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Human SAD1 Kinase Is Involved in UV-induced DNA Damage Checkpoint Function

2004; Elsevier BV; Volume: 279; Issue: 30 Linguagem: Inglês

10.1074/jbc.m404728200

1083-351X

Rui Lu, Hiroyuki Niida, Makoto Nakanishi,

Cancer-related Molecular Pathways

Checkpoint activation by DNA damage during G2 prevents activation of cyclin B/Cdc2 complexes, and as a consequence, mitotic entry is blocked. Although initiation and maintenance of G2 arrest are known to be regulated by at least two distinct signaling pathways, including those of p38MAPK and ataxia-telangiectasia-mutated (ATM)- and Rad3-related (ATR)-Chk1 in higher eukaryotes, the actual number of signaling pathways involved in this regulation is still elusive. In the present study, we identified human SAD1 (hsSAD1) by searching a sequence data base. The predicted hsSAD1 protein comprises 778 amino acids and shares significant homology with the fission yeast Cdr2, a mitosis-regulatory kinase, and Caenorhabditis elegans SAD1, a neuronal cell polarity regulator. HsSAD1 transcript was expressed ubiquitously with the highest levels of expression in brain and testis. HsSAD1 specifically phosphorylated Wee1A, Cdc25-C, and -B on Ser-642, Ser-216, and Ser-361 in vitro, respectively. Overexpression of hsSAD1 resulted in an increased phosphorylation of Cdc25C on Ser-216 in vivo. DNA damage induced by UV or methyl methane sulfonate but not by IR enhanced endogenous hsSAD1 kinase activity in a caffeine-sensitive manner and caused translocation of its protein from cytoplasm to nucleus. Overexpression of wild-type hsSAD1 induced G2/M arrest in HeLa S2 cells. Furthermore, UV-induced G2/M arrest was partially abrogated by the reduced expression of hsSAD1 using small interfering RNA. These results suggest that hsSAD1 acts as checkpoint kinase upon DNA damage induced by UV or methyl methane sulfonate. The identification of this new kinase suggests the existence of an alternative checkpoint pathway other than those of ATR-Chk1 and p38MAPK. Checkpoint activation by DNA damage during G2 prevents activation of cyclin B/Cdc2 complexes, and as a consequence, mitotic entry is blocked. Although initiation and maintenance of G2 arrest are known to be regulated by at least two distinct signaling pathways, including those of p38MAPK and ataxia-telangiectasia-mutated (ATM)- and Rad3-related (ATR)-Chk1 in higher eukaryotes, the actual number of signaling pathways involved in this regulation is still elusive. In the present study, we identified human SAD1 (hsSAD1) by searching a sequence data base. The predicted hsSAD1 protein comprises 778 amino acids and shares significant homology with the fission yeast Cdr2, a mitosis-regulatory kinase, and Caenorhabditis elegans SAD1, a neuronal cell polarity regulator. HsSAD1 transcript was expressed ubiquitously with the highest levels of expression in brain and testis. HsSAD1 specifically phosphorylated Wee1A, Cdc25-C, and -B on Ser-642, Ser-216, and Ser-361 in vitro, respectively. Overexpression of hsSAD1 resulted in an increased phosphorylation of Cdc25C on Ser-216 in vivo. DNA damage induced by UV or methyl methane sulfonate but not by IR enhanced endogenous hsSAD1 kinase activity in a caffeine-sensitive manner and caused translocation of its protein from cytoplasm to nucleus. Overexpression of wild-type hsSAD1 induced G2/M arrest in HeLa S2 cells. Furthermore, UV-induced G2/M arrest was partially abrogated by the reduced expression of hsSAD1 using small interfering RNA. These results suggest that hsSAD1 acts as checkpoint kinase upon DNA damage induced by UV or methyl methane sulfonate. The identification of this new kinase suggests the existence of an alternative checkpoint