In vitro Proteolytic Degradation of Bovine Brain Calcineurin by m-Calpain

Abstract

A major cause of neuronal dysfunction is due to altered Ca2+ regulation. An increase in Ca2+ influx can activate Ca2+-dependent enzymes including calpains, causing the proteolysis of its specific substrates. In the present study, calcineurin (CaN) was found to be proteolysed by a Ca2+-dependent cysteine protease, m-calpain. In the presence of Ca2+, the 60 kDa subunit (CaN A) was degraded to a 46 kDa immunoreactive fragment, whereas in the presence of Ca2+ /calmodulin (CaM) immunoreactive fragments of 48 and 54 kDa were observed. The beta-subunit (CaN B) was not proteolysed in either condition. The proteolysis of CaN A increased its phosphatase activity and rendered it totally CaM-independent after 10 min of proteolysis. The molecular weight of the proteolytic fragments suggested that the m-calpain cleaved CaN A in the CaN B binding domain. A CaM-overlay experiment revealed that the CaM-binding site was present only in the 54 kDa fragment produced by CaN A proteolysis in the presence of Ca2+ /CaM. Thus, the increase in CaN A phosphatase activity observed in many neuronal disorders, may be due to the action of calpain.

Increased expression of calcineurin in human colorectal adenocarcinomas

“…Recently, we observed higher expression of mcalpain, a Ca 2þ -activated cysteine protease in human colorectal adenocarcinomas [Lakshmikuttyamma et al, 2004c]. Furthermore, we demonstrated the in vitro proteolysis of brain CaN by m-calpain and the enhancement of its phosphatase activity after proteolysis [Lakshmikuttyamma et al, 2004a]. It may be possible that m-calpain plays a role in enhancing the expression of CaN in colon tumor.…”

“…CaN activity was assayed using p-nitrophenylphosphate (pNPP) as a substrate [Pallen and Wang, 1983;Lakshmikuttyamma et al, 2003Lakshmikuttyamma et al, , 2004a. The reaction mixture contained 50 mM Tris-HCl, pH 7.0, 1 mM Ni 2þ , 5 mg CaM, 3.4 mM pNPP, and CaN in a total volume of 1.0 ml.…”

“…The specificity of the bovine CaN polyclonal antibody was carried out as described previously [Seitz et al, 2002;Lakshmikuttyamma et al, 2003Lakshmikuttyamma et al, , 2004a. Bovine CaN polyclonal antibody is shown to cross-react with the human CaN (Fig.…”

Truncation and Activation of Calcineurin A by Calpain I in Alzheimer Disease Brain

“…3, A and B, during the homogenization procedure. Previous studies have demonstrated that calpain can cleave and activate CaN A in vitro (18,20,21). When CaN A is incubated with calpain I, CaN A is truncated into a 55-to 57-kDa transient form and then further into a 43-to 49-kDa form (18,21).…”

“…CaN A can also be activated by proteolytic cleavage of the autoinhibitory domain, resulting in a Ca 2ϩ /calmodulin-independent, active phosphatase (16,19,20). It has been reported that calpain I cleaves and activates CaN (18,21). Hence, the phosphatase activity of CaN is regulated by both Ca ϩ2 /calmodulin and calpain activation.…”