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The Effects of Calpain Inhibition on IkBα Degradation After Activation of PBMCs: Identification of the Calpain Cleavage Sites

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Abstract

Human peripheral blood mononuclear cells (PBMCs) were activated using anti-CD3/CD28 (HIT3A/CD28.2) resulting in degradation of IkBα, an inhibitor of NFkB, relative to unactivated cells. Degradation of IkBα began by 30 min and proceeded for at least 5 h. Calpeptin, a calpain inhibitor, inhibited IkBα degradation in a time- and dose-dependent manner. Furthermore, calpain inhibition increased IkBα levels compared to nonactivated controls. Recombinant IkBα 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 IkBα 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 IkBα degradation, a crucial event in T cell activation.

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  1. Kurt Schaecher

    Present address: Walter Reed Army Institute of Research, CDN1, Department of Immunology, Silver Springs, Maryland

Authors and Affiliations

  1. Departments of Neurology and Microbiology and Immunology, Medical University of South Carolina, Charleston, South Carolina

    Kurt Schaecher, Jean-Michel Goust & Naren L. Banik

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  1. Kurt Schaecher
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  2. Jean-Michel Goust
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Correspondence to Naren L. Banik.

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Schaecher, K., Goust, JM. & Banik, N.L. The Effects of Calpain Inhibition on IkBα Degradation After Activation of PBMCs: Identification of the Calpain Cleavage Sites. Neurochem Res 29, 1443–1451 (2004). https://doi.org/10.1023/B:NERE.0000026410.56000.dd

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