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Phosphorylation of a surface receptor bound urokinase-type plasminogen activator in a human metastatic carcinomatous
K Takahashi1, H C Kwaan, K Ikeo
1Department of Physiology, Shimane Medical University, Izumo, Japan.
Abstract:
The 32P-labeled urokinase (uPA) bound to surface receptors of Detroit 562 cells was immunoprecipitated by anti-uPA antibody. Amino acid analysis showed that tyrosines and serines were the acceptors. Inhibition of protein kinases greatly reduced the 32P incorporation, suggesting that the respective cellular src gene product and protein kinase C were involved in the phosphorylations. Proteins purified on chromatographic columns contained two forms of uPA, a high (HMW) and a low (LMW) molecular weight. Tyrosine-phosphorylation occurs in the HMW and A-chain. Such modifications might modulate the extracellular activities of uPA.
Insights
This study shows that urokinase (uPA) undergoes phosphorylation on tyrosine and serine residues in Detroit 562 cells. These modifications, involving protein kinases, may alter uPA's extracellular functions.
Area of Science:
- Biochemistry
- Cell Biology
- Molecular Oncology
Background:
- Urokinase (uPA) is a key enzyme in extracellular matrix remodeling.
- uPA's interaction with cell surface receptors influences its activity.
- Post-translational modifications can regulate protein function.
Purpose of the Study:
- To investigate the post-translational modifications of urokinase (uPA) in Detroit 562 cells.
- To identify the specific amino acid residues involved in uPA modification.
- To explore the role of protein kinases in uPA phosphorylation.
Main Methods:
- 32P-labeling of urokinase (uPA) bound to Detroit 562 cell surface receptors.
- Immunoprecipitation using anti-uPA antibody.
- Amino acid analysis to identify phosphorylation sites.
- Chromatographic purification of uPA forms.
- Inhibition of protein kinases to assess their involvement.
Main Results:
- Tyrosine and serine residues were identified as phosphorylation acceptors on uPA.
- Inhibition of protein kinases significantly reduced 32P incorporation into uPA.
- Two forms of uPA, high (HMW) and low (LMW) molecular weight, were purified.
- Tyrosine-phosphorylation was observed in the HMW form and the A-chain of uPA.
Conclusions:
- Urokinase (uPA) undergoes phosphorylation on tyrosine and serine residues in Detroit 562 cells.
- Cellular src gene product and protein kinase C are likely involved in uPA phosphorylation.
- These phosphorylation events may modulate the extracellular activities of uPA.