Polyoma large T antigen (LT) is the only viral gene product required for viral DNA replication. LT can be divided into two domains, one N-terminal (NT) spanning residues 1-260 and one C-terminal (CT) comprising approximately residues 264-785. NT is known to immortalize primary cells in a manner dependent on binding of pRB/p107. Here a CT construct comprising residues 264-785 was shown to have independent function in DNA replication. CT is entirely sufficient for driving viral DNA replication in vivo in growing mouse cells at a level approaching that of full-length LT. In contrast, CT is strikingly deficient for replication in serum-starved cells. However, this deficiency can be complemented by coexpression of NT. BrdUrd incorporation in transfected, starved cells showed that NT was sufficient for inducing S phase, suggesting a mechanism for complementation. By contrast, CT was unable to induce S phase when tested in the same assay. NT also promotes phosphorylation of sites in CT that are likely to be important for replication. Other DNA tumor virus gene products such as adenovirus E1A 12S and human papillomavirus 16 E7 could also complement CT for replication. Although NT, E1A 12S, and E7 all bind the retinoblastoma gene product (pRB) and p107, genetic analysis demonstrates an additional function, independent of that binding, is responsible for complementation.
Polyomavirus large T antigen has an N-terminal domain of approximately 260 amino acids which can immortalize primary cells but lacks sequences known to be required for DNA binding and replication. Treatment of full-length large T with either V8 protease or chymotrypsin yields an N-terminal fragment of 36 to 40 kDa and a C-terminal fragment of approximately 60 kDa. This finding suggests a division of the protein into two domains. Proteolysis experiments show that the N-terminal domain does not have strong physical association with the rest of the protein. It also does not self-associate. A construct expressing only the N-terminal 259 amino acids is sufficient for immortalization. The independently expressed N-terminal domain is multiply phosphorylated, although at a lower level than the same region in full-length large T. The 259-residue protein binds to both pRb and p107 with somewhat lower efficiency than the full-length protein.
Abstract Background Iron-deficiency anemia is the most prevalent form of anemia world-wide. The yeast Saccharomyces cerevisiae has been used as a model of cellular iron deficiency, in part because many of its cellular pathways are conserved. To better understand how cells respond to changes in iron availability, we profiled the yeast genome with a parallel analysis of homozygous deletion mutants to identify essential components and cellular processes required for optimal growth under iron-limited conditions. To complement this analysis, we compared those genes identified as important for fitness to those that were differentially-expressed in the same conditions. The resulting analysis provides a global perspective on the cellular processes involved in iron metabolism. Results Using functional profiling, we identified several genes known to be involved in high affinity iron uptake, in addition to novel genes that may play a role in iron metabolism. Our results provide support for the primary involvement in iron homeostasis of vacuolar and endosomal compartments, as well as vesicular transport to and from these compartments. We also observed an unexpected importance of the peroxisome for growth in iron-limited media. Although these components were essential for growth in low-iron conditions, most of them were not differentially-expressed. Genes with altered expression in iron deficiency were mainly associated with iron uptake and transport mechanisms, with little overlap with those that were functionally required. To better understand this relationship, we used expression-profiling of selected mutants that exhibited slow growth in iron-deficient conditions, and as a result, obtained additional insight into the roles of CTI6 , DAP1 , MRS4 and YHR045W in iron metabolism. Conclusion Comparison between functional and gene expression data in iron deficiency highlighted the complementary utility of these two approaches to identify important functional components. This should be taken into consideration when designing and analyzing data from these type of studies. We used this and other published data to develop a molecular interaction network of iron metabolism in yeast.
Toxicogenomics has provided innovative approaches to chemical screening, risk assessment, and predictive toxicology. If applied to ecotoxicology, genomics tools could greatly enhance the ability to understand the modes of toxicity in environmentally relevant organisms. Daphnia magna, a small aquatic crustacean, is considered a "keystone" species in ecological food webs and is an indicator species for toxicant exposure. Our objective was to demonstrate the potential utility of gene expression profiling in ecotoxicology by identifying novel biomarkers and uncovering potential modes of action in D. magna. Using a custom D. magna cDNA microarray, we identified distinct expression profiles in response to sublethal copper, cadmium, and zinc exposures and discovered specific biomarkers of exposure including two probable metallothioneins, and a ferritin mRNA with a functional IRE. The gene expression patterns support known mechanisms of metal toxicity and reveal novel modes of action including zinc inhibition of chitinase activity. By integrating gene expression profiling into an environmentally important organism, this study provides experimental support for the utility of ecotoxicogenomics.
