Human CLK2 Links Cell Cycle Progression, Apoptosis, and Telomere Length Regulation

Abstract

Mutations in the clk-2 gene of the nematode Caenorhabditis elegans affect organismal features such as development, behavior, reproduction, and aging as well as cellular features such as the cell cycle, apoptosis, the DNA replication checkpoint, and telomere length. clk-2 encodes a novel protein (CLK-2) with a unique homologue in each of the sequenced eukaryotic genomes. We have studied the human homologue of CLK-2 (hCLK2) to determine whether it affects the same set of cellular features as CLK-2. We find that overexpression of hCLK2 decreases cell cycle length and that inhibition of hCLK2 expression arrests the cell cycle reversibly. Overexpression of hCLK2, however, renders the cell hypersensitive to apoptosis triggered by oxidative stress or DNA replication block and gradually increases telomere length. The evolutionary conservation of the pattern of cellular functions affected by CLK-2 suggests that the function of hCLK2 in humans might also affect the same organismal features as in worms, including life span. Surprisingly, we find that hCLK2 is present in all cellular compartments and exists as a membrane-associated as well as a soluble form. Mutations in the clk-2 gene of the nematode Caenorhabditis elegans affect organismal features such as development, behavior, reproduction, and aging as well as cellular features such as the cell cycle, apoptosis, the DNA replication checkpoint, and telomere length. clk-2 encodes a novel protein (CLK-2) with a unique homologue in each of the sequenced eukaryotic genomes. We have studied the human homologue of CLK-2 (hCLK2) to determine whether it affects the same set of cellular features as CLK-2. We find that overexpression of hCLK2 decreases cell cycle length and that inhibition of hCLK2 expression arrests the cell cycle reversibly. Overexpression of hCLK2, however, renders the cell hypersensitive to apoptosis triggered by oxidative stress or DNA replication block and gradually increases telomere length. The evolutionary conservation of the pattern of cellular functions affected by CLK-2 suggests that the function of hCLK2 in humans might also affect the same organismal features as in worms, including life span. Surprisingly, we find that hCLK2 is present in all cellular compartments and exists as a membrane-associated as well as a soluble form. Identifying and studying the processes and the genes that are involved in determining the rate of aging is a challenging area of modern genetics. In particular, it would be of interest to determine whether the activity of specific genes limits human life span. Several epidemiological studies of centenarians are being carried out with this goal in mind under the hypothesis that there might be a genetic basis for the exceptional life span of very long lived individuals (1Perls T. Terry D.F. Silver M. Shea M. Bowen J. Joyce E. Ridge S.B. Fretts R. Daly M. Brewster S. Puca A. Kunkel L. Results Probl. Cell Differ. 2000; 29: 1-20Crossref PubMed Scopus (10) Google Scholar). However, given the pervasive evolutionary conservation of physiological processes among organisms, a practical approach to find genes that might be involved in human aging is to first investigate the genetic basis of aging in lower organisms. The nematode genetic model system, Caenorhabditis elegans, is being extensively used to this end, and a number of genes that have been identified in this organism for their effect on aging are now also being studied in vertebrates (2Hekimi S. Burgess J. Bussiere F. Meng Y. Benard C. Trends Genet. 2001; 17: 712-718Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 3Tissenbaum H.A. Guarente L. Dev. Cell. 2002; 2: 9-19Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). The clk-2 mutants of C. elegans display a pleiotropic phenotype (reviewed in Ref. 4Benard C. Hekimi S. Mech. Ageing Dev. 2002; 123: 869-880Crossref PubMed Scopus (5) Google Scholar) that includes a slowing down of numerous physiological processes, including embryonic and postembryonic development, behavioral rates, and reproduction (5Benard C. McCright B. Zhang Y. Felkai S. Lakowski B. Hekimi S. Development. 2001; 128: 4045-4055Crossref PubMed Google Scholar). clk-2 mutants also show an increase in life span that is particularly dramatic in combination with mutations in other genes, such as clk-1 and daf-2 (2Hekimi S. Burgess J. Bussiere F. Meng Y. Benard C. Trends Genet. 2001; 17: 712-718Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 6Lakowski B. Hekimi S. Science. 1996; 272: 1010-1013Crossref PubMed Scopus (425) Google Scholar). The clk-2 mutations are temperature-sensitive (5Benard C. McCright B. Zhang Y. Felkai S. Lakowski B. Hekimi S. Development. 