CRL4B^DCAF11 E3 ligase targets p21 for degradation to control cell cycle progression in human osteosarcoma cells

Cell cycle progression in mammals is strictly controlled by a number of cyclin-dependent kinases (CDKs) and CDK inhibitors (CKIs), the expression of which is often dysregulated in cancer cells. Our previous work revealed that Cullin 4B (CUL4B), a critical component of the Cullin4B-RING E3 ligase complex (CRL4B), is overexpressed in human osteosarcoma cells through an unknown mechanism. Here, we demonstrated that CUL4B forms an E3 ligase with RBX1 (RING-box 1), DDB1 (DNA damage binding protein 1), and DCAF11 (DDB1 and CUL4 associated factor 11) in human osteosarcoma cells. In vitro and in vivo ubiquitination analyses indicated that CRL4B^DCAF11 E3 ligase was able to specifically ubiquitinate a CDK inhibitor-p21^Cip1 at K16, K154, K161 and K163 but not at K75 and K141. Knocking down any component of the CRL4B^DCAF11 complex, including CUL4B, DDB1 or DCAF11, using short hairpin RNAs (shRNAs) attenuated the ubiquitination level of p21^Cip1, inhibited osteosarcoma cell proliferation, led to cell cycle arrest at S phase, and decreased colony formation rate. Taken together, our data suggest that the CRL4B^DCAF11 complex represents a unique E3 ligase that promotes the ubiquitination of p21^Cip1 and regulates cell cycle progression in human osteosarcoma cells.