pathway other than those of ATR-Chk1 and p38MAPK. Maintaining the genomic stability of a cell requires a complex network of cell cycle checkpoint mechanism(s) that prevent cell cycle progression when damage to DNA is encountered or key processes are not completed (1Melo J. Toczyski D. Curr. Opin. Cell Biol. 2002; 14: 237-245Google Scholar, 2O'Connell M.J. Walworth N.C. Carr A.M. Trends Cell Biol. 2000; 10: 296-303Google Scholar). After detection of specific DNA or DNA-protein structures, a signal(s) is transduced to effector molecules that implement checkpoint-dependent responses such as cell cycle arrest or apoptosis. In mammals, cell cycle arrest at the G1/S boundary in response to DNA damage is mediated through the transcriptional activation of DNA damage-inducible genes, including p21 cyclin-dependent kinase inhibitor, by the p53 tumor suppressor gene (3Kastan M.B. Zhan Q. el-Deiry W.S. Carrier F. Jacks T. Walsh W.V. Plunkett B.S. Vogelstein B. Fornace Jr., A.J. Cell. 1992; 71: 587-597Google Scholar, 4el-Deiry W.S. Tokino T. Velculescu V.E. Levy D.B. Parsons R. Trent J.M. Lin D. Mercer W.E. Kinzler K.W. Vogelstein B. Cell. 1993; 75: 817-825Google Scholar, 5Giaccia A.J. Kastan M.B. Genes Dev. 1998; 12: 2973-2983Google Scholar, 6Prives C. Hall P.A. J. Pathol. 1999; 187: 112-126Google Scholar, 7Vousden K.H. Cell. 2000; 103: 691-694Google Scholar). Although cells lacking functional p53 are completely defective at the G1 checkpoint upon DNA damage, they retain a checkpoint mechanism at G2/M, which may underlie the resistance of these cells to anticancer drugs (8Waldman T. Lengauer C. Kinzler K.W. Vogelstein B. Nature. 1996; 381: 713-716Google Scholar).Checkpoints have been investigated genetically in the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyhces pombe. In both, several Rad-related proteins are thought to participate in the signaling as well as monitoring processes that detect DNA damage or incomplete DNA replication (9Longhese M.P. Foiani M. Muzi-Falconi M. Lucchini G. Plevani P. EMBO J. 1998; 17: 5525-5528Google Scholar). Among these proteins, Rad3, a yeast homolog of human ATM 1The abbreviations used are: ATM, ataxia-telangiectasia-mutated; ATR, ATM- and Rad3-related; hsSAD1, human SAD1; MAPK, mitogen-activated protein kinase; AMPK, AMP-activated protein kinase; MARK, microtubule-affinity regulating kinase; MMS, methyl methane sulfonate; GST, glutathione S-transferase; GFP, green fluorescent protein; siRNA, small interfering RNA. 1The abbreviations used are: ATM, ataxia-telangiectasia-mutated; ATR, ATM- and Rad3-related; hsSAD1, human SAD1; MAPK, mitogen-activated protein kinase; AMPK, AMP-activated protein kinase; MARK, microtubule-affinity regulating kinase; MMS, methyl methane sulfonate; GST, glutathione S-transferase; GFP, green fluorescent protein; siRNA, small interfering RNA. or ATR, appears to play a central role in both DNA damage and DNA replication checkpoints (10Carr A.M. Curr. Opin. Genet. Dev. 1997; 7: 93-98Google Scholar). Furthermore, two checkpoint kinases, Chk1 and Chk2 (Cds1), are required for checkpoint responses at the G2/M phase by acting downstream of several rad gene products including Rad3 (11Walworth N. Davey S. Beach D. Nature. 1993; 363: 368-371Google Scholar, 12Murakami H. Okayama H. Nature. 1995; 374: 817-819Google Scholar). Fission yeast Chk1 and Cds1 phosphorylate Cdc25 at a serine residue within the region of the protein that binds to Rad24 and Rad25, fission yeast homologs of mammalian 14-3-3 protein, suggesting that these kinases act by regulating the binding of Cdc25 to Rad24 and Rad25 (13Furnari B. Blasina A. Boddy M.N. McGowan C.H. Russell P. Mol. Biol. Cell. 1999; 10: 833-845Google Scholar).Several lines of biochemical evidence suggest that mammalian homologs of Chk1 and Chk2 (Cds1), as well as fission yeast kinases, phosphorylate Cdc25-C on Ser-216 and act, at least in part, by regulating the binding of 14-3-3 to Cdc25-C (14Furnari B. Rhind N. Russell P. Science. 