Degradation of p57Kip2 mediated by SCFSkp2-dependent ubiquitylation

Abstract

The abundance of the cyclin-dependent kinase (CDK) inhibitor p57Kip2, an important regulator of cell cycle progression, is thought to be controlled by the ubiquitin-proteasome pathway. The Skp1/Cul1/F-box (SCF)-type E3 ubiquitin ligase complex SCFSkp2 has now been shown to be responsible for regulating the cellular level of p57Kip2 by targeting it for ubiquitylation and proteolysis. The elimination of p57Kip2 was impaired in Skp2-/- cells, resulting in abnormal accumulation of the protein. Coimmunoprecipitation analysis also revealed that Skp2 interacts with p57Kip2 in vivo. Overexpression of WT Skp2 promoted degradation of p57Kip2, whereas expression of a dominant negative mutant of Skp2 prolonged the half-life of p57Kip2. Mutation of the threonine residue (Thr-310) of human p57Kip2 that is conserved between the COOH-terminal QT domains of p57Kip2 and p27Kip1 prevented the effect of Skp2 on the stability of p57Kip2, suggesting that phosphorylation at this site is required for SCFSkp2-mediated ubiquitylation. Finally, the purified recombinant SCFSkp2 complex mediated p57Kip2 ubiquitylation in vitro in a manner dependent on the presence of the cyclin E-CDK2 complex. These observations thus demonstrate that the SCFSkp2 complex plays an important role in cell-cycle progression by determining the abundance of p57Kip2 and that of the related CDK inhibitor p27Kip1.

Fig. 1.

Degradation of p57^Kip2 by the proteasome pathway in HeLa cells. (A) Cells were incubated with 100 nM DEX for the indicated times, after which cell lysates were subjected to immunoblot analysis (IB) with Abs to p57^Kip2 or GSK-3β. (B) Cells were treated with 100 nM DEX for 8 h and then cultured for the indicated times in DEX-free medium in the presence of 0.1% DMSO, 50 μg/ml CHX, or 10 μM MG132. Cell lysates were then subjected to immunoblot analysis with Abs to p57^Kip2 or GSK-3β.

Fig. 2.

Accumulation of p57^Kip2 in Skp2^-/- MEFs as a result of its impaired elimination. (A) Schematic representation of the domain organization of mouse p57^Kip2 (mp57), human p57^Kip2 (hp57), and mouse p27^Kip1 (mp27). (B) Cell lysates prepared from WT, Skp2^-/-, or Skp2^-/-p27^-/- MEFs either in asynchronous culture (AS) or synchronized at G₀ phase or at S-G₂ phases were subjected to immunoblot analysis with Abs to p57^Kip2, p27^Kip1, Skp2, or HSP90. (C) Proliferating WT, Skp2^-/-, or Skp2^-/-p27^-/- MEFs were incubated for the indicated times in the presence of CHX (50 μg/ml), after which cell lysates were subjected to immunoblot analysis with Abs to p57^Kip2, p27^Kip1, or HSP90.

Fig. 3.

Association between p57^Kip2 and Skp2 in HeLa cells. (A) Cells were incubated with 100 nM DEX for 8 h and then with 10 μM MG132 for 6 h, after which cell lysates were subjected to IP with either rabbit Abs to p57^Kip2 or control rabbit IgG. The resulting precipitates were subjected to immunoblot analysis with Abs to p57^Kip2 or Skp2, as indicated. (B) After transfection (24 h) with expression vectors for the indicated proteins, cells were incubated for 6 h with MG132 (10 μM). Cell lysates were then subjected to IP with Abs to Myc. The immunoprecipitated proteins were subjected to immunoblot analysis with abs to Myc or HA.

Fig. 4.

Promotion by Skp2 of the degradation of p57^Kip2 in a CDK consensus phosphorylation site-dependent manner. (A) Schematic representation of the domain organization of mouse WT Skp2 and the Skp2(ΔNF) mutant. (B and C) After transfection (24 h) with expression plasmids for the indicated proteins, HEK293T cells were incubated for the indicated times with CHX (50 μg/ml). Cell lysates were then subjected to immunoblot analysis with Abs to Myc or GSK-3β.

Fig. 5.

Ubiquitylation of p57^Kip2 by the recombinant SCF^Skp2 complex in vitro. (A) The recombinant SCF^Skp2 complex purified from Sf21 insect cell lysates was analyzed by SDS/PAGE and staining with Coomassie brilliant blue. The positions of the various epitope-tagged recombinant proteins are indicated. (B-D) The recombinant SCF^Skp2 complex was assayed for the ability to mediate the ubiquitylation of p57^Kip2 (B), p27^Kip1 (C), or HIF-1α (D) in the presence of ATP, Uba1, UbcH5A, and GST-ubiquitin (Ub), and in the absence or presence of the recombinant cyclin E-CDK2 complex or Cks1. Reaction products were subjected to immunoblot analysis with Abs to p57^Kip2 (B), p27^Kip1 (C), or HPC4 (D).