The Mechanism About Post Translational-modification And Functional Dissection Study Of TRF1 And PinX1

Telomere is a specialized nucleoprotein complex which contains repetitive G-rich DNA sequences and partner proteins to protect the chromosomal ends from degradation and to prevent the chromosomal end-to-end fusion. Dysfunction of telomere leads to genome instability and higher incidence of cancer in the aged cells. Although the maintenance of telomere length is mainly performed by a specific reverse transcriptase named telomerase in most malignant cancer cells, telomere-associated proteins are essential for adequate maintenance of telomeric DNA. Telomere-associated proteins directly and indirectly interact with telomeric DNA and contribute to telomere structure and function. Moreover, mounting evidence suggests that telomeric associated proteins may play important roles in cell cycle progression. For examble, expression level of TRF1 is regulated during the cell cycle, besides, it is reported that TRF1 can interact with microtubule, EB1 and some spindle checkpoint proteins such as Madl and Nek2. These results indicate that TRF1 and its partner telomeric interacting proteins may function as important signals in cell cycle.The ubiquitin-proteasome system, the most important protein degradation pathway in eukaryotic cells, regulates a host of critical cellular functions such as cell cycle progression and apoptosis through mediating the selective and time-dependent degradation of short-lived regulatory proteins. Previous studies reported that TRF1 can be ubiquitiated by some E3 ligases such as FBX4 and RLIM, indicating the importance of ubiquitin-proteasome system in regulating telomeric associated proteins.Telomeres can be shorten by 50-150 base pairs (bp) per cell division owing to the end-replication problem of the chromosome, which leads to replicative senescence when the telomere length reaches a critical point in normal somatic cells. Most cancer cells elongate there telomeres by activating telomerase. However, there are some cancer cells cannot activate telomerase and use telomere homologous recombination to elongate telomeres, a mechanism termed alternative lengthening of telomeres (ALT). The presence of ALT-associated PML bodies (APBs) is a hallmark of ALT cells. It has been suggested that APBs may have an integral role in the ALT mechanism, but the precise mechanism of APBs formation remains to be elucidated. Previous studies revealed that TRFl and its sumoylation is essential for the formation of APBs. Our recent study also showed that PML3 can assist the recruitment of TRF1 to APBs and is essential for APBs formation, but the detailed mechanism of the APBs formation is unknown.The ubiquitin-proteasome system is one of the most important protein degradation pathway in eukaryotic cells. Ubiquitination of target proteins is a multistep process. After activation by E1 enzyme, ubiqui tin is transferred to an active cystine of a ubiquitin-conjugating enzyme (E2). A ubiquitin ligase (E3) then transfers ubiquitin from the E2 ubiquitin-conjugating enzyme to the target protein either by forming an E3-ubiquitin thioester intermediate in the case of HECT E3 ubiquitin ligases or by facilitating the transfer of ubiquitin directly from the E2 to the substrate for RING finger E3 ubiquitin ligases. The cullin subunit Cull functions as a molecular scaffold that interacts at the amino terminus with the adaptor subunit Skpl (S-phase kinase-associated protein 1) and at the carboxyl terminus with a RING-finger protein Rbxl and a specific E2 enzyme or ubiquitin conjugating enzyme. The F-box protein β-TrCP function as the variable component that binds Skp1, through the F-box domain, and the substrate, through its WD40 motif.Here, we show thatβ-TrCP interacts with TRF1 and promotes its ubiquition. We first identified 8 novel TRF1 interacting proteins from Hela cell lysates which stably express Flag-TRF1 using immunoprecipitation and protein mass spectrometry. We validated that TRF1 interacts withβ-TrCP in vivo. Overexpression ofβ-TrCP can decrease the expression of TRF1. Moreover, we carried out in vivo ubiquitination assay and the results showed thatβ-TrCP can promote ubiquitination of TRF1 in vivo. Further studies showed that overexpression ofβ-TrCP but not itsΔF-box mutant enhances the ubiquitination of TRF1 and promotes the turnover of endogenous TRF1, whereas depletion ofβ-TrCP decreases TRF1 degradation. Interestingly, we found in telomerase-negative cells, PML3 can specially protect TRF1 from degradation caused byβ-TrCP, andβ-TrCP can negatively regulate the APBs formation through interacting with TRF1. These findings indicate thatβ-TrCP is a novel E3 ligase for TRF1 and may facilitate APBs formation via regulating ubiquitination and degradation of TRF1.Human PinX1 was originally identified as a novel TRF1 interacting protein. PinXl is a widely expressed protein which notably contains two special domains: G-patch domain, which usually in RNA-binding proteins, and the telomerase inhibitor domain (TID). Suppression of telomerase activity by PinX1 is mediated by its direct interaction with hTERT and hTR. However, whether Gno1p, the yeast homolog of human PinX1, can suppress the telomerase ability is controversial.In addition to its telomeric localization and telomerase inhibitory function, PinX1 can be found in the nucleoli of human telomerase positive cells and enhance the accumulation of TRF1 in nucleolus. Moreover, our recent studies show that PinX1 can localize to chromosome periphery and outer plate of kinetochores during mitosis. Depletion of PinX1 results in lagging chromosome in mitosis and micronuclei in interphase. However, the precise regulatory mechanism of PinX1 remains elusive. Here we show that Plkl is a novel interacting protein of PinX1. We performed a yeast two-hybrid assay to search its interacting protein using full-length human PinX1 cDNA as bait. In all total 1×106 clones, we identified 19 positive clones from human testis cDNA library. Nucleotide sequencing revealed that one of these interactors encodes the N terminus of Plkl (21-217 aa). Our biochemical experiments demonstrate that Plkl can interact with and phosphorylate PinX1 in vitro and in vivo. Indirect immunofluorescence staining shows that both Plkl and PinX1 can localize to spindle in mitosis. PinX1 binds to the N-terminal domain of Plk1 through its 92-254 domain. In vitro phosphorylation assay showed that PinX1 is a novel phosphorylation substrate of Plk1. The phosphopeptide of full-length PinX1 was analyzed by MALDI-MS, and three serine and two threonine residues were identified. We generated PinX1 5A mutant which all five serines and threonines were replaced by alanine and phospho-mimicking PinXl mutant which five serines and threonines were replaced by asparagine. Overexpression of wild-type Plkl but not its kinase-defective mutant interrupts the stability of PinX1 through regulating ubiquitin-associated proteolytic degradation of PinX1. Depletion of Plkl by siRNA increases the protein level of PinX1. We validated that Plkl-associated phosphorylation of PinX1 is essential for Plkl-mediated degradation of PinX1. Moreover, expression of GFP-PinX1 resulted in a significant increase in cells bearing misaligned chromosomes. Our studies demonstrate that Plkl interacts with PinX1 and regulates its stability by phosphorylation and such phosphorylation of PinX1 by Plk1 is essential for faithful chromosome congression.