The Study Of Skp2 Involved In Cell Cycle Checkpoint Of Tumor Cells

Skp2 was identified by Zhang H as a S-phase kinase-associated protein 2 in 1995. Skp2 acts as a member of the F-box family of substrate-regulation subunits of SCF ubiquitin-protein ligase complexes in ubiquitin proteasome pathway. Degradation of a protein via ubiquitin-proteasome pathway involves two steps: generation of a polyubiquitin chain covalently conjugated to the protein by E1-E2-E3, and dergradation of the tagged protein by the 26S proteasome. The pathway plays an important role in a variety of basic cellular processes. Among these are regulation of cell cycle, control of neoplasia and metastasis, and modulation of the cellular response to stress. The SCF complex is an important E3s related to cell cycle, and it recognizes phosphorylated target protein and makes it degrade by proteasome. The COOH-terminus domain of Skp2 contains a conserved 40-amino acid motif called the F-box, which is required for binding to Skp1. The NH2- terminus domain of Skp2 contains substrate recognization motif and mediates interaction between SCFSkp2 complex and the phosphorylated substrate. The large numbers of these domains in different F-box proteins provide the basis for multiple substrate-specific ubiquitination pathways.To be interesting, SCFskp2 targets the cyclin-dependent kinase inhibitors such as p211WAFi/CIP1 and p27KIP1 p21 and p27 have been demonstrated to be pivotal regulators of G1/S and G2/M checkpoint. The G1/S checkpoint monitors the DNA replication and regulates the beginning of proliferation, and G2/M checkpoint is the later gatekeeper of maintaining integrity of genome and regulating malignant proliferation. They play an important role in generation and development of tumor. Recently, Skp2 was found to be oncogenic and overexpressed in many human cancers and tumor cell lines. Earlier studies revealed that skp2 was amplified and over-expressed in small-cell lung cancers and Skp2 amplification was associated with tumorigenesis. Down-regulation of Skp2 expression by means of an antisense oligonucleotide inhibited the growth of SCLC cells in culture and induced apoptosis in lung-cancer cells directly. These results suggest that the high expression of Skp2 would be associated with the state of cell proliferation.At present the molecular mechanism of Skp2 mediation tumorigenesis is unknown. Skp2 has been implicated in the ubiquitination and degradation of p27 and G1-S cell cycle progression. Latres et al found that Skp2 tumorigenic properties were not restricted to p27 proteolysis. p21 is not only the substrate of SCFSkp2 complex, but also the key regulator of G1/S and G2/M checkpoint, and numerous studies have been shown reduced or absent p21 in many tumor cell lines. On the basis of these observations, the relationship between the high expression of Skp2 and cell proliferation would be examined. How to Skp2 mediate the degradation of p21 and control the cell cycle checkpoint? How to the high expression of Skp2 correlate with low expression of p21? What is the molecular mechanism of Skp2 in p21 regulation? These questions remained to be answered.The present studies may challenge these questions mentioned above in following ways. (1) Inhibiting the expression of Skp2 by RNAi technology or antisense oligonucleotide were performed to examine the relationship between expression of Skp2 and cell proliferation. (2) To screen the molecule for Skp2 mediation cell cycle checkpoint, western blot assayed the lysate from HeLa cells transfected with Skp2 or antisense oligonucleotide. Furthermore, high Skp2 expression was indentified to correlate with low expression of p21. (3) PKA phosphrolylation of p21 was detected in vitro. Whether the phosphorylation affected the ubiquitination of p21 was investigated by co-immunoprecipitation. (4) By means of proteomics tools, the molecular mechanism of proteasome inhibitor, MG132, induced HL60 cell G2/M arrest was investigated.The primary results as follows:1. Skp2 involved in Gl/S and G2/M checkpoint control. Skp2 expression demonstrated a significant and direct correlat...