The effects of calpain inhibition on IkBα degradation after activation of PBMCs:: Identification of the calpain cleavage sites

Human peripheral blood mononuclear cells (PBMCs) were activated using anti-CD3/CD28 (HIT3A/CD28.2) resulting in degradation of IkBalpha, an inhibitor of NFkB, relative to unactivated cells. Degradation of IkBalpha began by 30 min and proceeded for at least 5 h. Calpeptin, a calpain inhibitor, inhibited IkBalpha degradation in a time- and dose-dependent manner. Furthermore, calpain inhibition increased IkBalpha levels compared to nonactivated controls. Recombinant IkBalpha was incubated with purified porcine m-calpain in the presence of 0.1% Triton X-100, and the degradation products were monitored by SDS-PAGE and sequenced. Most of the degradation products were peptides derived from calpain, but one was derived from IkBalpha cleaved between amino acids 50 and 51 ( glutamine and glutamic acid). The liberated fragment included the entire signal response domain (SRD), a region containing key serine and threonine residues necessary for phosphorylation by the IKKinase complex and sites required for ubiquitination. The results suggest that calpain plays an important role in IkBalpha degradation, a crucial event in T cell activation.