Phosphorylation-dependent regulation of cytosolic localization and oncogenic function of Skp2 by Akt/PKB

Abstract

Skp2 is an F-box protein that forms the SCF complex with Skp1 and Cullin-1 to constitute an E3 ligase for ubiquitylation. Ubiquitylation and degradation of the p27 is critical for Skp2-mediated cell cycle entry, and overexpression and cytosolic accumulation of Skp2 have been clearly associated with tumorigenesis although the functional significance of the latter has remained elusive. Here we show that the Akt/PKB interacts with and directly phosphorylates Skp2. We find that Skp2 phosphorylation by Akt triggers SCF complex formation and E3 ligase activity. Importantly, a phosphorylation-defective Skp2 mutant is drastically impaired in its ability to promote cell proliferation and tumorigenesis. Furthermore, we show that Akt-mediated phosphorylation triggers 14-3-3-β-dependent Skp2 relocalization to the cytosol, and we attribute a specific role to cytosolic Skp2 in the positive regulation of cell migration. Finally, we demonstrate that high levels of Akt activation correlate with Skp2 cytosolic accumulation in human cancer specimens. Our results therefore define a novel proto-oncogenic Akt/PKB-dependent signaling pathway. NIH Public Access Author ManuscriptNat Cell Biol. Author manuscript; available in PMC 2010 March 2. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptThe ubiquitin-proteasome system regulates the cell cycle through control of protein ubiquitylation and degradation 1,2 . One of the key ubiquitin ligases (E3 ligase) in this process is the Skp1/Cul-1/F-box (SCF) complex, which consists of Skp1, Cullin-1 (Cul-1), RBX1, as well as an F-box protein, all required for its E3 ubiquitin ligase activity. Disruption of this complex severely ablates its enzymatic activity 1,2 .Skp2 (S-phase kinase associated protein-2) is a SCF F-box protein and is responsible for substrate recognition 1,2 . It binds to p27 and targets it for ubiquitylation and degradation [3][4][5] .Overexpression of Skp2 induces cell cycle entry, and the degradation of p27 is required for Skp2-mediated cell cycle progression 6,7 . Skp2 deficiency displays elevated p27 protein levels and a profound impairment in proliferation accompanied by nuclear enlargement, polypoidy, and centrosome overduplication 8,9 . Overexpression of Skp2 is frequently observed in human cancers of diverse histology, while in most human cancers reduced level of p27 represents an adverse prognostic marker 1,2 . Skp2 cooperates with H-Ras G12V to transform primary rodent fibroblasts 10 . Overexpression of Skp2 in the T-cell compartment cooperates with N-Ras to induce T cell lymphomas 11 , while prostate specific expression of Skp2 leads to prostatic intraepithelial neoplasia (PIN) 12 . These observations suggest that Skp2 overexpression may contribute to tumorigenesis.Although substantial advances have been made in understanding the mechanisms that control its levels of expression, by contrast, the molecular mechanisms by which Skp2 activity within the SCF complex and its subcellular localization are regulated are currently unknown. Thi...

A Competitive Binding Mechanism between Skp1 and Exportin 1 (CRM1) Controls the Localization of a Subset of F-box Proteins

“…For example, Skp2/Fbxl1 localization is controlled by phosphorylation. It has been shown that phosphorylation of Skp2/Fbxl1 at Ser-72 during M phase may trigger its export to the cytoplasm affecting its ability to form functional SCF complexes (5,6). However, these observations remain controversial (7,8).…”

Hepatitis B virus X protein mutant upregulates CENP-A expression in hepatoma cells

“…Reduced HBx expression results in enhanced susceptibility of cells to anticancer drugs and induced proliferation suppression and apoptosis (28). HBx can increase SMYD3 expression, and SMYD3 upregulates c-myc in hepatoma; therefore we suggest that SMYD3-c-myc might be one of most important pathways in the development and progression of HBx-related HCC (29). HBx also plays a role in regulating a series of cell-signaling cascades, most notably in the Ras-and Raf-induced mitogen-activated protein kinase pathways, and in inactivation of tumor suppressive genes through promoter hypermethylation (30).…”

Phosphorylation by p38 Mitogen-Activated Protein Kinase Promotes Estrogen ReceptorαTurnover and Functional Activity via the SCFSkp2Proteasomal Complex

“…The SCF Skp2 complex is under tight bimodal regulation by the concerted actions of various kinases that modulate its activity by phosphorylating either its components (19,22,33) or its target proteins (26). Since there is compelling evidence for the requirement of substrate phosphorylation as a signal for SCF Skp2 -mediated protein turnover (57, 58), we have investigated the role of such posttranslational modifications in Skp2-mediated ER␣ turnover and identify the stress-activated kinase p38MAPK as a critical regulator.…”