2001; 128: 4045-4055Crossref PubMed Google Scholar, 7Ahmed S. Alpi A. Hengartner M.O. Gartner A. Curr. Biol. 2001; 11: 1934-1944Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar) and at 25 °C produce a lethal embryonic phenotype resulting in differentiated but highly disorganized embryos (5Benard C. McCright B. Zhang Y. Felkai S. Lakowski B. Hekimi S. Development. 2001; 128: 4045-4055Crossref PubMed Google Scholar). This is likely to be the null phenotype, since it is also produced by RNA interference at all temperatures. Extensive temperature shift experiments have demonstrated that clk-2 is required for embryonic development only during a narrow time window in which oocyte maturation, fertilization, the completion of meiosis, and the initiation of embryonic development occurs (5Benard C. McCright B. Zhang Y. Felkai S. Lakowski B. Hekimi S. Development. 2001; 128: 4045-4055Crossref PubMed Google Scholar). However, these events as well as subsequent embryonic development appear to proceed entirely normally until the 100-cell stage, after which aberrant development becomes apparent. Surprisingly, all clk-2 phenotypes, including the phenotypes observed in adults that are ∼1000 times larger than the eggs produced by the mother, are rescued by a maternal effect; i.e. homozygous mutant animals, issued from a heterozygous mother, appear wild type. This maternal rescue effect suggests that the presence of maternally provided clk-2 product might induce a self-maintained epigenetic state, although the possibility that the maternally provided clk-2 product can still function efficiently after extreme dilution cannot be excluded. A number of cellular phenotypes of clk-2 mutants have also been identified in addition to the organismal phenotypes described above (7Ahmed S. Alpi A. Hengartner M.O. Gartner A. Curr. Biol. 2001; 11: 1934-1944Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 8Gartner A. Milstein S. Ahmed S. Hodgkin J. Hengartner M.O. Mol. Cell. 2000; 5: 435-443Abstract Full Text Full Text PDF PubMed Scopus (417) Google Scholar). For example, the germ lines of clk-2 mutants do not respond normally to ionizing radiation. In the wild type, irradiation leads to cell cycle arrest in the mitotic phase of the germ line and to apoptotic cell death in the meiotic phase of the germ line. Both of these responses are abolished in clk-2 mutants. In addition, clk-2 mutants fail to respond with cell cycle arrest to treatment with hydroxyurea (HU), 1The abbreviations used are: HU, hydroxyurea; siRNA, small interfering RNA; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling; GST, glutathione S-transferase. a drug that blocks DNA replication, suggesting a defect in the S-phase replication checkpoint. Taken together, these cellular phenotypes suggest that clk-2 mutants are defective in important aspects of the normal cellular response to DNA damage. The C. elegans clk-2 gene encodes a protein of 877 amino acids that is similar to Saccharomyces cerevisiae Tel2p and has a unique homologue in every eukaryotic genome sequenced to date (5Benard C. McCright B. Zhang Y. Felkai S. Lakowski B. Hekimi S. Development. 2001; 128: 4045-4055Crossref PubMed Google Scholar). Yeast cells carrying the hypomorphic tel2-1 mutation grow slowly and have short telomeres (9Runge K.W. Zakian V.A. Mol. Cell. Biol. 1996; 16: 3094-3105Crossref PubMed Scopus (85) Google Scholar). The telomeres shorten gradually in the tel2 cells, reaching their shortest lengths only after ∼150 generations. In addition to affecting the length of telomeres, Tel2p has also been shown to be involved in the telomere position effect, contributing to silencing of subtelomeric regions. Mutations in other genes, such as tel1, that also affect telomere length do not result in abnormal telomere position effect, indicating that the telomere position effect defect in tel2 mutants is not a simple consequence of the altered telomere length (10Zakian V.A. Annu. Rev. Genet. 1996; 30: 141-172Crossref PubMed Scopus (170) Google Scholar). In contrast to the viable tel2–1 mutants, cells that fully lack Tel2p die rapidly with an abnormal cell morphology, which suggests that Tel2p also has telomere-independent functions in yeast (9Runge K.W. Zakian V.A. Mol. Cell. Biol. 1996; 16: 3094-3105Crossref PubMed Scopus (85) Google Scholar). Worm clk-2 mutants also have altered telomere length. In contrast to the phenotype in yeast, clk-2(qm37) mutants have lengthened telomeres, and transgenic expression of clk-2 shortens some telomeres (5Benard C. McCright B. Zhang Y. Felkai S. Lakowski B. Hekimi S. Development. 2001; 128: 4045-4055Crossref PubMed Google Scholar). Although yeast Tel2p can bind single- and double-stranded DNA and RNA under some in vitro conditions (11Kota R.S. Runge K.W. Nucleic Acids Res. 1998; 26: 1528-1535Crossref PubMed Scopus (29) Google Scholar, 12Kota R.S. Runge K.W. Chromosoma. 