SKP2 associates with p130 and accelerates p130 ubiquitylation and degradation in human cells

Abstract

p130 is a member of the retinoblastoma family of pocket proteins, which includes pRB and p107. Unlike pRB and p107, p130 protein levels decrease dramatically following its hyperphosphorylation starting in the mid-G1 phase of the cell cycle. However, the mechanism leading to p130 downregulation is unknown. We have found that the proteasome inhibitor, lactacystin, inhibited p130 downregulation in T98G cells progressing through the G1/S transition and S phase and that p130 is multiubiquitylated in 293 cells. We have previously shown that ectopic expression of both cyclin D and E induces phosphorylation and downregulation of p130. Since the SKP1/Cul1/SKP2 E3 ubiquitin ligase complex mediates ubiquitylation of substrates previously phosphorylated by cyclin-dependent kinases, we investigated the potential role of this ubiquitin ligase in mediating p130 downregulation. We found that p130 interacts with SKP1, Cul-1 and SKP2 in human 293 cells. We also found that ectopic coexpression of SKP2 and p130 leads to dose-dependent downregulation of p130, reduces p130 protein half-life and induces p130 ubiquitylation in these cells. Moreover, adenoviral-mediated expression of SKP2 accelerates downregulation of endogenous hyperphosphorylated p130 in mitogen-stimulated T98G cells and primary WI38 fibroblasts. We conclude that p130 is a substrate of the SCF SKP2 ubiquitin ligase and this E3 ligase regulates p130 abundance during the cell cycle.

The Evolutionarily Conserved C-terminal Domains in the Mammalian Retinoblastoma Tumor Suppressor Family Serve as Dual Regulators of Protein Stability and Transcriptional Potency

“…6A, p107 and RB levels were markedly diminished after CDK4 inhibition, whereas endogenous p130 levels were significantly increased. These data are consistent with the divergent regulation of the RB family during cell cycle progression in staged cells (28,29) and with experiments testing the effect of PD0332991 in asynchronously dividing hepatocellular carcinoma cells (30). Moreover, levels of p107 and p130 were significantly increased during a short duration (6 h) of MG132 treatment, whereas RB levels were modestly diminished.…”

“…For example, RB and p107 are active in cycling cells, whereas p130 functions predominately in quiescent cells that have exited from the cell cycle. Experiments performed with staged cells showed that steady state p130 levels peak in G 0 , in contrast to RB and p107, which increase as cells progress through G 1 (28,29). Consistently, CDK4 activity has opposite effects on p107 and p130 steady state levels; inhibition of the enzyme leads to diminished levels of p107 and higher levels of p130 (30).…”

“…1D wherein the images were acquired using identical parameters, suggesting that ES differentiation is correlated with reduced steady state expression. Variation in RB family abundance has been observed during cell cycle progression (24,28,29,41); therefore, we considered that RB family levels during differentiation might be associated with the differing cell cycle profiles for pluripotent ES cells compared with cells undergoing differentiation. As shown in Fig.…”

“…These differences suggest that cyclin-CDK activity may be key for regulation of RB family protein levels. At least for p130, proteasome-mediated turnover is known to contribute to cell cycleassociated changes (28,29). To assess the potential role of the IE in this process, we first measured the effect of CDK4 inhibition by PD0332991 on endogenous RB family members in asynchronously dividing U2OS cells.…”

Suppression of Anoikis by SKP2 Amplification and Overexpression Promotes Metastasis of Esophageal Squamous Cell Carcinoma

“…The most important substrates for SKP2 include p27 kip1 cyclin-dependent kinase inhibitor and cyclin E, both of which interact with cyclindependent kinase 2 to regulate G 1 -S transition (7). SKP2 has also been implicated in regulating the proteosome-mediated degradation of c-myc, p21 cip1 , p57 kip2 , and p130-Rb2 (8)(9)(10)(11)(12)(13). Elevated SKP2 expression has been shown in breast, colorectum, lung, and stomach carcinomas and lymphomas (14)(15)(16).…”