1997; 277: 1495-1497Google Scholar, 15Sanchez Y. Wong C. Thoma R.S. Richman R. Wu Z. Piwnica-Worms H. Elledge S.J. Science. 1997; 277: 1497-1501Google Scholar, 16Kaneko Y.S. Watanabe N. Morisaki H. Akita H. Fujimoto A. Tominaga K. Terasawa M. Tachibana A. Ikeda K. Nakanishi M. Oncogene. 1999; 18: 3673-3681Google Scholar). However, Chk1- and Chk2-deficient mice demonstrated critical differences in G2/M checkpoint function between yeasts and mammals. Chk1-deficient mice died at an early embryonic stage, and the Chk1-deficient cells showed severe impairment in DNA replication and DNA damage checkpoints (17Takai H. Tominaga K. Motoyama N. Minamishima Y.A. Nagahama H. Tsukiyama T. Ikeda K. Nakayama K. Nakanishi M. Genes Dev. 2000; 14: 1439-1447Google Scholar, 18Liu Q. Guntuku S. Cui X.S. Matsuoka S. Cortez D. Tamai K. Luo G. Carattini-Rivera S. DeMayo F. Bradley A. Donehower L.A. Elledge S.J. Genes Dev. 2000; 14: 1448-1459Google Scholar), whereas yeast Chk1 mutants remained viable and retained an apparently normal response to DNA replication blockage (11Walworth N. Davey S. Beach D. Nature. 1993; 363: 368-371Google Scholar). In addition, Chk2-deficient cells revealed the essential function of Chk2 in the regulation of p53 function but not in the initiation of G2/M arrest upon IR-induced DNA damage (19Hirao A. Kong Y.Y. Matsuoka S. Wakeham A. Ruland J. Yoshida H. Liu D. Elledge S.J. Mak T.W. Science. 2000; 287: 1824-1827Google Scholar, 20Takai H. Naka K. Okada Y. Watanabe M. Harada N. Saito S. Anderson C.W. Appella E. Nakanishi M. Suzuki H. Nagashima K. Sawa H. Ikeda K. Motoyama N. EMBO J. 2002; 21: 5195-5205Google Scholar). Thus, although the existence of mammalian homologs of yeast rad-related genes has suggested the conservation of checkpoint mechanisms, there appeared to be significant differences in the details of their regulation.Stress kinases including mitogen-activated protein kinase (MAPK) have also been reported to be involved in several cell cycle checkpoint systems. The MAPK signaling pathways serve to convey signals into the nucleus, thereby determining whether a cell undergoes division, cell cycle arrest, or apoptosis (21Lewis T.S. Shapiro P.S. Ahn N.G. Adv. Cancer Res. 1998; 74: 49-139Google Scholar, 22Widmann C. Gibson S. Jarpe M.B. Johnson G.L. Physiol. Rev. 1999; 79: 143-180Google Scholar, 23Schaeffer H.J. Weber M.J. Mol. Cell. Biol. 1999; 19: 2435-2444Google Scholar). Activation of p42MAPK is required for the G2/M transition in the maturation of Xenopus oocytes (24Palmer A. Gavin A.C. Nebreda A.R. EMBO J. 1998; 17: 5037-5047Google Scholar), and inactivation of this kinase releases these cells from G2 arrest at the time of fertilization (25Abrieu A. Fisher D. Simon M.N. Doree M. Picard A. EMBO J. 1997; 16: 6407-6413Google Scholar). MEK2 activity was recently reported to be necessary for G2 arrest in mammalian cells (26Abbott D.W. Holt J.T. J. Biol. Chem. 1999; 274: 2732-2742Google Scholar), whereas MEK1 activity is essential for the G2/M transition (27Wright J.H. Munar E. Jameson D.R. Andreassen P.R. Margolis R.L. Seger R. Krebs E.G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11335-11340Google Scholar). p38α has been reported to be involved in Cdc42-induced G1 arrest as well as at the spindle assembly checkpoint (28Molnar A. Theodoras A.M. Zon L.I. Kyriakis J.M. J. Biol. Chem. 1997; 272: 13229-13235Google Scholar, 29Takenaka K. Moriguchi T. Nishida E. Science. 