CD28 Costimulation Induces Transcription of SKP2 and CKS1, the Substrate Recognition Components of the Skp1-Cullin-F-box Ubiquitin Ligase, SCF^Skp2.

Cellular immune responses require expansion of antigen-specific T cell clones from the pool of resting T lymphocytes that perform immune surveillance. Regulation of this proliferative potential is critical for defense against pathogens and avoidance of autoimmunity. Costimulation through the T cell receptor and accessory molecule, CD28, triggers replication of T lymphocytes in response to antigen that is mediated via both IL-2-dependent and IL-2-independent mechanisms. This event is associated with decreased abundance of the cyclin-dependent kinase inhibitor p27^kip1 and increased cyclin D/cdk4 and cyclin E/cdk2 enzymatic activity. We have previously reported that in contrast to p27^kip1 protein, the abundance of P27KIP mRNA transcript is unchanged in response to TCR/CD28 T cell activation. Ubiquitin-targeted degradation by the proteasome regulates the abundance of p27^kip1 protein and ubiquitination of p27^kip1 occurs in primary human T cells activated through the TCR and CD28. Ubiqutination of p27^kip1 is mediated by a Skp1-Cullin-F-box (SCF) family ubiquitin ligase, SCF^skp2. The SCF^skp2 holoenzyme is comprised of the core components Roc1, Cul1, Skp1, and the substrate recognititon components, an F-box (cyclin F homology) protein, Skp2, that in cooperation with a smaller subunit, Cks1, mediates recognition of p27^kip1 substrate. Targeted deletion of the murine Skp2 or Cks1 genes results in accumulation of p27^kip1 in multiple cell types. T cell proliferation in response to TCR/CD3-plus-CD28 costimulation is reduced in Skp2^−/− mice in comparison to wild-type controls, confirming the role of Skp2 in CD28 costimulation mediated degradation of p27^kip1 and T cell proliferation. Here, we examined the mechanisms that regulate Skp2 and Cks1 abundance in primary T lymphocytes in response to TCR/CD3-plus-CD28 costimulation. Using primary human T lymphocytes we observed that the SCF^skp2 core components Roc1, Cul1 and Skp1 are constitutively expressed and their levels remain unchanged upon TCR/CD3-plus-CD28 costimulation. In contrast, TCR/CD3-plus-CD28 costimulation lead to striking induction of SKP2 and CKS1 mRNA, and this event is dependent on PI3K/PKB and MEK/ERK signaling pathways. We determined that the SKP2 promoter lies directly upstream of the translation start site and contains binding sites for SP1, Elk-1 and E2F transcription factors. Mutagenesis of SP1 and Elk-1 sites abrogated TCR/CD3-plus-CD28-mediated SKP2 promoter-driven reporter activity, whereas mutagenesis of E2F site enhanced reporter activity, suggesting that SKP2 promoter may act as a node of integration for mitogenic and anti-mitogenic signals. Thus, in primary T lymphocytes CD28 costimulation can directly regulate cell cycle progression by inducing transcription of the substrate recognition components of SCF^skp2 ubiquitin ligase that targets p27^kip1 for degradation.