1999; 108: 278-290Crossref PubMed Scopus (22) Google Scholar), a functional worm CLK-2::GFP fusion protein accumulates predominantly in the cytoplasm (5Benard C. McCright B. Zhang Y. Felkai S. Lakowski B. Hekimi S. Development. 2001; 128: 4045-4055Crossref PubMed Google Scholar). These findings, together with the broad pleiotropy observed mostly in worms, but also in yeast, suggest that clk-2 and tel2 mutations affect telomere length indirectly. Cell Culture—All cells were grown in high glucose Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (plus nonessential medium amino acids for HT-1080 and SK-HEP-1) at 37 °C in an atmosphere of 5% CO2 and 95% air. Construction of the Plasmid pLXSH-hclk2 and Establishment of a Stable Cell Line Overexpressing hCLK2—A cDNA clone hk02952 (insert size 4337 bp), containing the full-length hclk2 cDNA sequence, as well as parts of intronic sequences (1929–2171, 2288–2456, and 2812–3434) from DNA this cDNA as a that the to and to were by and a to produce and A from the of C. Mol. Cell. Biol. PubMed Scopus Google Scholar) to produce A from the of to produce Stable cell lines were described PubMed Scopus Google Scholar). the were used to cells S. A. J. PubMed Google Scholar), and produced were used to the cell line after in and until were The were and to the cell cells were with the as described T. Cell Res. 1999; PubMed Scopus Google Scholar) and in at the cells were together as a which the hCLK2 cell lines in a Construction of Plasmid and Establishment of a Stable HT-1080 Line with hclk2 full-length cDNA containing and by and the of to produce The DNA HT-1080 cells the were in after and until were were and that and hCLK2 in an were grown in the presence of to hCLK2 expression and grown in the of to on hCLK2 that the of expression of hCLK2 in the cell line on the of the of hCLK2 by RNA were by and were as described J. A. T. 2001; PubMed Scopus Google Scholar). The of the for hclk2 as siRNA, siRNA, For each well of a of were to the treatment on and were on of were used to The first as A to of the hclk2 clone amino acids of hCLK2, the expression A protein of the size in and by on a This protein and to the a to of the hclk2 clone amino acids of hCLK2, the expression A protein of the size in and from This protein and to to hCLK2 by the The terminal of each the in vitro hCLK2, a at the size of in cell and a of the same size in cells hCLK2 This is not by of the this of the with the protein but not with other including the of this is in cells as with The serum from the and used for this cells were and and in of and The cells were to in and at 37 Cell were by and the of protein protein of protein were on or and to The were in 5% at temperature for and with the at °C at the to and the were in at temperature for The were with or at temperature for by the by were with a and were on the were in for in for and at temperature for with by or for the cells were with for and under a were with and The pattern observed not by the or the In addition, the observed pattern of the with the protein but not with other including were in at the times the cells were and with a Cell were at in The the cells were by and A of also and the cells were at times treatment Cell by the by with a death of in a the of DNA from cells by and of DNA by and at 37 °C The DNA on at for and by to a The used as a to The in for at by an in and at 37 The in at °C for and at temperature of as described S. S. S. Res. Google Scholar). to cells were with and in at a of were on at until 95% of the cells were on The were to and at for to the The in of containing 25 with of containing the same and at for the resulting were in of The resulting from the to at for at °C to the that and The for at to the that the and and the The and were in of protein from each by by Overexpression of hCLK2 in high of hCLK2 expression in cells, we used a hCLK2 to a of cell lines and cell from of cells with the hCLK2 or the A high of hCLK2 expression in all of the cell lines and not In every the cells hCLK2 not show with not We however, that the rate of J. L. In Cell Dev. Biol. PubMed Scopus Google Scholar) cells hCLK2 the line indicating that rate is to the of We used cells for all subsequent of the function of cell lines not display on and their phenotype not studied rate of cells is affected by the of expression of cells hCLK2 and cells were at a of in a the time cells were and the number of cells were a cells were at a of in a and by or the at a of Cell were as For the and of experiments are the of hCLK2 investigate the of a of function of we used the J. A. T. 2001; PubMed Scopus Google Scholar). cells were with siRNA, for a gene that is not normally in human cells, or the same of The of hCLK2 and the cell number were for treatment and The that the cells were with siRNA, the of hCLK2 by and until at in a of the expression of In addition, the expression of not affected by specific siRNA, siRNA, or hclk2 treatment cellular in contrast to treatment with