1998; 280: 599-602Google Scholar). p38 has also been proposed to play an important role in G2 arrest upon DNA damage induced by IR (30Wang X. McGowan C.H. Zhao M. He L. Downey J.S. Fearns C. Wang Y. Huang S. Han J. Mol. Cell. Biol. 2000; 20: 4543-4552Google Scholar) and UV (31Bulavin D.V. Higashimoto Y. Popoff I.J. Gaarde W.A. Basrur V. Potapova O. Appella E. Fornace Jr., A.J. Nature. 2001; 411: 102-107Google Scholar). Interestingly, p38 binds and phosphorylates Cdc25-B and -C at serines 309 and 361 and at serine 216, respectively, and phosphorylation of these residues is required for binding to 14-3-3 proteins (31Bulavin D.V. Higashimoto Y. Popoff I.J. Gaarde W.A. Basrur V. Potapova O. Appella E. Fornace Jr., A.J. Nature. 2001; 411: 102-107Google Scholar). Therefore, the mechanism controlling G2/M checkpoint activation after DNA damage is complex, involving at least two different biochemical systems that target Cdc25-B and -C phosphatases. Following DNA damage, different kinases integrate signals from the damaged DNA and other damaged cellular components to regulate Cdc25 inactivation.Fission yeast Cdr2 was first identified as a kinase that regulates the onset of mitosis through regulating Wee1 protein kinase (32Breeding C.S. Hudson J. Balasubramanian M.K. Hemmingsen S.M. Young P.G. Gould K.L. Mol. Biol. Cell. 1998; 9: 3399-3415Google Scholar, 33Kanoh J. Russell P. Mol. Biol. Cell. 1998; 9: 3321-3334Google Scholar). Cdr2 associates with the N-terminal regulatory domain of Wee1 in cell lysates and phosphorylates Wee1 in vitro. Thus, it is possible that a mammalian homolog(s) of Cdr2 could also function in mitotic regulation under genotoxic stress. In the present study, we identified human cDNA corresponding to homolog protein that showed sequence similarity to fission yeast Cdr2. From the results of a search of protein databases using the predicted amino acid sequences, we have found an additional kinase with the highest similarity in Caenorhabditis elegans, called SAD1 (34Crump J.G. Zhen M. Jin Y. Bargmann C.I. Neuron. 2001; 29: 115-129Google Scholar), and we thus termed our protein human SAD1. HsSAD1 phosphorylated Wee1A, Cdc25-C, and Cdc25-B on Ser-642, Ser-216, and Ser-361, respectively. More interestingly, hsSAD1 was activated and translocated into nuclei in response to UV- or methyl methane sulfonate (MMS)-induced DNA damage but not to IR-induced DNA damage. These results suggest that hsSAD1 may be involved in the regulation of G2/M arrest in response to UV- or MMS-induced DNA damage.MATERIALS AND METHODSCloning of Human SAD1 cDNA—The data base of the NCBI (National Center for Biotechnology Information) was searched for sequences that show homology to a protein sequence corresponding to the entire region of SpCdr2. A human clone, NP003948, obtained from the Human Genome Project, was thereby identified. In accordance with the nucleotide sequence information on the NP003948 clone, the hsSAD1 probe was generated by PCR using a set of primers: 5′-CCA TTA TGC GTG TCC AGA GGT-3′ and 5′-TCA GGA GGC TCT GGC AAT CTG-3′. The entire cDNA for hsSAD1 was cloned by conventional screening of a λ phage library of human testis cDNA (Stratagene) with the hsSAD1 probe. The hsSAD1 cDNA contains an in-frame stop codon upstream of the first ATG in the predicted open reading frame. For generation of pcDNA3.1Myc/His6hsSAD1, we first performed PCR with the 5′ primer 5′-TTT GAA TTC GCC ACC ATG TCG TCC GGG GCC AAG GAG-3′ (S-1), the 3′ primer 5′-AAA TCT AGA GGG CAG AGG GGT CCC GTT GGT-3′ (AS-1), and hsSAD1 cDNA as the template. The resulting PCR product was digested with EcoRI and XbaI and then ligated into pcDNA3.1/Myc-HisA (Invitrogen). The plasmid encoding the kinase-dead mutant of hsSAD1 was generated by a two-step PCR with S1 and AS-1, the inner primers 5′-TTG ATG