Activation of the Cdc2·cyclin B kinase is a pivotal step of mitotic initiation. This step is mediated principally by the dephosphorylation of residues threonine 14 (Thr14) and tyrosine 15 (Tyr15) on the Cdc2 catalytic subunit. In several organisms homologs of the Wee1 kinase have been shown to be the major activity responsible for phosphorylating the Tyr15 inhibitory site. A membrane-bound kinase capable of phosphorylating residue Thr14, the Myt1 kinase, has been identified in the frog Xenopus laevis and more recently in human. In this study, we have examined the substrate specificity and cell cycle regulation of the human Myt1 kinase. We find that human Myt1 phosphorylates and inactivates Cdc2-containing cyclin complexes but not complexes containing Cdk2 or Cdk4. Analysis of endogenous Myt1 demonstrates that it remains membrane-bound throughout the cell cycle, but its kinase activity decreased during M phase arrest, when Myt1 became hyperphosphorylated. Further, Cdc2·cyclin B1 was capable of phosphorylating Myt1 in vitro, but this phosphorylation did not affect Myt1 kinase activity. These findings suggest that human Myt1 is negatively regulated by an M phase-activated kinase and that Myt1 inhibits mitosis due to its specificity for Cdc2·cyclin complexes. Activation of the Cdc2·cyclin B kinase is a pivotal step of mitotic initiation. This step is mediated principally by the dephosphorylation of residues threonine 14 (Thr14) and tyrosine 15 (Tyr15) on the Cdc2 catalytic subunit. In several organisms homologs of the Wee1 kinase have been shown to be the major activity responsible for phosphorylating the Tyr15 inhibitory site. A membrane-bound kinase capable of phosphorylating residue Thr14, the Myt1 kinase, has been identified in the frog Xenopus laevis and more recently in human. In this study, we have examined the substrate specificity and cell cycle regulation of the human Myt1 kinase. We find that human Myt1 phosphorylates and inactivates Cdc2-containing cyclin complexes but not complexes containing Cdk2 or Cdk4. Analysis of endogenous Myt1 demonstrates that it remains membrane-bound throughout the cell cycle, but its kinase activity decreased during M phase arrest, when Myt1 became hyperphosphorylated. Further, Cdc2·cyclin B1 was capable of phosphorylating Myt1 in vitro, but this phosphorylation did not affect Myt1 kinase activity. These findings suggest that human Myt1 is negatively regulated by an M phase-activated kinase and that Myt1 inhibits mitosis due to its specificity for Cdc2·cyclin complexes. The cyclin-dependent kinases (Cdks) 1The abbreviations used are: Cdk, cyclin-dependent kinase; PAGE, polyacrylamide gel electrophoresis; DTT, dithiothreitol. are a family of highly conserved serine/threonine kinases that mediate many of the cell cycle transitions that occur during duplication. Each of these Cdk catalytic subunits associates with a specific subset of regulatory subunits, termed cyclins, to produce a distinct Cdk·cyclin kinase complex that, in general, functions to execute a specific cell cycle event (1Nigg E.A. Bioessays. 1995; 17: 471-480Crossref PubMed Scopus (803) Google Scholar). The placement of a specific Cdk·cyclin complex function with a particular cell cycle transition has led to the identification of key downstream substrates as well as upstream regulatory mechanisms. Well characterized Cdk·cyclin-regulated cell cycle transitions that have been identified thus far include Cdk4·cyclin D and Cdk6·cyclin D complexes with the pRB-gated G1/S transition, Cdk2·cyclin E and Cdk2·cyclin A complexes with the initiation and progression of DNA replication and Cdc2·cyclin A and Cdc2·cyclin B complexes with the initiation of mitosis (reviewed in Refs. 2Nasmyth K. Science. 1996; 274: 1643-1645Crossref PubMed Scopus (327) Google Scholar, 3Stillman B. Science. 1996; 274: 1659-1664Crossref PubMed Scopus (436) Google Scholar, 4Sherr C.J. Science. 1996; 274: 1672-1677Crossref PubMed Scopus (5029) Google Scholar). Activation of the Cdk·cyclin kinases during these transitions is controlled by a variety of regulatory mechanisms. For the Cdc2·cyclin B complex, inhibition of kinase activity during S phase and G2 is accomplished by phosphorylation of Cdc2 residues Thr14 and Tyr15, which are positioned within the ATP-binding cleft (5Gould K.L. Nurse P. Nature. 1989; 342: 39-45Crossref PubMed Scopus (953) Google Scholar, 6Krek W. Nigg E.A. EMBO J. 1991; 10: 3331-3341Crossref PubMed Scopus (266) Google Scholar, 7Jin P. Gu Y. Morgan D.O. J. Cell Biol. 1996; 134: 963-970Crossref PubMed Scopus (253) Google Scholar). Phosphorylation of Thr14and/or Tyr15 is believed to suppress catalytic activity by disrupting the orientation of the ATP molecule present within this cleft (8Atherton-Fessler S. Parker L.L. Geahlen R.L. Piwnica-Worms H. Mol. Cell. Biol. 1993; 13: 1675-1685Crossref PubMed Scopus (153) Google Scholar, 9De Bondt H.L. Rosenblatt J. Jancarik J. Jones H.D. Morgan D.O. Kim S.-H. Nature. 1993; 363: 595-602Crossref PubMed Scopus (844) Google Scholar). The abrupt dephosphorylation of these residues by the Cdc25 phosphatase results in the rapid activation of Cdc2·cyclin B kinase activity and downstream mitotic events (10Dunphy W.G. Trends Cell Biol. 1994; 4: 202-207Abstract Full Text PDF PubMed Scopus (255) Google Scholar). While phosphorylation/dephosphorylation of the conserved Tyr15site in Cdk2 likely plays an important role in regulating the G1/S transition, the role that Cdk2 Thr14phosphorylation plays is less clear (11Gu Y. Turck C.W. Morgan D.O. Nature. 1993; 366: 707-710Crossref PubMed Scopus (724) Google Scholar, 12Sebastian B. Kakizuka A. Hunter T. Proc. Natl. Acad. Sci. U. S. A.. 1993; 90: 3521-3524Crossref PubMed Scopus (215) Google Scholar). Phosphorylation of the corresponding inhibitory tyrosine residue in Cdk4 has also been observed (13Terada Y. Tatsuka M. Jinno S. Okayama H. Nature. 1995; 376: 358-362Crossref PubMed Scopus (181) Google Scholar, 14Iavarone A. Massague J. Nature. 