周期蛋白依赖激酶抑制因子p21Cip1的研究进展

ｐ２１Ｃｉｐ１是广泛的ＣＤＫ－ｃｙｃｌｉｎ抑制因子。它的表达受ｐ５３的正调控,也可不依赖于ｐ５３。它与ＰＣＮＡ结合抑制ＤＮＡ的复制,同时又允许ＤＮＡ损伤的切除修复。近两年的研究还表明它与细胞分化密切相关,但它与癌症的相关性并不确定。     

Use of the two-hybrid system to identify Rab-interacting proteins

The yeast two-hybrid system has been useful for identifying many partners and effectors of small GTPases of the Rab family. We describe here such a screen using Rab6, a protein involved in the regulation of intracellular transport at the level of the Golgi apparatus, as bait.     

TOK-1, a novel p21Cip1-binding protein that cooperatively enhances p21-dependent inhibitory activity toward CDK2 kinase

A p21(Cip1/Waf1/Sdi1) is known to act as a negative cell-cycle regulator by inhibiting kinase activity of a variety of cyclin-dependent kinases. In addition to binding of the cyclin-dependent kinase to the N-terminal region of p21, p21 is also bound at its C-terminal region by proliferating cell nuclear antigen (PCNA), SET/TAF1, and calmodulin, indicating the versatile function of p21. In this study, we cloned cDNA encoding a novel protein named TOK-1 as a p21 C-terminal-binding protein by a two-hybrid system. Two splicing isoforms of TOK-1, TOK-1alpha and TOK-1beta, comprising 322 and 314 amino acids, respectively, were co-localized with p21 in nuclei and showed a similar expression profile to that of p21 in human tissues. TOK-1alpha, but not TOK-1beta, directly bound to the C-terminal proximal region of p21, and both were expressed at the G(1)/S boundary of the cell cycle. TOK-1alpha also preferentially bound to an active form of cyclin-dependent kinase 2 (CDK2) via p21, and these made a ternary complex in human cells. Furthermore, the results of three different types of experiments showed that TOK-1alpha enhanced the inhibitory activity of p21 toward histone H1 kinase activity of CDK2. TOK-1alpha is thus thought to be a new type of CDK2 modulator.     

AKT/PKB phosphorylation of p21Cip/WAF1 enhances protein stability of p21Cip/WAF1 and promotes cell survival.

p21(Cip1/WAF1) (p21), a p53-inducible protein, is a critical regulator of cell cycle and cell survival. p21 binds to and inhibits both the DNA synthesis regulator proliferating cell nuclear antigen and cyclin A/E-CDK2 complexes. Recently, p21 has also been shown to be a positive regulator of cell cycle progression as p21 is necessary for the assembly and activation of cyclin D1-CDK4/6 complexes. Furthermore, elevated p21 protein levels have been observed in various aggressive tumors as well as linked to chemoresistance. Here we demonstrate that p21 is directly phosphorylated by AKT/PKB, a survival kinase that is hyperactivated in many late stage tumors. Two sites (Thr(145) and Ser(146)) in the carboxyl terminus of p21 are phosphorylated by AKT/PKB in vitro and in vivo. Phosphorylation of Thr(145) inhibits PCNA binding, whereas phosphorylation of Ser(146) significantly increases p21 protein stability. Glioblastoma cell lines with activated AKT/PKB show enhanced p21 stability, and they are more resistant to taxol-mediated toxicity. Finally, AKT/PKB controls the assembly of cyclin D1-CDK4 complexes through modulation of p21 and cyclin D1 levels. These data imply that enhanced levels of p21 in tumors are due, in part, to phosphorylation by activated AKT/PKB. Furthermore, they suggest that one mechanism of AKT/PKB regulation of tumor cell survival and/or proliferation is to stabilize p21 protein.     

5-aza-2'-deoxycytidine activates the p53/p21Waf1/Cip1 pathway to inhibit cell proliferation

In addition to its demethylating function, 5-aza-2'-deoxycytidine (5-aza-CdR) also plays an important role in inducing cell cycle arrest, differentiation, and cell death. However, the mechanism by which 5-aza-CdR induces antineoplastic activity is not clear. In this study, we found that 5-aza-CdR at limited concentrations (0.01-5 microm) induces inhibition of cell proliferation as well as increased p53/p21(Waf1/Cip1) expression in A549 cells (wild-type p53) but not in H1299 (p53-null) and H719 cells (p53 mutant). The p53-dependent p21(Waf1/Cip1) expression induced by 5-aza-CdR was not seen in A549 cells transfected with the wild-type human papilloma virus type-16 E6 gene that induces p53 degradation. Furthermore, deletion analysis and site-directed mutagenesis of the p21 promoter reveals that 5-aza-CdR induces p21(Waf1/Cip1) expression through two p53 binding sites in the p21 promoter. Finally, 5-aza-CdR-induced p21(Waf1/Cip1) expression was dependent on DNA damage but not on DNA demethylation as demonstrated by comet assay and bisulfite sequencing, respectively. Our data provide useful clues for judging the therapeutic efficacy of 5-aza-CdR in the treatment of human cancer cells.     

