteins and kinases involved is essential for deciphering cellular responses to growth factors.
Purpose of the Study:
- To investigate the effect of Platelet-Derived Growth Factor (PDGF) on protein phosphorylation in Swiss mouse 3T3 cells.
- To identify the specific protein(s) phosphorylated by PDGF and the type of kinase involved.
- To compare the effects of PDGF with Epidermal Growth Factor (EGF) on protein phosphorylation.
Main Methods:
- Utilized membrane preparations from Swiss mouse 3T3 cells.
- Employed [gamma-32P]ATP to measure 32P incorporation into proteins.
- Analyzed protein phosphorylation using sodium dodecyl sulfate (NaDodSO4) polyacrylamide gel electrophoresis.
- Determined the role of tyrosine-specific protein kinase activity.
Main Results:
- PDGF stimulated the phosphorylation of a Mr approximately 170,000 protein via an endogenous tyrosine-specific protein kinase.
- EGF also stimulated phosphorylation of a similar Mr protein, predominantly a Mr 160,000 component.
- PDGF preferentially phosphorylated Mr 180,000 and 160,000 components, while EGF predominantly phosphorylated the Mr 160,000 component.
- Optimal activity required Mg2+ or Mn2+ and occurred at specific PDGF and ATP concentrations.
Conclusions:
- Both PDGF and EGF activate a common phosphotyrosine-specific protein kinase.
- The Mr 170,000 protein, with its Mr 180,000 and 160,000 components, is a target for both growth factors.
- A shared signaling mechanism may underlie the increased DNA synthesis and cell growth stimulated by PDGF and EGF, potentially similar to pathways activated by viral oncogenes.