Abstract
The regulation of the cell cycle by the ubiquitin-proteasome system is dependent on the activity of E3 ligases. Skp2 (S-phase kinase associated protein-2) is the substrate recognition subunit of the E3 ligase that ubiquitylates the cell cycle inhibitors p21(cip1) and p27(kip1) thus promoting cell cycle progression. Increased expression of Skp2 is frequently observed in diseases characterized by excessive cell proliferation, such as cancer and neointima hyperplasia. The stability and cellular localization of Skp2 are regulated by Akt, but the molecular mechanisms underlying these effects remain only partly understood. The scaffolding protein Ezrin-Binding Phosphoprotein of 50 kDa (EBP50) contains two PDZ domains and plays a critical role in the development of neointimal hyperplasia. Here we report that EBP50 directly binds Skp2 via its first PDZ domain. Moreover, EBP50 is phosphorylated by Akt on Thr-156 within the second PDZ domain, an event that allosterically promotes binding to Skp2. The interaction with EBP50 causes cytoplasmic localization of Skp2, increases Skp2 stability and promotes proliferation of primary vascular smooth muscle cells. Collectively, these studies define a novel regulatory mechanism contributing to aberrant cell growth and highlight the importance of scaffolding function of EBP50 in Akt-dependent cell proliferation.
Highlights
The PDZ scaffolding protein Ezrin-Binding Phosphoprotein of 50 kDa (EBP50) promotes Akt-dependent cell proliferation through the S-phase kinase associated protein-2 S-phase kinase-associated protein 2 (Skp2)
Consistent with our previous report [23], we found that Skp2 interacts with EBP50 in coimmunoprecipitation experiments (Fig. 1B)
We characterized the details of the interaction between Skp2 and the first PDZ domain of EBP50 by NMR. 1H,15N correlation spectra (HSQC) of PDZ1 both free (Fig. 1E, in black) and bound to the C-terminal 22 amino acids of Skp2 (Fig. 1E, in red) were acquired
Summary
The PDZ scaffolding protein EBP50 promotes Akt-dependent cell proliferation through the S-phase kinase associated protein-2 Skp. The interaction with EBP50 causes cytoplasmic localization of Skp, increases Skp stability and promotes proliferation of primary vascular smooth muscle cells. These studies define a novel regulatory mechanism contributing to aberrant cell growth and highlight the importance of scaffolding function of EBP50 in Akt-dependent cell proliferation. One of the critical E3 ligases involved in cell cycle progression is the Skp1/Cul-1/Rbx-1/Skp complex Within this complex, S-phase kinase-associated protein 2 (Skp2) is the substrate recognition subunit that binds the cell cycle inhibitors p21cip and p27kip and promotes their ubiquitylation and proteasomal degradation [3,4,5,6]. We reported that EBP50 expression increases in restenotic vessels upon endoluminal injury [23, 24] and participates in inti-
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