1997; 387: 417-421Crossref PubMed Scopus (336) Google Scholar). It has been proposed that Thr14/Tyr15 phosphorylation functions to permit a cell to attain a critical concentration of inactive Cdk·cyclin complexes, which, upon activation, induces a rapid and complete cell cycle transition (15Solomon M.J. Glotzer M. Lee T.H. Philippe M. Kirschner M.W. Cell. 1990; 63: 1013-1024Abstract Full Text PDF PubMed Scopus (560) Google Scholar). There is evidence in mammalian cells that Thr14/Tyr15 phosphorylation also functions, in part, to delay Cdk activation after DNA damage (7Jin P. Gu Y. Morgan D.O. J. Cell Biol. 1996; 134: 963-970Crossref PubMed Scopus (253) Google Scholar, 13Terada Y. Tatsuka M. Jinno S. Okayama H. Nature. 1995; 376: 358-362Crossref PubMed Scopus (181) Google Scholar, 16Lock R.B. Ross W.E. Cancer Res. 1990; 50: 3761-3766PubMed Google Scholar, 17Poon R.Y.C. Jiang W. Toyoshima H. Hunter T. J. Biol. Chem. 1996; 271: 13283-13291Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 18Wang Q. Fan S.F. Eastman A. Worland P.J. Sausville E.A. O'Connor P.M. J. Natl. Cancer Inst. 1996; 88: 956-965Crossref PubMed Scopus (454) Google Scholar). The Schizosaccharomyces pombe wee1 gene product was the first kinase identified that is capable of phosphorylating Tyr15 in Cdc2 (19Parker L.L. Atherton-Fessler S. Lee M.S. Ogg S. Falk J.L. Swenson K.I. Piwnica-Worms H. EMBO J. 1991; 10: 1255-1263Crossref PubMed Scopus (164) Google Scholar). Homologs of the Wee1 kinase have been subsequently identified and biochemically characterized from a wide range of species including human, mouse, frog, Saccharomyces cerevisiae, and Drosophila (20Igarashi M. Nagata A. Jinno S. Suto K. Okayama H. Nature. 1991; 353: 80-83Crossref PubMed Scopus (157) Google Scholar, 21Parker L.L. Piwnica-Worms H. Science. 1992; 257: 1955-1957Crossref PubMed Scopus (559) Google Scholar, 22Watanabe N. Broome M. Hunter T. EMBO J. 1995; 14: 1878-1891Crossref PubMed Scopus (373) Google Scholar, 23Honda R. Tanaka H. Ohba Y. Yasuda H. Chromosome Res. 1995; 3: 300-308Crossref PubMed Scopus (23) Google Scholar, 24Mueller P.R. Coleman T.R. Dunphy W.G. Mol. Biol. Cell. 1995; 6: 119-134Crossref PubMed Scopus (270) Google Scholar, 25Booher R.N. Deshaies R.J. Kirschner M.W. EMBO J. 1993; 12: 3417-3426Crossref PubMed Scopus (290) Google Scholar, 26Campbell S.D. Sprenger F. Edgar B.A. O'Farrell P.H. Mol. Biol. Cell. 1995; 6: 1333-1347Crossref PubMed Scopus (55) Google Scholar). In vertebrate systems, where Thr14 in Cdc2 is also phosphorylated, the Wee1 kinase was capable of phosphorylating Cdc2 on Tyr15, but not Thr14, indicating that another kinase was responsible for Thr14 phosphorylation (21Parker L.L. Piwnica-Worms H. Science. 1992; 257: 1955-1957Crossref PubMed Scopus (559) Google Scholar, 27McGowan C.H. Russell P. EMBO J. 1993; 12: 75-85Crossref PubMed Scopus (398) Google Scholar). Direct evidence for the existence of a kinase activity that phosphorylated Cdc2 on Thr14 in the membrane fractions ofXenopus egg extracts has been reported (28Kornbluth S. Sebastian B. Hunter T. Newport J. Mol. Biol. Cell. 1994; 5: 273-282Crossref PubMed Scopus (102) Google Scholar). TheXenopus gene encoding this membrane-associated kinase, the Myt1 kinase, has been isolated, and its gene product was shown to be capable of phosphorylating Thr14 and, to a lessor extent, Tyr15 in Cdc2 (29Mueller P.R. Coleman T.R. Kumagai A. Dunphy W.G. Science. 1995; 270: 86-90Crossref PubMed Scopus (548) Google Scholar). A human Myt1 homolog displaying similar properties has been recently identified (30Liu F. Stanton J.J. Wu Z. Piwnica-Worms H. Mol. Cell. Biol. 1997; 17: 571-583Crossref PubMed Scopus (273) Google Scholar) (see "Results"). An apparently unrelated, non-membrane-associated Thr14 kinase activity has also been identified in bovine thymus cytosol (31Matsuura I. Wang J.H. J. Biol. Chem. 1996; 271: 5443-5450Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). While Xenopus Myt1 clearly plays an important role in regulating the rapid early embryonic cell cycles, its role in cells that have distinct G1 and G2 phases as well as growth and checkpoint controls is not known. To investigate the role of Cdk Thr14 phosphorylation in somatic cell cycle control, we have initiated a biochemical analysis of the human Myt1 kinase. The results presented reveal that Myt1, unlike Wee1, exhibits a restricted substrate specificity; it was capable of phosphorylating Thr14 only on Cdc2·cyclin and not Cdk2·cyclin complexes. We also found that endogenous Myt1 kinase activity was reduced during a drug-induced M-phase arrest, concomitant with hyperphosphorylation of the Myt1 protein, implying that Myt1 may be negatively regulated by phosphorylation. A 325-base pair DNA fragment, corresponding to an internal region of EST clones 335241 and 336264, was polymerase chain reaction-generated using oligos 5′-AGCAGCCTCTCCAGCAACTGG-3′ and 5′-CAGAGAAGACCATGGAGTTCC-3′ (5′ and 3′ primers, respectively) and the first strand cDNA synthesis of fetal brain RNA (Clonetech) as a template. This DNA fragment was32P-labeled by random priming (Pharmacia) and used as a probe to screen approximately 7 × 105 clones of a human placental Lambda ZAPII cDNA library (Stratagene). Of nine positives isolated, clone 16 was found to contain the longest cDNA insert. The complete DNA sequence of this 1.98-kilobase pair insert was determined using an ABI sequencer. Frozen pellets of Sf9 cells containing baculovirus-expressed proteins were thawed and lysed by Dounce homogenization in 10 mm Hepes, pH 7.5, 1 mmEDTA, 1% Nonidet P-40, 1 mm DTT, and protease inhibitors (1 mm Pefabloc (Boehringer), 5 μg/ml aprotinin, 5 μg/ml leupeptin) at 4 °C, followed by ultracentrifugation for 30 min at 42,000 rpm in a TLA45 rotor at 4 °C. The clarified lysates were aliquoted and stored at −80 °C. Combinations of various Cdk, cyclin, Wee1, and Myt1 lysates were mixed together with an ATP regeneration system (5 × stock: 0.5 mg/ml creatine phosphokinase, 10 mm ATP, 350 mm phosphocreatine, 20 mm