Stoichiometry of cyclin A-cyclin-dependent kinase 2 inhibition by p21Cip1/Waf1

Progression through the eukaryotic cell cycle is regulated by phosphorylation, which is catalyzed by cyclin-dependent kinases. Cyclin-dependent kinases are regulated through several mechanisms, including negative regulation by p21 (variously called CAP20, Cip1, Sdi1, and WAF1). It has been proposed that multiple p21 molecules are required to inhibit cyclin-dependent kinases, such that p21 acts as a sensitive buffer of cyclin-dependent kinase activity or as an assembly factor for the complexes formed by the cyclins and cyclin-dependent kinases. Using purified, full-length proteins of known concentration (determined by absorbance) and cyclin A-Cdk2 of known activity (calibrated with staurosporine), we find that a 1:1 molar ratio of p21 to cyclin A-Cdk2 is able to inhibit Cdk2 activity both in the binary cyclin A-Cdk2 complex and in the presence of proliferating cell nuclear antigen (PCNA). Our results indicate that the mechanism of p21 inhibition of cyclin A-Cdk2 does not involve multiple molecules of bound p21.     

Use of altered specificity mutants to probe a specific protein-protein interaction in differentiation: the GATA-1:FOG complex

GATA-1 and FOG (Friend of GATA-1) are each essential for erythroid and megakaryocyte development. FOG, a zinc finger protein, interacts with the amino (N) finger of GATA-1 and cooperates with GATA-1 to promote differentiation. To determine whether this interaction is critical for GATA-1 action, we selected GATA-1 mutants in yeast that fail to interact with FOG but retain normal DNA binding, as well a compensatory FOG mutant that restores interaction. These novel GATA-1 mutants do not promote erythroid differentiation of GATA-1- erythroid cells. Differentiation is rescued by the second-site FOG mutant. Thus, interaction of FOG with GATA-1 is essential for the function of GATA-1 in erythroid differentiation. These findings provide a paradigm for dissecting protein-protein associations involved in mammalian development.     

Search for protein-protein interaction partners of peroxiredoxin 6 with the yeast two-hybrid system

Peroxiredoxins (Prx) are a family of nonselenium peroxidases that are involved in cell defense against oxidative stress and in redox regulation of intracellular signaling. Mammalian peroxiredoxin 6 (Prx6) belongs to the 1-Cys Prx subfamily. The protein--protein interactions of human Prx6 were studied using a yeast two-hybrid system. Hybrid plasmid pHybLex/Zeo/Prx6, which directed synthesis of a chimeric protein consisting of the DNA-binding domain (BD) of LexA and a Prx6 sequence, was used to screen a two-hybrid cDNA library Hybrid Hunter (Invitrogen). The screening identified two potential interaction partners of Prx6: the calcium-activated cysteine endopeptidase calpain and the p50RhoGAP protein of the family of Sec14-like proteins. The possibility for the interactions observed in the two-hybrid system to occur in oxidative stress in vivo is discussed.     

Use of peptides from p21 (Waf1/Cip1) to investigate PCNA function in Xenopus egg extracts

Cell-free systems derived from unfertilized Xenopus eggs have been particularly informative in the study of the regulation and biochemistry of DNA replication. We have developed a Xenopus-based system to analyze proliferating cell nuclear antigen (PCNA)-specific effects on the functional properties of egg extracts. To do this, we have coupled peptides derived from p21 (Waf1/Cip1) to beads and used these to deplete PCNA from Xenopus egg extracts. The effect on various aspects of DNA replication can be analyzed after the readdition of PCNA and other purified proteins. Using this system, we have shown that replication of single-stranded M13 DNA is entirely dependent upon PCNA. By adding exogenous T7 DNA polymerase to PCNA-depleted extracts, we have uncoupled processive DNA replication from PCNA activity and so created an experimental system to analyze the dependence of postreplicative processes on PCNA function. We have shown that successful chromatin assembly is specifically dependent on PCNA. However, systems for analyzing the far more complex mechanisms required for the replication of nuclear double-stranded DNA have proved so far to be refractory to specific PCNA depletion.     

