Cloning,sequencing, and expression of the genomic DNA encoding the protein phosphatase cdc25 in <Emphasis Type="Italic">Dictyostelium discoideum</Emphasis>

A genomic DNA (Dd-cdc25) encoding the protein phosphatase cdc25 was isolated from the cellular slime mold Dictyostelium discoideum. The Dd-cdc25 DNA sequence, with a length of 2,958 bp, encodes a protein consisting of 986 amino acid (aa) residues. The sequence shares significant identities with cdc25 from human, mouse, Xenopus, Drosophila, and Shizosaccharomyces pombe, particularly at the C-terminal region including the catalytic site for phosphatase activity. The deduced Dictyostelium cdc25 protein (Dd-cdc25) has the highest molecular mass (109.9 kDa) in several cdc25 species so far reported and contains four regions consisting of unusually long asparagine repeats (22–31) in the sequence. Unexpectedly, however, Western blot analysis using a specific antibody raised against the C terminus (aa 892–986) of Dd-cdc25 demonstrated that the protein exists as a short form (56 kDa), which has the C-terminal active site of phosphatase, during the course of Dictyostelium development. The Western blot analysis also revealed marked changes in the phosphorylated state of the Dd-cdc25, coupling with cellular development.Electronic Supplementary Material Supplementary material to this paper is available in electronic form at The sequence reported in this paper has been deposited in the DDBJ/EMBL/GenBank database with the accession number AB039883Edited by N. Satoh     

Cloning and characterization of a cdc25 phosphatase from mouse lymphocytes

Members of the cdc25 phosphatase family are proposed to function as important regulators of the eukaryotic cell cycle, particularly in the induction of mitotic events. A new cdc25 tyrosine phosphatase, cdc25M1, has been cloned from a mouse pre-B cell cDNA library and characterized. The cdc25M1 protein consists of 465 amino acids with a predicted relative molecular mass (M_r) of 51 750. Over the highly conserved carboxyl terminal region, the amino acid sequence similarity to the human cdc25 C or Hs1 isoform is 89%, while the overall similarity is 67%. The phosphatase active site is located within residues 367–374. Tissue expression of the cdc25M1 was highest in mouse spleen and thymus by northern blot analysis. The cdc25M1 mRNA was detected in a number of cloned mouse lymphocyte cell lines including both CD8⁺ and CD4⁺ cells. cdc25M1 mRNA was shown to be cell cycle-regulated in T cells following interleukin-2 (IL-2)-stimulation. Accumulation of cdc25M1 mRNA occured at 48 h after IL-2 stimulation, when lymphocytes were progressing from S phase to G₂/M phase of the cell cycle. This pattern of expression is in contrast to that observed for other protein tyrosine phosphatases expressed in T lymphocytes including CD45, LRP, SHP, and PEP. The elevation in cdc25M1 mRNA level occurred concomittant to the appearance of the hyperphosphorylated form of p34^cdc2 protein kinase. A purified, bacterial-expressed recombinant cdc25M1 phosphatase domain catalyzed the dephosphorylation of p-nitrophenol phosphate, as well as [³²P-Tyr] and [³²P-Ser/Thr]-containing substrates. Preincubation of p34^cdc2 kinase with cdc25M1 activated its histone H1 kinase activity in vitro. These results suggest that cdc25M1 may be involved in regulating the proliferation of mouse T lymphocytes following cytokine stimulation, through its action on p34^cdc2 kinase.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number L16926.     

The <Emphasis Type="Italic">alp1-1315</Emphasis> mutation of the tubulin-folding cofactor D gene delays the mitosis initiation in <Emphasis Type="Italic">cdc25-22</Emphasis> mutant cells of <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis>

Tubulin-folding cofactor D is necessary for the assembly of tubulin heterodimers and, possibly, plays additional roles in the cell. The effects of cofactor D, microtubules, and/or tubulin dimers on the mitosis initiation were studied in Schizosaccharomyces pombe. It was found for the first time that S. pombe cells with the alp1-1315 and cdc25-22 mutations remained highly viable at 36°C for 8 h, in contrast to cells with the alp1-1315 mutation alone. The progression of cdc25-22 alp1-1315 cells through mitosis after a cell division arrest at 36°C was described. When transferred to 25°C, cdc25-22 alp1-1315 cells displayed a lag of approximately 30 min in Plo1-GFP appearance in the spindle pole body (SPB), 1 h in chromosome condensation, and 75 min in spindle formation. Thus, the initiation of mitosis in cdc25-22 alp1-1315 cells was delayed as compared with cdc25-22 cells. Since treatment of cdc25-22 cells with a microtubule-destabilizing drug during an arrest is known to cause a premitotic arrest with low activity of the mitosis-promoting factor (MPF), it was assumed that an impaired integrity of microtubules and/or lack of tubulin dimers in the nucleus were responsible for the delayed mitosis initiation in cdc25-22 alp1-1315 cells and in cdc25-22 cells treated with a microtubule-destabilizing drug. The progression through mitosis after a cdc25-22 arrest was extremely slow in cdc25-22 alp1-1315 cells, which was attributed to the de novo formation of tubulin dimers.     