Hepes, pH 7.5, 10 mm MgCl2) for 25 min at 25 °C. To determine the Cdk·cyclin kinase activity in these extracts, 1 μl of the lysate was incubated with 9 μl of a histone H1 kinase mix (50 mm Tris, pH 7.5, 10 mm MgCl2, 1 mm DTT, 5 μg histone H1, 50 μm ATP, 1 μCi of [γ-32P]ATP (3000 Ci/mmol) for 10–15 min at 25 °C. This reaction was stopped with SDS sample buffer, resolved by SDS-PAGE, and analyzed by autoradiography. The coding regions of Myt1, full-length Wee1 (clone 1E-12 encoding p98/100Wee1 (22Watanabe N. Broome M. Hunter T. EMBO J. 1995; 14: 1878-1891Crossref PubMed Scopus (373) Google Scholar), provided by Dr. Tony Hunter, Salk Institute), and cyclins were cloned into the baculovirus transfer vector pAcoG such that the expressed protein contained an N-terminal EE-epitope (32Rubinfeld B. Souza B. Albert I. Munemitsu S. Polakis P. J. Biol. Chem. 1995; 270: 5549-5555Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar). The monomeric Cdc2 in Fig. 3 C contained a C-terminal EE-tag and the cyclin B1 complexed with Cdc2-AF in Fig. 2 A contained an N-terminal glutathioneS-transferase fusion. All other Cdc2 and Cdk2 subunits used in this study contained a C-terminal HA tag and have been described previously (11Gu Y. Turck C.W. Morgan D.O. Nature. 1993; 366: 707-710Crossref PubMed Scopus (724) Google Scholar, 33Desai D. Gu Y. Morgan D.O. Mol. Biol. Cell. 1992; 3: 571-582Crossref PubMed Scopus (195) Google Scholar). The coding region of Wee1Δ, which corresponds to the p49Wee1 truncated protein (20Igarashi M. Nagata A. Jinno S. Suto K. Okayama H. Nature. 1991; 353: 80-83Crossref PubMed Scopus (157) Google Scholar), was polymerase chain reaction-generated using the full-length Wee1 clone 1E-12 as a template. Cdk·cyclin complexes were obtained by mixing and incubating individual Cdk and cyclin lysates together prior to purification, except in the case of Cdk4·cyclin D1, which was obtained by co-infecting Sf9 cells with Cdk4 and cyclin D1-expressing baculoviruses. Additionally, Sf9 lysates containing Cdk-activating kinase (Cdk7 and cyclin H) (34Fisher R.P. Morgan D.O. Cell. 1994; 78: 713-724Abstract Full Text PDF PubMed Scopus (568) Google Scholar) had been added to Cdc2-wild type and cyclin B1 lysates during the activation incubation. Baculovirus-expressed proteins were immunoaffinity purified from Sf9 cell lysates using anti-Glu-Glu (EE) antibody-coupled agarose beads (32Rubinfeld B. Souza B. Albert I. Munemitsu S. Polakis P. J. Biol. Chem. 1995; 270: 5549-5555Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar) or affinity-purified using glutathione-agarose beads (Sigma).Figure 2In vitro phosphorylation of Cdk·cyclin complexes by Myt1. A, purified Myt1 or Wee1Δ (p49Wee1) kinases were incubated either alone (lanes 10 and11) or in combination with purified Cdc2·cyclin B1 (lanes 1–3), Cdc2-AF·cyclin B1 (lanes 4–6), or Cdc2(K−)·cyclin B1 (lanes 7–9) complexes in an in vitro kinase reaction containing [γ-32P]ATP. The reaction was subjected to SDS-PAGE and analyzed by autoradiography. The basis of the low level of32P-labeled Cdc2 in the untreated wild-type Cdc2·cyclin B1 kinase reaction (lane 1) is unknown, but a similar observation has been reported by Watanabe et al. (22Watanabe N. Broome M. Hunter T. EMBO J. 1995; 14: 1878-1891Crossref PubMed Scopus (373) Google Scholar).B, purified Cdk2·cyclin A (lanes 1–3), Cdk2·cyclin E (lanes 4–6), and Cdk4·cyclin D1 (lanes 7 and 8) complexes were incubated either alone or in combination with purified Myt1 or Wee1Δ (p49Wee1) in anin vitro kinase reaction and analyzed as in panel A. C, cyclin-dependent phosphorylation of Cdc2 by Myt1. Affinity-purified Cdc2·cyclin B1 complexes (lanes 1–3), monomeric Cdc2 (lanes 4–6), monomeric Cdc2 plus monomeric cyclin B1 (lanes 7–9), or monomeric cyclin B1 (lanes 10 and 11) proteins were incubated either alone or in combination with purified Myt1 or Wee1Δ (p49Wee1) in an in vitro kinase reaction and analyzed as in panel A. The faint band in lane 11 may represent an endogenous insect Cdk that co-purified with the human cyclin B1 protein.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In vitro 32P-labeling reactions were performed by preincubating 1 μl (1–7 μg) of purified Cdk·cyclin complexes in 4 μl of KAB (50 mm Tris, pH 7.5, 10 mmMgCl2, 10 mm DTT) containing 25 μm ATP for 20 min at 25 °C, followed by addition of 0.5 μl p49Wee1 (0.78 mg/ml) or Myt1 (2.2 mg/ml) in 4.5 μl of KAB containing 5 μCi of [γ-32P]ATP (3000 Ci/mmol) and incubating at room temperature for 20 min. The reaction was stopped with sample buffer and resolved by 12% SDS-PAGE. Mammalian cell lines CEM, U2-0S, and C-33A used in this study were obtained from the American Type Culture Collection. Various Myt1-containing DNA fragments were cloned into a derivative of mammalian expression vector pcDNA3 (Invitrogen) that contained a 5′-HA epitope-coding sequence such that the expressed Myt1 proteins contained an N-terminal HA epitope tag. The expressed Myt1 proteins are designated as follows: Myt1-1b, residues 10–499; Myt1-1A, residues 75–499; Myt1-K−, residues 10–499 containing a Lys139 to Ala139 change that was created by site-directed mutagenesis; Myt1-ΔT.M., residues 10–499 containing a deletion of the transmembrane domain (residues 379–397) 2R. N. Booher, P. S. Holman, and A. Fattaey, unpublished data. (30Liu F. Stanton J.J. Wu Z. Piwnica-Worms H. Mol. Cell. Biol. 1997; 17: 571-583Crossref PubMed Scopus (273) Google Scholar). The S100 and P100 fractions were prepared from Myt1-expressing cell lines that had been lysed by Dounce homogenization in an ice-chilled hypotonic buffer (10 mm Hepes, pH 7.5, 5 mm KCl, 1.5 mm MgCl2, protease inhibitors). This lysate was ultracentrifuged for 30 min at 40,000 rpm in a TLA45 rotor at 4 °C to obtain the S100 supernatant. The membrane-bound proteins in the P100 fractions were solubilized by extracting this fraction in the same buffer containing 