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pp60src is an endogenous substrate for calpain in human blood platelets
A Oda1, B J Druker, H Ariyoshi
1Department of Surgery, Beth Israel Hospital, Boston, Massachusetts 02115.
The Journal of Biological Chemistry
|June 15, 1993
Summary
Calpain cleaves pp60src, a tyrosine kinase in platelets, suggesting a regulatory role in platelet function. This cleavage, observed in response to calcium, is inhibited by calpain inhibitors and EGTA.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Calpain is a ubiquitous protease with an undetermined physiological role.
- Identifying endogenous substrates of calpain is crucial for understanding its function.
- pp60src, a major tyrosine kinase in platelets, is known to be cleaved during purification.
Purpose of the Study:
- To investigate whether pp60src is an endogenous substrate of calpain in platelets.
- To determine the effect of calpain activation on pp60src cleavage and associated kinase activity.
Main Methods:
- Western blotting of platelet lysates with anti-pp60src antibody.
- Activation of platelet calpain using calcium ionophore A23187 or dibucaine.
- Inhibition of calpain activity using calpeptin and EGTA.
- Analysis of pp60src distribution in cytosolic and particulate fractions.
- Assay of pp60src-associated enolase kinase activity.
Main Results:
- Calpain activation led to dose- and time-dependent cleavage of actin-binding protein, talin, and pp60src.
- pp60src cleavage was inhibited by calpeptin and EGTA, confirming calpain's role.
- Cleaved pp60src species were found exclusively in the cytosolic fraction.
- pp60src-associated enolase kinase activity was reduced following cleavage.
Conclusions:
- pp60src is an endogenous substrate of calpain in platelets.
- Calpain-mediated cleavage of pp60src may regulate its kinase activity.
- This finding provides insights into the physiological roles of calpain in platelet signaling.