Identification And Functional Characterization Of Telomere Associated Centrosomal Protein 1(TACP1)

Telomeres are specialized protective structures at the extreme ends of eukaryotic chromosomes, which consist of tandem repetitive DNA sequences and binding proteins. Telomeres undergo shortening following each cell division due to the "end replicative problem" of conventional DNA replication machinery. It was hypothesized that cells lose the capacity of division and senesence ensues and subsequently apoptosis occurs when the telomere length of one or more chromosomes reaches a critical point. Telomere dysfunction is associated with chromosomal instability which is implicated in ageing and carcinogenesis. Telomerase is a ribonucleoprotein complex with reverse transcriptase activity. Telomerase synthesizes telomeric DNA repeats using its integral RNA component as template and adds them onto chromosome ends to replenish the telomere loss incurred during DNA replication. The vast majority of mammalian cells maintain their telomere length by telomerase. However, most somatic tissues and primary cultured cells do not have active telomerase activity while over 85% tumors and immortalized cell strains have remarkably increased telomerase activity. Therefore, telomerase is postulated to take a critical part in development and perhaps progression of human cancer.There is increasing evidence suggesting that both the maintenance of telomere length and structure and telomere elongation by telomerase are regulated by telomericbinding proteins. To date, three different types of telomeric binding proteins have been described: double-stranded telomeric DNA binding proteins and their associated factors;single-stranded telomeric DNA binding proteins and their associated factors;and certain components of the general DNA damage response pathways that also localize at telomeres. Among these, telomeric repeat binding factor 1 (TRFl) is the first identified human telomeric binding protein purified by ion exchange and affinity chromatography with the [TTAGGG]27 probe. TRFl binds to the duplex telomeric repeats as homodimers to promote the T-loop formation at telomere ends to facilitate telomeric shielding. TRFl is proposed to be an inhibitor of telomerase, acting in cis to limit the elongation of chromosome ends. Long-term overexpression of TRFl in telomerase-positive cells results in a gradual and progressive telomere shortening while telomere elongation is induced by expression of a dorminant-negative TRFl mutant to inhibit binding of endogenous TRFl to telomeres. TRFl forms complex with other telomeric proteins such as Tin2, Tankyrase, PinXl and Potl to shelter telomeric ends. Tankyrasel, a poly(ADP-Ribose) polymerase, catalyses TRFl poly(ADP-ribosy)lation and releases TRFl from telomeres, opening up the telomeric complex and allowing access to telomerase. TRFl and Tankyrase will translocate to centrosome and/or spindle pole during mitosis. Moreover, overexpression of TRFl induces mitotic entry in cells with short telomeres. Though these findings indicate their involvement of mitotic regulation, the functional relevance of TRFl-tankyrasel during mitosis and the underlying mechanism remain unclear.The centrosome, the principal microtubule organizing center (MTOC) in nearly all higher eukaryotes, has a pivotal role in regulating cell division in mitotic cells. During mitosis, the centrosome governs assembly and orientation of the bipolar mitotic spindle that is essential for correct chromosome segregation and cytokinesis. Centrosome aberration may compromise the fidelity of cell division and cause chromosome instability. The spindle pole body (SPB), the yeast centrosome equivalent, is a laminar structure associated with the nuclear envelope that nucleates nuclear spindle microtubules during division. Centrosomes of vertebrates and yeast, although morphologically distinct, share several conserved components including the large coiled-coil protein known as Spell Op in S. cerevisiae, Pcpl in S. Pombe andpericentrin/kendrin in vertebrates. More recently, a molecular component has just been identified, which interacts with both telomeric binding protein spTazl (an ortholog of human TRF1) and centrosomal protein Pcpl in fission yeast. It was of great interest to test whether similar mechanism acts to link telomeres to the mitotic centrosome in human cells.In present study, a novel TRF1 interacting protein was identified from mitotic HeLa cell lysates by employing immunoafrlnity isolation coupled with mass spectrometry (MS). The peptides of this protein generated from MS matched the previously characterized open reading frame AAH03618 in human protein database that encodes a 593 amino acid protein with predicted molecular mass of 68,041 Da. Computational analysis show that AAH03618 contains one putative PH domain in its amino-terminal half while two extended coiled coil domains in its carboxyl-terminal half which forms a SMC-like domain. Detailed sequence alignment reveals the fission yeast's centrosomal protein Pcpl is the closest ortholog of the protein. We therefore refer to the protein as Telomere Associated Centrosomal Protein 1 (TACPl) since it distinguishes from other TRF1 binding proteins and locates to the centrosome. Then, the full-length of TACPl coding sequence was amplified from human testis cDNA library by polymerase chain reaction and was subcloned into appropriate mammalian expressing vectors. Truncated deletion mutants were also created according to the predicted functional domains. The biochemical interaction in vivo between TRF1 and TACPl was validated by reciprocal immunoprecipitation. Deletion analysis indicated that the C-terminal coiled-coil/SMC domain of TACPl was required for TRF1 binding. Immunofluorescence studies revealed TRF1 and TACPl colocated in interphase nuclei with a dot/speckle pattern of distribution. In late prophase/earliest prometaphase, TACPl emigrated toward the centrosome while TRF1 remained associated with chromosomes without apparent centrosomal distribution. Both TRF1 and TACPl localized to centrosomes when cell cycle progressed to metaphase and anaphase. Immuno-electron microscopic study of late prophase cells revealed that TACPl localized to the pericentrioles. The temporal order of TRF1 and TACPl centrosomal localization and truncated deletion analysis revealed the centrosomal localization domain of TACPl resided within the first coiled-coil domain and TACPl specified thelocalization of TRF1 to the centrosome. The recuitment of TRF1 to centrosome by TACPl was confirmed by siRNA depletion of TACPl since the centrosome localization of TRF1 was diminished in TACPl-depleted cells. In addition, the distribution of tankyrasel to the centrosome was also eliminated in TACP1-depleted cells. Notably, TACP1 knockdown by siRNA resulted in multiplicity of spindle pole and misalignment of chromosomes in mitotic cells. The evolutionary conservancy of TACPl in amino acid sequence and function suggested it facilitates the mitotic regulation by providing a functional link between telomere and the centrosome in human cells.To further delineate the biological ruction, we characterized the expression profile of TACPl and its regulation during cell cycle progression. A stable cell line expressing GFP-tagged TACPl was established and used for determination of the protein levels by semi-quantitive Western blot analysis with an anti-GFP antibody. The results indicated TACP1 was tightly regulated in a cell-cycle dependent fasion, with a nadir at Gl/S and a zenith at G2/M, similar to most centrosomal proteins. The down-regulation of TACP1 following mitotic exit might be degraded through proteasomal pathway. Two-dimensional electropherosis and dephosphorylation assay revealed that TACP1 was specifically phosphorylated during mitosis, which was possibly responsible for TACPl translocation and function in centrosome. Thr221 and Thr457 were identified as two potential phosphorylation sites by mass spectrometric analyses of immunoisolated protein from mitotic cells. Bioinformatic analysis suggests that the putative kinases responsible for the aforementioned phosphorylation are Nek2A and Plkl, respectively. The Gl/S arrest observed in TACP1-overexpressing suggested a function of TACPl in regulation of Gl/S transition. Neverthelessly, further investigation on the mechanism of TACPl phosphorylation and identification of TACP1 partners in centrosome are in urgent need to clarify its regulation and role in cell-cycle progression.