1% Nonidet P-40 for 30 min at 4 °C. The remaining insoluble material was removed by ultracentrifugation for 30 min at 40,000 rpm in a TLA45 rotor at 4 °C. Synchronization of CEM cells by drug treatments was performed by culturing cells for 16–23 h in the presence of either 5 mmhydroxyurea (Calbiochem), 1 mm mimosine (Sigma), or 50 ng/ml nocodazole (Calbiochem). U2-OS cells were treated with 125 ng/ml nocodazole. Myt1 peptides N2 (MPMPTEGTPPPLSGC, residues 10–23), C1 (CNSEPPRGSFPSFEPRN, residues 472–487), and C2 (CRNLLSMFEDTLDPT, residues 486–499) were coupled to activated keyhole limpet hemocyanin (Pierce) (via the included cysteine residues) and injected into rabbits (two per peptide), resulting in the respective sera N2-1, N2-2, C1-1, C1-2, C2-1, and C2-2. Mouse antibodies against Myt1 protein were raised by injecting three mice with purified Myt1 (residues 9–499). Monoclonal antibody 12CA5 was used to detect HA-tagged proteins. Mammalian cells were lysed in ELB (50 mmHepes, pH 7.4, 250 mm NaCl, 0.1% Nonidet-P40, 1 mm DTT) with protease inhibitors and clarified by microcentifugation at 8200 × g for 15 min. Myt1 was immunoprecipitated by incubating 2 μl of anti-Myt1 antibody serum in 0.2–0.4 ml of ELB containing 1 mg of lysate for 30 min at 4 °C, followed by addition of 25 μl of protein A-Sepharose (1:1 slurry, Pierce). After 1 h at 4 °C, the beads were pelleted, washed three times with ELB, and once with KAB. To assess kinase activity, 10 μl of Cdc2·cyclin B1 kinase reaction (1.5 μl of Cdc2·cyclin B1 Sf9 lysates mix, see above, in 8.5 μl of KAB containing 7.5 μm ATP) was added to the beads and incubated for 45 min at room temperature with periodic mixing. Additionally, 10 μm final concentration of the corresponding peptide was included during kinase assays of C-terminal antibody immunoprecipitates. This reaction was then assayed for histone H1 kinase activity by adding 5 μl of a histone H1 kinase mix (50 mm Tris, pH 7.5, 10 mm MgCl2, 1 mm DTT, 5 μg of histone H1, 1 μCi of [γ-32P]ATP (3000 Ci/mmol)) and incubating for 15 min at 30 °C with agitation. This reaction was stopped with SDS sample buffer, resolved by SDS-PAGE, and analyzed by autoradiography. For quantitation, the labeled bands were excised, and32P-incorporation was determined by Cerenkov counting. The mean of individual kinase assays performed on two or three aliquots of the same immunoprecipitation was determined. Clarified mammalian cell lysates, immunoprecipitates, and Sf9 cell lysates were fractionated by SDS-PAGE (Novex) and transferred to Immobilon polyvinylidene difluoride membranes (Millipore) using a Mini Trans-Blot cell (Bio-Rad). The filters were blocked with Blotto (15% nonfat dry milk in TBST (0.15m NaCl, 0.1% Tween 20, and 10 mm Tris, pH 8.0)), washed with TBST, and incubated with primary antibodies (1:1000) and secondary antibodies, horseradish peroxidase-conjugated goat anti-mouse or horseradish peroxidase-conjugated goat anti-rabbit (1:10,000 dilution, Bio-Rad), in blocking solutions. Immunodetection was performed using an enhanced chemiluminescence system (Amersham Corp.). Analysis of Myt1 proteins in Xenopus interphase and mitotic egg extracts was performed essentially as described by Stukenberg et al.(35Stukenberg T.P. Lustig K.D. McGarry T.J. King R.W. Kuang J. Kirschner M.W. Curr. Biol. 1997; 7: 338-348Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar) except that the Myt1 proteins were produced from pcDNA3-containing Myt1 plasmids in a coupled transcription/translation system (TNT, Promega) in the absence of radiolabel. To identify a human homolog of the Xenopus MYT1 gene, we performed a TBLASTN search of the EST data base using the amino acid sequence encoded byXenopus MYT1 as the query (29Mueller P.R. Coleman T.R. Kumagai A. Dunphy W.G. Science. 1995; 270: 86-90Crossref PubMed Scopus (548) Google Scholar). As expected, the majority of high scoring matches were protein kinases, owing to the extensive conservation of residues within the catalytic domain of protein kinases (36Hanks S.K. Quinn A.M. Hunter T. Science. 1988; 241: 42-52Crossref PubMed Scopus (4104) Google Scholar). However, two nearly identical human ESTs (dbEST Id: 336264 and 335241) were found to match significantly amino acid tracts within the C-terminal (noncatalytic domain) 130 residues of the XenopusMyt1 kinase. A full-length cDNA clone was isolated and found to encode the recently described human Myt1 kinase (30Liu F. Stanton J.J. Wu Z. Piwnica-Worms H. Mol. Cell. Biol. 1997; 17: 571-583Crossref PubMed Scopus (273) Google Scholar). To analyze human Myt1 activity, we examined its ability to inactivate Cdk·cyclin complexes in a cell-free extract system. We used the baculovirus expression system to produce insect cell extracts that individually contained high levels of either human Cdk, cyclin, Wee1, or Myt1 proteins. It has been shown previously that Cdk·cyclin kinase activation can be reconstituted upon mixing individual Cdk and cyclin-containing lysates (33Desai D. Gu Y. Morgan D.O. Mol. Biol. Cell. 1992; 3: 571-582Crossref PubMed Scopus (195) Google Scholar); thus, addition of an inhibitory activity would be expected to block this activation. We first assessed the ability of this assay system to detect Wee1 activity. Lysates containing wild-type Cdc2 were mixed with cyclin B1 lysates and with either mock-infected or Wee1-containing lysates. After a brief incubation, an aliquot of this lysate mixture was removed and Cdc2·cyclin B1 kinase activity was determined by performing a histone H1 kinase reaction. As shown in Fig. 1 A, addition of Wee1 lysates effectively inhibited Cdc2·cyclin B1 kinase activation. The Wee1 inhibitory activity was dependent on the presence of the Tyr15 residue in Cdc2, since both Cdc2-F15 (Tyr15 replaced with phenylalanine) and Cdc2-AF (Thr14 and Tyr15 replaced with alanine and phenylalanine) proteins complexed to cyclin B1 retained full