GCC ACC GCC TGA CCC GTG ATG C-3′ and 5′-GCA TCA CGG GTC AGG CGG TCG CCA TCA A-3′, and pcDNA3.1Myc/His6hsSAD1 as the template.Northern Blot Analysis—Polyadenylated RNAs isolated from various human tissues (Clontech) were subjected to Northern blot hybridization as described previously (47Fujita F. Taniguchi Y. Kato T. Narita Y. Furuya A. Ogawa T. Sakurai H. Joh T. Itoh M. Delhase M. Karin M. Nakanishi M. Mol. Cell. Biol. 2003; 23: 7780-7793Google Scholar).Production of Recombinant Proteins—Baculoviruses expressing Myc- and His6-tagged hsSAD1 were generated by introducing the EcoRI/PmeI fragment from pcDNA3.1Myc/His6hsSAD1 into pVL1392, which was digested with EcoRI and SmaI. One microgram of pVL1392hsSAD1Myc/His6 was cotransfected into Sf9 cells with 2.5 μg of linearized baculovirus DNA (BaculoGold; Pharmingen).Generation of Polyclonal Antibodies against hsSAD1 Protein—Rabbit polyclonal antibodies to hsSAD1 were generated with purified hsSAD1 expressed in Sf9 cells. Sera were tested for reactivity against purified hsSAD1Myc/His6 expressed in Sf9 and asynchronized A172 cell extracts by Western blotting. Antisera were affinity-purified on CNBr-activated Sepharose 4B (Amersham Biosciences) coupled with purified hsSAD1Myc/His6, and the concentration was adjusted to 0.1 mg/ml.Kinase Assay—Myc/His6-tagged hsSAD1 protein expressed in Sf9 cells was precipitated with antibody to the Myc epitope tag (Medical and Biological Laboratories Co., Ltd.). The kinase activity of the immunoprecipitated proteins was measured by incubation for 30 min at 30 °C in a reaction mixture (30 μl) containing 50 mm Tris-HCl (pH 7.5), 10 mm MgCl2, 10 mm MnCl2, 1 mm dithiothreitol, 10 μm ATP, 185 kBq of [γ-32P]ATP (222 TBq/mmol; PerkinElmer Life Sciences), and GST-fused Wee1A, Cdc25-B, or -C fragment as substrate. The reaction was terminated by the addition of 30 μlof2× sample buffer. The supernatant was subjected to SDS-polyacrylamide gel electrophoresis and autoradiography. For endogenous hsSAD1 kinase assay, A172 cells were treated with UV, MMS, x-ray, or hydroxyurea. The treated cells were then lysed in an immunoprecipitation kinase buffer (50 mm Hepes, pH 8.0, 150 mm NaCl, 25 mm EGTA, 1 mm EDTA, 0.1% Tween 20, and 10% glycerol) containing a mixture of protease inhibitors (20 μg/ml soybean trypsin inhibitor, 2 μg/ml aprotinin, 5 μg/ml leupeptin, and 100 μg/ml phenylmethylsulfonyl fluoride) and phosphatase inhibitors (50 mm NaF and 0.1 mm Na3VO4). The cell lysates were immunoprecipitated with α-hsSAD1 antibody, and the immunoprecipitates were assayed for kinase activity using GST-fused Cdc25-C fragment as a substrate.Indirect Immunofluorescence Analysis—A172 cells were treated with UV or x-ray and then fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, and incubated with either antibodies to hsSAD1 (1:100 dilution) or normal rabbit IgG (1:1000 dilution). Immune complexes were detected with anti-rabbit IgG conjugated with Alexa 488 antibody (1:1000 dilution) for 1 h in the dark.Transfection and Fluorescence-activated Cell Sorter Analysis—HeLa S2 cells (5 × 105 cells/10-cm dish) were cotransfected with 2 μg of pcDNA3.1Myc/His6hsSAD1 and 1 μg of pME18S-GFP using a Mirus-TransIT-LT1 kit (Mirus). Thirty hours after transfection, cells were harvested and fixed in 70% ice-cold ethanol. The fixed cells were stained with propidium iodide and analyzed by flow cytometry to determine the DNA content of green fluorescent protein (GFP)-negative (not transfected) and GFP-positive (transfected) cells. The cell cycle profiles were analyzed by ModiFitLT model programs according to the manufacturer's instructions.Quantitative Real-time PCR—Quantitative real-time PCR was performed using an ABI7700 sequence detector (PerkinElmer