siRNA, which only a effect The effect on rate until after which time the cells to from the treatment and increase in cell death or other were indicating that the arrest not the consequence of to the cells were also by cell to DNA not The cells with hclk2 not appear to have in phase of the cell Overexpression of hCLK2 to by or DNA by the in the germ line of C. elegans, clk-2 mutations affect the response to ionizing and to DNA replication block by HU, we the response of cells hCLK2 to of apoptotic cell death as well as to and The cells hCLK2 not show increase in to apoptotic but were hypersensitive to of oxidative which leads to of PubMed Scopus Google Scholar), and which leads to the of the highly M. J. PubMed Scopus Google Scholar) The cells were also hypersensitive to the DNA that the cell death observed we the cells the Cell Res. PubMed Scopus (85) Google Scholar), which of in of the of the DNA of apoptotic A increase in the number of observed in cells with the that produced cell death with and hydroxyurea We have also the response of cells and that the cells for hCLK2 not show increase in to apoptotic not is whether a in hCLK2 has effect on the of the cells to the used or whether the arrest produced by treatment the of Overexpression of hCLK2 investigate whether hclk2 affects telomere length in human cells, as it in S. cerevisiae and in C. elegans, we the telomere length of cells hCLK2 and of cells by We the telomere length at during The telomere length of the cells hCLK2 gradually at an rate of it in the cells do not appear to increase telomere length not hCLK2 in of the determine the of hCLK2, we used to and hCLK2 in The of hCLK2 to be to be by this with hCLK2 However, in cells hCLK2, the to be in the the cytoplasm and the The same also observed in cell line HT-1080 the the and with a number of We the of hCLK2 by treatment with and apoptotic which result in DNA replication inhibition and oxidative in the of hCLK2 were observed not whether this of hCLK2 a result of we hCLK2 in the HT-1080 cell line S. PubMed Scopus Google Scholar) under an in these cells produced the same expression at all of a not an of of hCLK2, we carried out and We that and hCLK2 in cells were present in all including in the includes and Surprisingly, the of hCLK2 in each were This for the of hCLK2 and the in cells that the an of protein is on the for each this that the of hCLK2 to other is very similar in all hCLK2 Both and of the hCLK2 protein is since it is present in the hCLK2 in the we used to the and from For the of the hCLK2 cannot be by and is in the However, for the of hCLK2 were in the and which is with the of hCLK2 in the treatment is of these also that hCLK2 can be with the in all of is also from there is in size soluble and membrane-associated CLK-2 from Yeast and to of mutants of the S. cerevisiae homologue of have the gene product Tel2p in the of telomere length and subtelomeric silencing as well as in an function for cell (9Runge K.W. Zakian V.A. Mol. Cell. Biol. 1996; 16: 3094-3105Crossref PubMed Scopus (85) Google Scholar). In worms, clk-2 mutations have been shown to affect numerous processes, including organismal features such as embryonic development, behavioral rates, and reproduction B. Hekimi S. Science. 1996; 272: 1010-1013Crossref PubMed Scopus (425) Google Scholar, S. Lakowski B. PubMed Google Scholar) as well as cellular features such as the apoptotic death and mitotic arrest responses to irradiation and DNA replication block (5Benard C. McCright B. Zhang Y. Felkai S. Lakowski B. Hekimi S. Development. 2001; 128: 4045-4055Crossref PubMed Google Scholar, 7Ahmed S. Alpi A. Hengartner M.O. Gartner A. Curr. Biol. 2001; 11: 1934-1944Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 8Gartner A. Milstein S. Ahmed S. Hodgkin J. Hengartner M.O. Mol. Cell. 2000; 5: 435-443Abstract Full Text Full Text PDF PubMed Scopus (417) Google Scholar) (reviewed in Ref. 4Benard C. Hekimi S. Mech. Ageing Dev. 2002; 123: 869-880Crossref PubMed Scopus (5) Google Scholar). The broad pleiotropy observed in clk-2 mutants suggests in worms, the function of CLK-2 important cellular processes such as cell cycle apoptosis, and telomere length the pattern of observed in C. elegans might result from some of specific to this this we have the function of the human clk-2 homologue in human We find that overexpression of the hCLK2 protein decreases the time and that down the expression of hCLK2 with arrest We also find that overexpression of hCLK2 in an apoptotic response to oxidative stress and treatment but not to other that induce apoptosis we find that overexpression of hCLK2 but increases telomere length These that CLK-2 and affect the same set of cellular processes in yeast, worms, and humans and suggest the possibility that it also affect in humans the same set of organismal processes that are affected in worms, including life span. hclk2 and of yeast, the tel2-1 mutation a in telomere length (9Runge K.W. Zakian V.A. Mol. Cell. Biol. 1996; 16: 3094-3105Crossref PubMed Scopus (85) Google Scholar). In worms, however, the clk-2(qm37) mutation an of telomeres (5Benard C. McCright B. Zhang Y. Felkai S. Lakowski B. Hekimi S. Development. 