用酵母双杂交系统研究胰岛素样生长因子-1突变体与其结合蛋白-3的相互作用

为研究胰岛素样生长因子 1(IGF1)及其突变体与IGF结合蛋白 3(IGFBP3)的相互作用 ,针对IGF1的第 3、4、15、16位氨基酸残基 ,采用定点突变的方法构建了 [Y15L16 ]IGF1和 [Q3A4Y15L16 ]IGF1。然后分别将IGF1/IGF1突变体和IGFBP3cDNA克隆至酵母表达载体pGBT9和pACT2中 ,利用酵母双杂交技术检测IGF1/IGF1突变体和IGFBP3之间的相互作用。结果表明用酵母双杂交系统检测IGF1与其结合蛋白的结合力是可行的 ,构建的这两个IGF1突变体与IGFBP3的结合力 ,与天然IGF1相比 ,结合力大大减小     

Prolonged induction of p21Cip1/WAF1/CDK2/PCNA complex by epidermal growth factor receptor activation mediates ligand-induced A431 cell growth inhibition

Proliferation of some cultured human tumor cell lines bearing high numbers of epidermal growth factor (EGF) receptors is paradoxically inhibited by EGF in nanomolar concentrations. In the present study, we have investigated the biochemical mechanism of growth inhibition in A431 human squamous carcinoma cells exposed to exogenous EGF. In parallel, we studied a selected subpopulation, A431-F, which is resistant to EGF-mediated growth inhibition. We observed a marked reduction in cyclin-dependent kinase-2 (CDK2) activity when A431 and A431-F cells were cultured with 20 nM EGF for 4 h. After further continuous exposure of A431 cells to EGF, the CDK2 activity remained at a low level and was accompanied by persistent G1 arrest. In contrast, the early reduced CDK2 activity and G1 accumulation in A431-F cells was only transient. We found that, at early time points (4-8 h), EGF induces p21Cip1/WAF1 mRNA and protein expression in both EGF-sensitive A431 cells and EGF-resistant A431-F cells. But only in A431 cells, was p21Cip1/WAF1 expression sustained at a significantly increased level for up to 5 d after addition of EGF. Induction of p21Cip1/WAF1 by EGF could be inhibited by a specific EGF receptor tyrosine kinase inhibitor, tyrphostin AG1478, suggesting that p21Cip1/WAF1 induction was a consequence of receptor tyrosine kinase activation by EGF. We also demonstrated that the increased p21Cip1/WAF1 was associated with both CDK2 and proliferating cell nuclear antigen (PCNA). Taken together, our results demonstrate that p21Cip1/WAF1 is an important mediator of EGF-induced G1 arrest and growth inhibition in A431 cells.     

Ubiquitination of p21Cip1/WAF1 by SCFSkp2: substrate requirement and ubiquitination site selection

Multiple proteolytic pathways are involved in the degradation of the cyclin-dependent kinase inhibitor p21(Cip1/WAF1). Timed destruction of p21(Cip1/WAF1) plays a critical role in cell-cycle progression and cellular response to DNA damage. The SCF(Skp2) complex (consisting of Rbx1, Cul1, Skp1, and Skp2) is one of the E3 ubiquitin ligases involved in ubiquitination of p21(Cip1/WAF1). Little is known about how SCF(Skp2) recruits its substrates and selects particular acceptor lysine residues for ubiquitination. In this study, we investigated the requirements for SCF(Skp2) recognition of p21(Cip1/WAF1) and lysine residues that are ubiquitinated in vitro and inside cells. We demonstrate that ubiquitination of p21(Cip1/WAF1) requires a functional interaction between p21(Cip1/WAF1) and the cyclin E-Cdk2 complex. Mutation of both the cyclin E recruitment motif (RXL) and the Cdk2-binding motif (FNF) at the N terminus of p21(Cip1/WAF1) abolishes its ubiquitination by SCF(Skp2), while mutation of either motif alone has minimal effects, suggesting either contact is sufficient for substrate recruitment. Thus, SCF(Skp2) appears to recognize a trimeric complex consisting of cyclin E-Cdk2-p21(Cip1/WAF1). Furthermore, we show that p21(Cip1/WAF1) can be ubiquitinated at four distinct lysine residues located in the carboxyl-terminal region but not two other lysine residues in the N-terminal region. Any one of these four lysine residues can be targeted for ubiquitination in the absence of the others in vitro, and three of these four lysine residues are also ubiquitinated in vivo, suggesting that there is limited specificity in the selection of ubiquitination sites. Interestingly, mutation of the carboxyl-terminal proline to lysine enables ubiquitin conjugation at the carboxyl terminus of the substrate both in vitro and in vivo. Thus, our results highlight a unique property of the ubiquitination enzymatic reaction in that substrate ubiquitination site selection can be remarkably diverse and occur in distinct spatial areas.