Biosystems) according to the manufacturer's instructions. IQSYBR Green Supermix (Bio-Rad) was incubated with a set of specific primers for HsSAD1: 5′-CCA TCA AGA TCG TGA ACC GG-3′ (sense) and 5′-CCT TCA TCA GCA CCG ACT CC-3′ (antisense). The results were analyzed using ABI Primer Sequence Detector Software v1.6.3 (PerkinElmer Biosystems) and normalized by cyclophilin products.Small Interfering RNA (siRNA) Transfection—siRNA duplexes were 21 bp including a 2-deoxynucleotide overhang. The coding strand of hsSAD1 siRNA was 5′-GUU CUU CCG CCA GAU UGU GdTdT-3′. GFP siRNA was used as a control. HeLa S2 cells (2 × 105) were cultured in a 3.5-mm dish for 24 h prior to transfection. Cells were transfected with each siRNA with the use of the Oligofectamine 2000 reagent (Invitrogen). The transfection was done twice with 24-h intervals, and the transfected cells were then treated with UV irradiation at 72 h after the first transfection.RESULTS AND DISCUSSIONIdentification of Human SAD1 Kinase—Recent studies of fission yeast have revealed that stress-activated protein kinases influence mitotic control. Of these, Nim1/Cdr1 was identified as a negative regulator of Wee1 protein kinase (35Coleman T.R. Tang Z. Dunphy W.G. Cell. 1993; 72: 919-929Google Scholar, 36Parker L.L. Walter S.A. Young P.G. Piwnica-Worms H. Nature. 1993; 363: 736-738Google Scholar, 37Wu L. Russell P. Nature. 1993; 363: 738-741Google Scholar). Deletion of nim1+ causes defects in response to nutritional deprivation and a cell elongation phenotype that is suppressed by wee1 mutations. Another Nim1 family of serine/threonine kinases, Cdr2, shares homology with Nim1/Cdr1 and modulates phosphorylation of Cdc2 on tyrosine 15 through regulation of Wee1, probably in a manner similar to Nim1/Cdr1 (32Breeding C.S. Hudson J. Balasubramanian M.K. Hemmingsen S.M. Young P.G. Gould K.L. Mol. Biol. Cell. 1998; 9: 3399-3415Google Scholar, 33Kanoh J. Russell P. Mol. Biol. Cell. 1998; 9: 3321-3334Google Scholar). Thus, we speculated that a mammalian homolog of Cdr kinases may regulate stress responses, such as cell cycle arrest, in mammalian cells. To identify cDNAs that encode protein kinases homologous to Cdr kinases, we searched an expressed sequence tag data base using a protein sequence of fission yeast Cdr2. Human and murine expressed sequence tags (human NP003948 and murine XP145303) corresponding to previously unidentified proteins were detected. A full-length cDNA corresponding to the human expressed sequence tag was obtained by conventional screening of a λ phage library of human testis cDNA and then sequenced. The protein encoded by the open reading frame comprises 778 amino acids, with a predicted molecular mass of 87 kDa, and exhibits 44.5% overall sequence identity to S. pombe Cdr2. There are also highly conserved homologs termed SAD1 in C. elegans (34Crump J.G. Zhen M. Jin Y. Bargmann C.I. Neuron. 2001; 29: 115-129Google Scholar) and in the ascidian Halocynthia roretzi, which have overall sequence identities of 52.7 and 59.4%, respectively, and which have been identified as having kinase regulating neuronal polarity. Thus, we have termed this gene human SAD1.Phylogenetic analysis placed human SAD1 and fission yeast Cdr2 in the MARK subfamily of the Snf1/AMPK group (Fig. 1B). Although MARKs do not phosphorylate Cdc25-C at Ser-216 or Cdc25-B at Ser-361 to a comparable extent, C-TAK1 (32% identity to hsSAD1), which is a member of the MARK family of kinases, is involved in the regulation of Cdc25-C function through phosphorylating its Ser-216 residue (38Peng C.Y. Graves P.R. Ogg S. Thoma R.S. Byrnes III, M.J. Wu Z. Stephenson M.T. Piwnica-Worms H. Cell Growth & Differ. 