The effects of calpain inhibition on IkB alpha 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 IkB alpha, an inhibitor of NFkB, relative to unactivated cells. Degradation of IkB alpha began by 30 min and proceeded for at least 5 h. Calpeptin, a calpain inhibitor, inhibited IkB alpha degradation in a time- and dose-dependent manner. Furthermore, calpain inhibition increased IkB alpha levels compared to nonactivated controls. Recombinant IkB alpha 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 IkB alpha 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 IkB alpha degradation, a crucial event in T cell activation.
Schaecher
Kurt
K
Department of Neurology, Medical University of South Carolina, Charleston, South Carolina 29425, USA.
Goust
Jean-Michel
JM
Banik
Naren L
NL
D000595:Amino Acid Sequence; D001483:Base Sequence; D018106:CD28 Antigens / Q000097:blood; D017252:CD3 Complex / Q000097:blood; D002154:Calpain / Q000037:antagonists & inhibitors* / Q000097:blood; D002478:Cells, Cultured; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004151:Dipeptides / Q000494:pharmacology; D006801:Humans; D020672:I-kappa B Proteins / Q000097:blood* / Q000235:genetics; D007700:Kinetics; D007963:Leukocytes, Mononuclear / Q000378:metabolism*; D008213:Lymphocyte Activation / Q000502:physiology; D008214:Lymphocytes / Q000187:drug effects / Q000378:metabolism*; D008969:Molecular Sequence Data; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000097:blood; D015345:Oligonucleotide Probes
D018106:CD28 Antigens; D017252:CD3 Complex; D015853:Cysteine Proteinase Inhibitors; D004151:Dipeptides; D020672:I-kappa B Proteins; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D015345:Oligonucleotide Probes; D000072000:NF-KappaB Inhibitor alpha; C071482:calpeptin; D002154:Calpain