Abstract
E2F-1-activated transcription promotes cell cycle progression and apoptosis. These functions are regulated by several factors including the E2F-1-binding protein MDM2 and the retinoblastoma protein pRb. Using a yeast two-hybrid screen we have identified the MDM2-related protein, MDMX, as an E2F-1-binding protein. In these studies we find that coexpression of MDMX with E2F-1 results in degradation of the MDMX protein. Although this proteolytic degradation can be blocked by the protease inhibitors bafilomycin A(1), N-acetyl-Leu-Leu-Norleu-AL, and N-acetyl-Leu-Leu-Met-AL, MDMX degradation is not inhibited by lactacystin, suggesting that degradation occurs by a proteasome-independent mechanism. Using an E2F-1 deletion mutant (E2F-1(180-437)) we show that E2F-1-targeted degradation of MDMX does not require the E2F-1 DNA binding domain and therefore is independent of E2F-1-driven transcription. We also find that this transcriptionally inactive E2F-1 mutant is capable of degrading the MDMX-related protein MDM2 and the MDMX isoform MDMX-S. Mapping of the E2F-1 C terminus reveals that neither a previously characterized C-terminal MDM2 binding domain nor the pRb binding domain on E2F-1 is required for MDMX and MDM2 degradation.
Highlights
The E2F-1 transcription factor is a regulator of both cell cycle progression and apoptosis
This proteolytic degradation can be blocked by the protease inhibitors bafilomycin A1, N-acetyl-LeuLeu-Norleu-AL, and N-acetyl-Leu-Leu-Met-AL, MDMX degradation is not inhibited by lactacystin, suggesting that degradation occurs by a proteasome-independent mechanism
In this study we provide evidence that the E2F-1 protein is capable of targeting the MDM2, MDMX, and MDMX-S proteins for proteolytic degradation
Summary
The E2F-1 transcription factor is a regulator of both cell cycle progression and apoptosis. The ability of E2F-1 to regulate transcription is in turn controlled by the retinoblastoma protein (pRb), which inhibits E2F-1 activity by binding to the C-terminal transactivation domain of E2F-1 [3,4,5]. MDM2 inhibits p53-driven transcription by multiple mechanisms, including direct binding and blocking of the p53 transactivation domain [20, 21]. As a result of this ability to inhibit p53, MDM2 plays a role in promoting transformation [24] Consistent with this notion, increased MDM2 protein levels have been observed in a broad spectrum of human tumors [25]. MDMX can bind p53 and block its ability to activate transcription and induce apoptosis, MDMX protein can increase the stability of p53 protein by binding MDM2 through a conserved C-terminal RING finger domain [28].
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