1998; 9: 197-208Google Scholar).The distribution of SAD1 mRNA in various normal human tissues was examined by Northern blot analysis. The SAD1-specific probe recognized a 3.5-kb mRNA that was expressed ubiquitously with the highest levels of expression in the brain and testis. It is noteworthy that the hybridization pattern was somewhat distinct from those observed in MARK1 or MARK2, the levels of expression of which were relatively low in testis (39Drewes G. Ebneth A. Preuss U. Mandelkow E.M. Mandelkow E. Cell. 1997; 89: 297-308Google Scholar). Therefore, the higher expression of hsSAD1 in testis may reflect its physiological function in cell cycle regulation.Northern blot analysis with the hsSAD1 cDNA probe revealed that a hsSAD1 transcript was readily detected at the G1/S boundary and the steady-state level of hsSAD1 mRNA slightly increased as cells approached the M phase and declined during the next G1 phase (data not shown). However, the hsSAD1 protein level detected with a specific antibody was relatively constant throughout the cell cycle (Fig. 1D). hsSAD1 Phosphorylated Wee1 on Ser-642, Cdc25-C on Ser-216, and Cdc25-B on Ser-361 in Vitro—Since fission yeast Cdr2 phosphorylates Wee1, we next determined whether this is also the case with hsSAD1. HsSAD1 protein produced in Sf9 cells phosphorylated the GST-fused human wild-type Wee1A fragment but not its S642A mutant produced in Escherichia coli (Fig. 2). The kinase-dead hsSAD1 mutant (K59A) failed to phosphorylate the wild-type Wee1A fragment. Phosphorylation of Xenopus Wee1 on Ser-549 corresponding to mouse Ser-642 is important for binding of 14-3-3 protein and for maximal activation (40Lee J. Kumagai A. Dunphy W.G. Mol. Biol. Cell. 2001; 12: 551-563Google Scholar). Given that fission yeast Mik1 and Wee1 have been proposed as targets of DNA damage and as DNA replication checkpoints (41O'Connell M.J. Raleigh J.M. Verkade H.M. Nurse P. EMBO J. 1997; 16: 545-554Google Scholar, 42Raleigh J.M. O'Connell M.J. J. Cell Sci. 2000; 113: 1727-1736Google Scholar, 43Rhind N. Russell P. Mol. Cell. Biol. 2001; 21: 1499-1508Google Scholar), hsSAD1 phosphorylation of Wee1A on Ser-642 suggests the involvement of this protein at these checkpoint functions. Furthermore, wild-type hsSAD1, but not the kinase-dead mutant, effectively phosphorylated GST-Cdc25-C. HsSAD1 phosphorylated the wild-type GST-Cdc25-C fragment but not the S216A mutant. Wild-type hsSAD1 also phosphorylated the GST-Cdc25-B fragment to the same extent as that of Cdc25-C. HsSAD1 kinase phosphorylated the GST-Cdc25-B S309A mutant as effectively as the wild-type fragment, whereas it failed to phosphorylate GST-Cdc25-B S361A. Thus, hsSAD1 specifically phosphorylates Cdc25-C and Cdc25-B on Ser-216 and Ser-361 in vitro, respectively. Given that phosphorylation of Cdc25-C and -B on Ser-216 and Ser-361 inhibits its phosphatase activity, these results suggested that hsSAD1 may regulate cell cycle arrest upon DNA damage or DNA replication blockage through two independent mechanisms: inhibition of Cdc25-C and -B and activation of Wee1A. Notably, the Ser-642 of Wee1A, Ser-216 of Cdc25-C, and Ser-361 of Cdc25-B have also been found to act as substrates of Chk1 (16Kaneko Y.S. Watanabe N. Morisaki H. Akita H. Fujimoto A. Tominaga K. Terasawa M. Tachibana A. Ikeda K. Nakanishi M. Oncogene. 