activity in the presence of Wee1 lysates. When Myt1 lysates were combined together with Cdc2 and cyclin B1-containing lysates, a dramatic reduction in Cdc2 kinase activation was also observed (Fig. 1 B). Furthermore, Myt1 lysates inhibited both Cdc2-A14 and Cdc2-F15 activation, indicating that Myt1 can phosphorylate either Thr14 or Tyr15inhibitory sites in Cdc2; although it appears that Myt1 preferentially phosphorylates Thr14 since Cdc2-A14 lysates consistently retained some histone H1 kinase activity. To determine whether Myt1 can inactivate Cdk2·cyclin complexes, we measured the kinase activity of Cdk2 that had been activated in the presence of Myt1-containing lysates. We used the Cdk2-AF mutant as a reference to control for any non-Thr14/Tyr15phosphorylation inhibitory activity that may be present in the Myt1 lysate. As shown in Fig. 1 C, an equivalent amount of histone H1 kinase activity was present in wild-type Cdk2 and Cdk2-AF lysates that had been mixed with either cyclin A or cyclin E lysates in the presence of Myt1, indicating that neither Cdk2·cyclin A nor Cdk2·cyclin E complexes were inactivated by Myt1. However, when Cdc2 was substituted for Cdk2 in the cyclin A complex, Myt1 significantly reduced histone H1 kinase activity, as compared with mock-treated or the Cdc2-AF control lysate (Fig. 1 D). Thus, the Cdk subunit, not the cyclin subunit, is a key determinant for whether a particular Cdk·cyclin complex is recognized by the Myt1 kinase. To analyze Myt1 substrate specificity further, various combinations of affinity purified Myt1, Wee1, and Cdk·cyclin kinases were incubated together in an in vitro kinase reaction containing [γ-32P]ATP followed by SDS-PAGE analysis of the32P-labeled proteins. As shown in Fig. 2 A, both purified Myt1 and Wee1 were capable of phosphorylating wild-type Cdc2 as well as a kinase-deficient Cdc2 mutant (K−), each of which was complexed with cyclin B1. The Cdc2-AF mutant was not32P-labeled by Myt1 or Wee1, indicating that these kinases were phosphorylating Cdc2 on residue Thr14 and/or Tyr15. Direct phosphoamino acid analysis and immunoblot analysis using anti-Cdc2 phospho-specific antibodies showed that human Myt1, while having some Tyr15 activity, preferentially phosphorylated Cdc2 on Thr14 (data not shown), consistent with previous studies of human and Xenopus Myt1 (29Mueller P.R. Coleman T.R. Kumagai A. Dunphy W.G. Science. 1995; 270: 86-90Crossref PubMed Scopus (548) Google Scholar,30Liu F. Stanton J.J. Wu Z. Piwnica-Worms H. Mol. Cell. Biol. 1997; 17: 571-583Crossref PubMed Scopus (273) Google Scholar). When Cdk2·cyclin complexes were used as substrates in these kinase reactions, we found that Myt1 failed to phosphorylate Cdk2 that was complexed with cyclins A or E, as well as Cdk4 complexed with cyclin D1 (Fig. 2 B). In contrast, Wee1 readily phosphorylated Cdk2 complexed with cyclins A or E, but not Cdk4 complexed with cyclin D1, as has been previously demonstrated (22Watanabe N. Broome M. Hunter T. EMBO J. 1995; 14: 1878-1891Crossref PubMed Scopus (373) Google Scholar). Thus, using either a crude lysate system or an in vitro kinase reaction with purified components, we consistently observed that Myt1 only phosphorylated and inactivated Cdk·cyclin complexes in which Cdc2 was the catalytic subunit. To assess whether Myt1 phosphorylation of Cdc2 requires cyclin association, we performed a Myt1 kinase reaction using monomeric Cdc2 as substrate. As shown in Fig. 2 C, Myt1 did not phosphorylate monomeric Cdc2. However, reconstitution of the Cdc2·cyclin B1 complex, by preincubating monomeric Cdc2 with monomeric cyclin B1, enabled Myt1 to phosphorylate Cdc2, indicating that Myt1 can only phosphorylate Cdc2 that is complexed with a cyclin subunit. The truncated Wee1 kinase was capable of32P-labeling monomeric Cdc2, but this phosphorylation was enhanced when the cyclin-bound form was reconstituted, which is consistent with previous studies (37Parker L.L. Sylvestre P.J. Byrnes M.J. Liu F. Piwnica-Worms H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9638-9642Crossref PubMed Scopus (71) Google Scholar). To examine the regulation of endogenous Myt1, rabbit polyclonal antibodies were raised against three different peptides corresponding to Myt1 residues 10–23, 472–487, and 486–499. These antibodies are designated N2-1, C1-1, and C2-1, respectively. When used for immunoblotting of total cell lysates, cytosolic fractions, and detergent-solubilized membrane fractions prepared from asynchronously growing C-33A human cells, each of the anti-Myt1 antibodies recognized a 66-kDa membrane-associated protein (Fig. 3). This 66-kDa protein comigrated with baculovirus-expressed human Myt1 as well as Myt1 that was produced in an in vitrotranscription/translation-coupled reaction (data not shown). In Xenopus mitotic extracts, the electrophoretic mobility of Xe-Myt1 is drastically decreased, concomitant with Myt1 hyperphosphorylation and decreased kinase activity (29Mueller P.R. Coleman T.R. Kumagai A. Dunphy W.G. Science. 