Constitutive degradation of IκBα in human T lymphocytes is mediated by calpain
Activation-induced induction of transcription factor NFκB in T lymphocytes is regulated by its inhibitor IκBα. NFκB activation has been demonstrated to occur either by phosphorylation on serine residues 32 and 36 of the inhibitor, IκBα, followed by ubiquitination and degradation of the inhibitor by the 26S proteasome, or by a proteasome-independent mechanism involving tyrosine phosphorylation, but not degradation. However, the mechanism underlying constitutive regulation of the levels of the inhibitor, IκB, in primary human T lymphocytes, remains to be fully delineated.
We demonstrate here, the involvement of a proteasome-independent pathway for constitutive regulation of IκBα levels in primary human T lymphocytes. Pretreatment with a cell permeable calpain inhibitor, E64D, but not with a proteasome specific inhibitor, lactacystin, blocks stimulus-independent IκBα degradation in primary human T cells. However, E64D pre-treatment fails to impact on IκBα levels following stimulation with either TNFα or pervanadate. Other isoforms of the inhibitor, IκBβ, and IκBγ, appear not to be subject to a similar ligand-independent regulation. Unlike the previously reported decline in ligand-induced degradation of IκBα in T cells from the elderly, constitutive degradation does not exhibit an age-associated decline, demonstrating proteasome-independent regulation of the activity.
Our studies support a role for an E64D sensitive protease in regulating constitutive levels of IκBα in T cells, independent of the involvement of the 26S proteasome, and suggests a biological role for constitutive degradation of IκBα in T cells.