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PEST sequences do not influence substrate susceptibility to calpain proteolysis
M Molinari1, J Anagli, E Carafoli
1Institute of Biochemistry, Swiss Federal Institute of Technology, Zurich.
The Journal of Biological Chemistry
|February 3, 1995
Summary
Calmodulin (CaM) binding to the Ca2+ pump is critical for its proteolysis by mu-calpain (mu-CANP). Mutations affecting PEST scores did not alter susceptibility, indicating CaM binding, not PEST regions, dictates mu-CANP activity.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- The plasma membrane Ca2+ ATPase (PMCA) is a crucial ion pump involved in calcium homeostasis.
- Calpain (CANP) are calcium-dependent proteases that play roles in cellular signaling and degradation.
- The interaction between PMCA and calpains is not fully understood, particularly regarding regulatory elements like PEST sequences and calmodulin binding.
Purpose of the Study:
- To investigate the role of PEST scores and the calmodulin (CaM)-binding region in the susceptibility of the plasma membrane Ca2+ ATPase to mu-calpain (mu-CANP) proteolysis.
- To determine if PEST-rich sequences or the CaM-binding domain are primary recognition sites for mu-CANP.
- To elucidate the impact of CaM binding or phosphorylation on the interaction between the Ca2+ pump and mu-CANP.
Main Methods:
- Site-directed mutagenesis was used to alter PEST scores in domains surrounding the CaM-binding region of the Ca2+ ATPase.
- Synthetic peptides corresponding to high PEST score sequences were synthesized.
- Proteolysis assays were performed using purified mu-CANP and the Ca2+ ATPase, with and without CaM or phosphorylation of the CaM-binding domain.
Main Results:
- Mutations that reduced the PEST score of domains near the CaM-binding region did not affect the Ca2+ pump's susceptibility to mu-CANP.
- Synthetic peptides representing high PEST score regions did not inhibit proteolysis, suggesting they are not the primary mu-CANP recognition sites.
- An accessible CaM-binding region was found to be critical for Ca2+ pump proteolysis by mu-CANP.
- CaM binding or phosphorylation of the CaM-binding domain significantly decreased the rate of proteolysis.
Conclusions:
- The PEST score of surrounding domains is not a critical determinant for Ca2+ pump proteolysis by mu-CANP.
- The accessibility of the CaM-binding region is essential for mu-CANP to recognize and cleave the Ca2+ pump.
- CaM binding or phosphorylation acts as a protective mechanism, reducing the susceptibility of the Ca2+ pump to mu-CANP-mediated degradation.