1995; 270: 86-90Crossref PubMed Scopus (548) Google Scholar). To determine whether human Myt1 exhibits similar cell cycle regulation, we examined Myt1 in lysates prepared from unsynchronized human CEM cells as well as cells that had been arrested at G1, S, and M phases of the cell cycle by treatment with mimosine, hydroxyurea, and nocodazole, respectively. Immunoblot analysis using anti-Myt1 C-terminal peptide antibodies (C1-1 and C2-1) showed that Myt1 shifted to a slower migrating form in mitotic lysates (Fig. 4 A). The main contribution to this modification is phosphorylation, since phosphatase treatment of the mitotic lysate increased the migration of Myt1 to nearly that observed in G1- and S-phase extracts (Fig. 4 B). At least five laddering Myt1 forms were detectable in the phosphatase-treated mitotic lysate, indicating that multiple residues are phosphorylated during mitosis. Further, immunoblot analysis of cytosolic and detergent-extracted membrane fractions prepared from nocodazole-arrested CEM cells revealed that human Myt1 remained membrane-associated during M-phase arrest (Fig. 4 C). Interestingly, anti-Myt1 antibody N2-1 failed to recognize the slower migrating Myt1 protein in mitotic lysates but was capable of detecting Myt1 in lysates prepared from unsynchronized as well as mimosine, or hydroxyurea-arrested CEM cells (Fig. 4 A). Phosphatase treatment of the mitotic lysate alleviated this Myt1 masking effect, suggesting that this antibody recognized only unphosphorylated Myt1 (data not shown). Consistent with the immunoblot results, this N-terminal antibody was incapable of immunoprecipitating mitotic Myt1 protein (Fig. 5 A). Since Myt1 likely spans the endoplasmic reticulum membrane2(30Liu F. Stanton J.J. Wu Z. Piwnica-Worms H. Mol. Cell. Biol. 1997; 17: 571-583Crossref PubMed Scopus (273) Google Scholar), it is possible that membrane association is required for Myt1 hyperphosphorylation observed during mitosis. To investigate this question, we established a U2-OS cell line that stably expressed a non-membrane-associated Myt1 mutant, Myt1ΔT.M., which lacks the 19-residue transmembrane domain (see "Experimental Procedures"). This Myt1ΔT.M.-expressing cell line was arrested in mitosis with nocodazole, and the soluble (S100) and detergent-solubilized particulate (P100) fractions were prepared. Immunoblot analysis of these fractions revealed that the S100-containing Myt1ΔT.M. protein exhibited a reduced mobility shift similar to that seen for the endogenous Myt1 protein (Fig. 4 D) present in mitotic extracts. This result indicates that a kinase(s) exists in the cytosol that is capable of phosphorylating Myt1 during mitosis, independent of Myt1 association with endoplasmic reticulum membranes. We cannot, however, rule out the possibility that this unidentified kinase is also present in the endoplasmic reticulum lumen. To characterize the kinase activity of endogenous human Myt1, anti-Myt1 antibody immunoprecipitates from CEM cell extracts were tested for their ability to inactivate various Cdk·cyclin complexes. Fig. 5 A shows that the C-terminal anti-Myt1 antibodies were capable of immunoprecipitating Myt1 from lysates prepared from nonsynchronized and mitotic cells, whereas, as described above, the N-terminal antibody failed to recognize mitotic Myt1. Consistent with our analysis of recombinant Myt1, anti-Myt1 immunoprecipitates dramatically reduced the histone H1 kinase activity of Cdc2·cyclin B1 and Cdc2-F15·cyclin B1 complexes, partially inhibited Cdc2-A14·cyclin B1 complexes, and had no effect on the Cdc2-AF·cyclin B1 complex (Fig. 5 B). Likewise, anti-Myt1 immunoprecipitates failed to inactivate Cdk2·cyclin A and Cdk2·cyclin E complexes (Fig. 5 C). These results demonstrate that the endogenous Myt1 kinase phosphorylates preferentially Cdc2 on residue Thr14 and does not phosphorylate Cdk2. To determine if the kinase activity of human Myt1 was cell cycle-dependent, we compared the kinase activity of Myt1 immunoprecipitates isolated from nonsynchronized and nocodazole-arrested CEM cells. Using two different C-terminal Myt1 peptide antibodies, Myt1 immunoprecipitated from mitotic extracts had approximately 40–50% reduced kinase activity (Fig. 6 A). Analysis of Myt1 immunoprecipitates using an N-terminal Myt1 peptide antibody showed that Myt1 was active in asynchronous, as well as mimosine- and hydroxyurea-arrested cells (Fig. 6 C). As discussed above, this N-terminal peptide antibody failed to immunoprecipitate Myt1 from nocodazole-arrested cells. These results strongly suggest that Myt1 is active during G1, S, and G2 but becomes inactive during M phase, concomitant with Myt1 hyperphosphorylation. The similarity of cell cycle-dependent kinase activity and phosphorylation state for both human and Xenopus Myt1 suggested that Myt1 kinases may be regulated by a conserved mechanism. As an initial test of this possibility we compared the electrophoretic mobility of human Myt1 that had been incubated in Xenopus interphase and mitotic egg extracts. As shown in Fig. 7 A,Xenopus mitotic extracts contained an activity that drastically reduced the electrophoretic mobility of each of the four human Myt1 proteins tested. The reduced mobility observed for Myt1 truncation mutants lacking the N-terminal 57 residues (-1A,lane 4) and C-terminal 206 residues2 indicates that phosphorylated residues responsible for the Myt1 mobility shift are not contained exclusively within either of these regions. Since the Xenopus mitotic extracts were derived from interphase egg extracts that had been induced into an M-phase state by addition of exogenous cyclin B1, it is possible that Cdc2·cyclin B1 may phosphorylate Myt1 directly, perhaps acting as an autocatalytic feedback mechanism such as has been proposed for XenopusCdc25 and Wee1 (24Mueller P.R. Coleman T.R. Dunphy W.G. Mol. Biol. Cell. 1995; 6: 119-134Crossref PubMed Scopus (270) Google Scholar, 38Izumi T. Maller J.L. Mol. Biol. Cell. 1995; 6: 215-226Crossref PubMed Scopus (111) Google Scholar). To address this possibility, we first determined whether Cdc2·cyclin B1 could affect the electrophoretic mobility of a kinase-defective Myt1 (Myt1-K−). Sf9 lysates containing Cdc2·cyclin B1 were incubated together with a Myt1-K− lysate in the presence of an ATP regeneration system. Immunoblot analysis of this reaction showed that the Myt1-K− protein migrated with a reduced mobility after Cdc2·cyclin B1 treatment (Fig. 7 B), albeit to a lesser degree than observed for Myt1 in mitotic extracts derived from human CEM cells (data not shown). Interestingly, Fig. 7 B also shows that the Myt1 N2-1 antibody exhibited a reduced ability to recognize Myt1 that had been phosphorylated by the Cdc2·cyclin B1 kinase. The direct phosphorylation of Myt1 by Cdc2·cyclin B1 was