1999; 18: 3673-3681Google Scholar), suggesting that their functions overlap at several cell cycle checkpoints.Fig. 2HsSAD1 specifically phosphorylates GST-Wee1A, GST-Cdc25-C, and GST-Cdc25-B on Ser-642, Ser-216, and Ser-361, respectively. Wild-type (wt) and kinase-dead mutant (kd) Myc-hsSAD1 were purified from Sf9 cells, and each was assayed for its ability to phosphorylate purified GST-fused wild-type or mutant Wee1A, Cdc25-C, or Cdc25-B fragments as indicated on the top (Kinase activity; upper panel). The amount of substrate was confirmed by Coomassie Brilliant Blue staining (CBB staining; lower panel). The amount of wild-type and kinase-dead mutant Myc-hsSAD1 proteins was also determined by immunoprecipitation (IP)-immunoblotting analysis using α-Myc antibody (bottom panel). FL, full-length.View Large Image Figure ViewerDownload (PPT)Overexpression of hsSAD1 Enhanced the Phosphorylation of Cdc25C on Ser-216 in Vivo—We examined whether hsSAD1 could phosphorylate Cdc25C on Ser-216 in vivo. HeLa cells were transfected with either wild-type hsSAD1 or kinase-dead mutant (K59A), and the phosphorylation of endogenous Cdc25C on Ser-216 was determined by the immunoblotting using antibodies specific to phospho-Cdc25C on Ser-216. The phosphorylated form of Cdc25C on Ser-216 was significantly increased in wild-type hsSAD1 transfected cells. The Cdc25C protein levels were constant in wild-type, kinase-dead, and control vector transfected cells (Fig. 3). Thus, these results suggested that hsSAD1 could phosphorylate Cdc25C on Ser-216 in vivo.Fig. 3Overexpression of hsSAD1 enhances phosphorylation of Cdc25C on Ser-216 in vivo. Asynchronous HeLaS2 cells were transfected with wild-type (WT) hsSAD1, kinase-dead (KD) mutant, or vector alone. Forty-eight hours after transfection, cells were harvested, and cell lysates were subjected to immunoblotting using antibodies specific to Cdc25C, phospho-Cdc25C on Ser-216 (P-S216), or hsSAD1.View Large Image Figure ViewerDownload (PPT)UV-induced, but Not IR-induced, DNA Damage Activates hsSAD1—We next determined the effect of DNA damage on hsSAD1 kinase activity. A172 glioma cells were treated with x-ray, UV, MMS, or hydroxyurea, and the endogenous hsSAD1 was assayed for its kinase activity using the GST-Cdc25-C fragment as a substrate. The kinase activity in the anti-hsSAD1 immunoprecipitates increased ∼5-fold at 10 min after UV irradiation or MMS treatment but not after x-ray irradiation or hydroxyurea treatment, although the hsSAD1 protein level was not affected by these treatments (Fig. 4A). The induction was apparent as early as 10 min after treatment, and the level remained constant for up to 120 min. The kinase activity was not detected when the Cdc25-C mutant (S216A) was used as a substrate (data not shown). Thus, these results suggested that kinase activation of hsSAD1 occurs at the DNA damage checkpoint upon UV or MMS treatment.Fig. 4Activation of hsSAD1 in response to DNA damage induced by UV or MMS, but not by IR, is caffeine-sensitive. A, asynchronous A172 cells were treated with UV (70 J/m2), MMS (50 μg/ml), x-ray (10 grays), or hydroxyurea (HU, 5 mm) and harvested at the indicated intervals. The cell lysates were then immunoprecipitated with anti-hsSAD1 antibody (α-HsSAD1) or normal rabbit IgG (control), and the resultant precipitates were subjected to kinase assay using the GST-Cdc25-C fragment as a substrate. The cell lysates were also subjected to immunoblotting using anti-hsSAD1 antibody. B, asynchronous A172 cells were treated with 10 mm caffeine for 2 h before treatment with UV. The treated cells were then harvested after a 30-min interval, and the hsSAD1 activity was measured using GST-Cdc-25C as a substrate as described in A. CBB, Coomassie Brilliant Blue. C, caffeine did not directly inhibit hsSAD1 kinase activity. Wild-type Myc-hsSAD1 protein purified from insect cells was subjected to in vitro kinase assay in the presence of the indicated concentrations of caffeine using GST-fused Cdc25-C fragment as a substrate.View Large Image Figure ViewerDownload (PPT)The UV-induced Activation of hsSAD1 Is Sensitive to Caffeine—Caffeine has been shown to override the DNA damage checkpoint (44Wang S.W. Norbury C. Harris A.L.