In animal cells there are several regulatory complexes which interact with 20S proteasomes and give rise to functionally distinct proteasome complexes. gamma-Interferon upregulates three immuno beta catalytic subunits of the 20S proteasome and the PA28 regulator, and decreases the level of 26S proteasomes. It also decreases the level of phosphorylation of two proteasome alpha subunits, C8 (alpha7) and C9 (alpha3). In the present study we have investigated the role of phosphorylation of C8 by protein kinase CK2 in the formation and stability of 26S proteasomes. An epitope-tagged C8 subunit expressed in mammalian cells was efficiently incorporated into both 20S proteasomes and 26S proteasomes. Investigation of mutants of C8 at the two known CK2 phosphorylation sites demonstrated that these are the two phosphorylation sites of C8 in animal cells. Although phosphorylation of C8 was not absolutely essential for the formation of 26S proteasomes, it did have a substantial effect on their stability. Also, when cells were treated with gamma-interferon, there was a marked decrease in phosphorylation of C8, a decrease in the level of 26S proteasomes, and an increase in immunoproteasomes and PA28 complexes. These results suggest that the down-regulation of 26S proteasomes after gamma-interferon treatment results from the destabilization that occurs after dephosphorylation of the C8 subunit.

Normal human fibroblasts undergo a limited number of divisions in culture and progressively they reach a state of irreversible growth arrest, a process termed as replicative senescence. The proteasome is the major cellular proteolytic machinery, the function of which is impaired during replicative senescence. However, the exact causes of its malfunction in these conditions are unknown. Using WI38 fibroblasts as a model for cellular senescence we have observed reduced levels of proteasomal peptidase activities coupled with increased levels of both oxidized and ubiquitinated proteins in senescent cells. We have found the catalytic subunits of the 20 S complex and subunits of the 19 S regulatory complex to be down-regulated in senescent cells. This is accompanied by a decrease in the level of both 20 S and 26 S complexes. Partial inhibition of proteasomes in young cells caused by treatment with specific inhibitors induced a senescence-like phenotype, thus demonstrating the fundamental importance of the proteasome for retaining cellular maintenance and homeostasis. Stable overexpression of ␤ 1 and ␤ 5 subunits in WI38 established cell lines was shown to induce elevated expression levels of ␤ 1 subunit in ␤ 5 transfectants and vice versa.

The cystic fibrosis transmembrane conductance regulator (CFTR) protein is a member of the ABC superfamily of transporter proteins. Recently, crystal structures of intact, prokaryotic members of this family have been described. These structures suggested that ATP binding and hydrolysis occurs at two sites formed at the interface between their nucleotide binding domains (NBDs). In contrast to the prokaryotic family members, the NBDs of CFTR are asymmetric (both structurally and functionally), and previous to the present studies, it was not clear whether both NBDs are required for ATP hydrolysis. In order to assess the relative roles of the two NBDs of human CFTR, we purified and reconstituted NBD1 and NBD2, separately and together. We found that NBD1 and NBD2 by themselves exhibited relatively low ATPase activity. Co-assembly of NBD1 and NBD2 exhibited a 2-3-fold enhancement in catalytic activity relative to the isolated domains and this increase reflected enhanced ATP turnover (V max ). These data provide the first direct evidence that heterodimerization of the NBD1 and NBD2 domains of CFTR is required to generate optimal catalytic activity.