confirmed by including [γ-32P]ATP in an in vitro kinase reaction that contained immunoprecipitated Myt1 and purified Cdc2·cyclin B1 (Fig. 7 C). Phosphoamino acid analysis showed that Cdc2·cyclin B1 phosphorylated Myt1 on both threonine and serine residues (data not shown). Using an Sf9 lysate system, we examined whether the activity of Myt1 was affected by Cdc2·cyclin B1 phosphorylation. A Myt1-containing lysate was first mixed with a Cdc2-AF·cyclin B1 lysate, followed by subsequent addition of wild-type Cdc2·cyclin B1. The latter Cdc2·cyclin B1 complex was isolated by immunoprecipitation and its kinase activity was determined by performing a histone H1 kinase assay. We observed that phosphorylation of Myt1 by Cdc2-AF·cyclin B1 had no affect on its ability to inactivate Cdc2 kinase activity (data not shown). Hence, phosphorylation by Cdc2 does not appear to directly block Myt1 catalytic activity. In this study we report several biochemical properties of the human Myt1 kinase. We found that human Myt1 was capable of phosphorylating and inactivating Cdc2 associated with cyclin A or cyclin B1. In contrast, however, Myt1 was unable to phosphorylate or inactivate Cdk·cyclin complexes that function earlier in the cell cycle. In particular, Myt1 did not phosphorylate Cdk4·cyclin D1, Cdk2·cyclin E, or Cdk2·cyclin A complexes. The inability of Myt1 to phosphorylate Cdk4 was not unexpected since Cdk4 contains an alanine residue at the corresponding Thr14 position. Since Myt1 readily phosphorylated Cdc2·cyclin A complexes, Myt1 specificity appears to be determined principally by the Cdk subunit. In vivo, Cdk2 Tyr15 is phosphorylated to much greater extent than Thr14 (11Gu Y. Turck C.W. Morgan D.O. Nature. 1993; 366: 707-710Crossref PubMed Scopus (724) Google Scholar, 12Sebastian B. Kakizuka A. Hunter T. Proc. Natl. Acad. Sci. U. S. A.. 1993; 90: 3521-3524Crossref PubMed Scopus (215) Google Scholar), consistent with our in vitro results that Myt1 does not phosphorylate Cdk2 on Thr14. It is possible, however, that Myt1 can phosphorylate Cdk2 that has been initially phosphorylated on Tyr15, as has been previously suggested (11Gu Y. Turck C.W. Morgan D.O. Nature. 1993; 366: 707-710Crossref PubMed Scopus (724) Google Scholar). Alternatively, additional kinases may be responsible for phosphorylating Thr14 in Cdk2, such as a non-membrane-associated Thr14 kinase activity that is present in bovine thymus cytosol (31Matsuura I. Wang J.H. J. Biol. Chem. 1996; 271: 5443-5450Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). In either case, the significantly greater extent of Tyr15 phosphorylation on endogenous Cdk2 suggests that Wee1, or another Wee1-like kinase, is the major kinase activity that suppresses Cdk2·cyclin kinase activity during the G1/S transition. The kinase activity of human Myt1 was decreased during M-phase arrest. This decreased activity correlated with hyperphosphorylated and electrophoretically slower forms of Myt1. Indeed, phosphorylation of the high number of TP and SP amino acid doublets in both human andXenopus Myt1, 10 and 12, respectively, could account for the dramatic Myt1 hyperphosphorylation observed during M phase. Many of these TP/SP doublets conform to the Cdc2·cyclin B1 phosphorylation consensus site (39Songyang Z. Blechner S. Hoagland N. Hoekstra M.F. Piwnica-Worms H. Cantley L.C. Curr. Biol. 1994; 4: 973-982Abstract Full Text Full Text PDF PubMed Scopus (543) Google Scholar), consistent with our observation that Cdc2·cyclin B1 readily phosphorylated Myt1 in vitro. Our finding that an anti-Myt1 N-terminal peptide antibody recognized interphase but not M-phase Myt1, or Myt1 phosphorylated by Cdc2·cyclin B1 in vitro, strongly suggests that a residue near the N terminus is phosphorylated during mitosis. Which kinase(s) phosphorylates Myt1 during mitosis? Our in vitro data demonstrate that Cdc2·cyclin B1 can phosphorylate Myt1 on sites that reduce its mobility on SDS-polyacrylamide gels. While this phosphorylation did not affect Myt1 kinase activity, we cannot rule out the possibility that, in vivo, Cdc2 phosphorylation of Myt1 may be a requisite for further modifications that directly block Myt1 catalytic activity. Additionally, the partial reduction in Myt1 gel mobility induced by Cdc2·cyclin B1 phosphorylation suggests that an additional kinase(s) may phosphorylate Myt1. Further experiments will be required to discern the mechanism of Myt1 regulation during the cell cycle. We thank Brian Karlak and Eric Vermaas for assistance in identifying the initial human Myt1 clones. We are also very grateful to the following: Dr. Janice Williams for generously providing purified Cdk2·cyclin A and Cdk2·cyclin E complexes; Dr. David Morgan (University of California, San Francisco) for providing numerous Cdc2 and Cdk2 clones and recombinant baculoviruses; Dr. Emma Lees (DNAX) for providing cyclin clones; Dr. Todd Stukenberg (HMS) for providing Xenopus extracts and cyclins; Drs. Michelle Garrett, Nancy Pryer, and Abdallah Fanidi for helpful discussions; David Lowe and Drs. Jim Litts and Barbara Belisle for expressing and purifying the recombinant baculovirus proteins used throughout this study.
The expression of polyomavirus large T antigen in stably transfected C2 myoblast cells inhibits terminal differentiation without inducing a transformed phenotype. In the present work, we report on the lifting of this inhibition by a mutation that prevents polyomavirus large T antigen from binding to the product of the retinoblastoma susceptibility gene (p105 RB). In contrast with cells containing wild-type large T, those with the Rb binding site mutant large T showed the same up-regulation of myosine heavy chain and myogenin mRNA expression as control cells. Furthermore, we correlate the cell cycle alteration induced by polyomavirus large T antigen expression with the inability of the cells to undergo terminal differentiation.