2001; 128: 4045-4055Crossref PubMed Google Scholar). In the cells, we now find that overexpression of hCLK2 increases telomere length This suggests that a of function of the gene hclk2 would shorten telomeres, since it is the in the yeast tel2-1 but to the clk-2(qm37) mutant in the that the effect of overexpression of Tel2p in yeast has not been can we these possibility is that the clk-2(qm37) mutation is not a simple mutation but also a of function that in telomere by the on worm telomeres, is that the effect of clk-2 on telomeres is of worm telomeres, which can be with that are specific to the parts of terminal (5Benard C. McCright B. Zhang Y. Felkai S. Lakowski B. Hekimi S. Development. 2001; 128: 4045-4055Crossref PubMed Google Scholar), it observed that some telomeres, but not were by overexpression of This that telomeres indicating that the effect of clk-2 on telomere length is telomere length is in worms, the DNA is from at a of a of telomeres is the telomeres of a cell were affected this would not be the other the cells a cell type. is that the protein is involved in a of processes that on telomere length and that this to might on the organism or cell type. that the telomere is very which suggests that is the of length and not other such as of telomeres 2002; PubMed Google Scholar). hclk2 and ionizing irradiation a increase in apoptosis is observed in the meiotic phase of the germ line of (7Ahmed S. Alpi A. Hengartner M.O. Gartner A. Curr. Biol. 2001; 11: 1934-1944Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 8Gartner A. Milstein S. Ahmed S. Hodgkin J. Hengartner M.O. Mol. Cell. 2000; 5: 435-443Abstract Full Text Full Text PDF PubMed Scopus (417) Google Scholar). This however, is mostly abolished in clk-2 mutants. We have not a to irradiation in cells However, we have a increase in to that induce apoptosis by oxidative This is since the by which irradiation is the of normal DNA replication Google Scholar), and treatment with this arrests the mitotic cell cycle in the germ line of but not in clk-2 mutants (7Ahmed S. Alpi A. Hengartner M.O. Gartner A. Curr. Biol. 2001; 11: 1934-1944Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). We find that hCLK2 cells are hypersensitive to and apoptotic death in response to treatment with this be however, that is also an Google Scholar) and that effect in might be similar to that of other that we have not suggesting that an of the cells to or that can DNA such as of and it is that the to respond to irradiation and in not a specific defect in a DNA but is the result of a to oxidative stress a to respond to oxidative hclk2 and Cell that down hCLK2 with treatment arrests the cell cycle and that hCLK2 shortens cell This that the activity of hCLK2 is for cell cycle and that the of hCLK2 is for cell cycle at in the cells do not appear to arrest in phase of the cycle, hCLK2 is not with of the that cells to from phase to the such as DNA However, cannot the possibility that the activity of hCLK2 DNA to of the cell cycle as a In worms, in of the described with hCLK2, of function of clk-2 leads to a to arrest the cell cycle in response to irradiation and However, in the of an of the function of it is to on the of these since we cannot which aspects of the function of CLK-2 have been and which have been in the temperature-sensitive mutants that have been hCLK2 in to hCLK2 can affect processes, we have cellular and Surprisingly, we find that hCLK2 is present in and hCLK2 is present as a soluble and a membrane-associated form. can have cellular For example, is in the the and the J. Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). yeast is and A. M. Mol. Biol. Cell. 2002; PubMed Scopus Google Scholar). some can on For example, and can be or M. Rev. Mol. Cell. Biol. 2002; PubMed Scopus Google Scholar). However, to there is of a protein present in cellular compartments at the same time and in similar In hCLK2 is with the since it cannot be by which the of membrane-associated hCLK2 is from that of the soluble form. hCLK2 has it is that it with an protein with a the of the function of hCLK2 and might we have to the pleiotropy of and broad cellular also that the amino of hCLK2 is not well This suggests that this protein not high which might be to protein of function is carried out in the but not very specific with other possibility is that hCLK2 in a of The of each of as well as the function of Both soluble and membrane-associated hCLK2 bind a the and in the are small and with which would to the of soluble hCLK2, which would bind the of the We for the We the DNA for the clone