Distinct Pools of cdc25C Are Phosphorylated on Specific TP Sites and Differentially Localized in Human Mitotic Cells

Background

The dual specificity phosphatase cdc25C was the first human cdc25 family member found to be essential in the activation of cdk1/cyclin B1 that takes place at the entry into mitosis. Human cdc25C is phosphorylated on Proline-dependent SP and TP sites when it becomes active at mitosis and the prevalent model is that this phosphorylation/activation of cdc25C would be part of an amplification loop with cdk1/cyclin B1.

Methodology/Principal Findings

Using highly specific antibodies directed against cdc25C phospho-epitopes, pT67 and pT130, we show here that these two phospho-forms of cdc25C represent distinct pools with differential localization during human mitosis. Phosphorylation on T67 occurs from prophase and the cdc25C-pT67 phospho-isoform closely localizes with condensed chromosomes throughout mitosis. The phospho-T130 form of cdc25C arises in late G2 and associates predominantly with centrosomes from prophase to anaphase B where it colocalizes with Plk1. As shown by immunoprecipitation of each isoform, these two phospho-forms are not simultaneously phosphorylated on the other mitotic TP sites or associated with one another. Phospho-T67 cdc25C co-precipitates with MPM2-reactive proteins while pT130-cdc25C is associated with Plk1. Interaction and colocalization of phosphoT130-cdc25C with Plk1 demonstrate in living cells, that the sequence around pT130 acts as a true Polo Box Domain (PBD) binding site as previously identified from in vitro peptide screening studies. Overexpression of non-phosphorylatable alanine mutant forms for each isoform, but not wild type cdc25C, strongly impairs mitotic progression showing the functional requirement for each site-specific phosphorylation of cdc25C at mitosis.

Conclusions/Significance

These results show for the first time that in human mitosis, distinct phospho-isoforms of cdc25C exist with different localizations and interacting partners, thus implying that the long-standing model of a cdc25C/cdk1 multi-site auto amplification loop is implausible.     

Oncogenic potential of a C.elegans cdc25 gene is demonstrated by a gain-of-function allele

In multicellular organisms, developmental programmes must integrate with central cell cycle regulation to co-ordinate developmental decisions with cell proliferation. Hyperplasia caused by deregulated proliferation without significant change to other aspects of developmental behaviour is a probable step towards full oncogenesis in many malignancies. CDC25 phosphatase promotes progression through the eukaryotic cell cycle by dephosphorylation of cyclin-dependent kinase and, in humans, different cdc25 family members have been implicated as potential oncogenes. Demonstrating the direct oncogenic potential of a cdc25 gene, we identify a gain-of-function mutant allele of the Caenorhabditis elegans gene cdc-25.1 that causes a deregulated proliferation of intestinal cells resulting in hyperplasia, while other aspects of intestinal cell function are retained. Using RNA-mediated interference, we demonstrate modulation of the oncogenic behaviour of this mutant, and show that a reduction of the wild-type cdc-25.1 activity can cause a failure of proliferation of intestinal and other cell types. That gain and loss of CDC-25.1 activity has opposite effects on cellular proliferation indicates its critical role in controlling C.elegans cell number.     

Regulation of the cdc25 protein during the cell cycle in Xenopus extracts.

The cdc25 protein is a highly specific tyrosine phosphatase that triggers mitosis by dephosphorylating the cdc2 protein kinase. Using Xenopus extracts, we have found that the cdc25 protein is active at a low level throughout interphase. Near the onset of mitosis, the cdc25 protein undergoes a marked elevation in phosphatase activity that coincides with an extensive phosphorylation of the protein in its N-terminal region. In vitro dephosphorylation of this hyperphosphorylated form of cdc25 reduces its phosphatase activity back to the interphase level. Moreover, treatment of interphase Xenopus extracts with okadaic acid, a phosphatase inhibitor that accelerates the entry into mitosis, elicits both the premature hyperphosphorylation of cdc25 and the stimulation of its cdc2-specific tyrosine phosphatase activity. These experiments demonstrate the existence of a cdc25 regulatory system consisting of both a stimulatory kinase that phosphorylates a putative regulatory domain of the cdc25 protein and an inhibitory serine/threonine phosphatase that counteracts this kinase activity.     

Decreased UV mutagenesis in cdc8, a DNA replication mutant of Saccharomyces cerevisiae.

Summary A DNA replication mutant of yeast, cdc8, was found to decrease UV-induced reversion of lys2-1, arg4-17, tyr1 and ura1. This effect was observed with all three alleles of cdc8 tested. Survival curves obtained following UV irradiation in cdc8 rad double mutants show that cdc8 is epistatic to rad6, as well as to rad1; cdc8 rad51 double mutants seem to be more sensitive than the single mutants. Since UV-induced reversion in cdc8 rad1 and cdc8 rad51 double mutants is like that of the cdc8 single mutants, we conclude that CDC8 plays a direct role in error-prone repair. To test whether CDC8 codes for a DNA polymerase, we have purified both DNA polymerase I and DNA polymerase II from cdc8 and CDC+ cells. The purified DNA polymerases from cdc8 were no more heat labile than those from CDC+, suggesting that CDC8 is not a structural gene for either enzyme.     

Cytokinin controls the cell cycle at mitosis by stimulating the tyrosine dephosphorylation and activation of p34cdc2-like H1 histone kinase

In excised pith parenchyma from Nicotiana tabacum L. cv. Wisconsin Havana 38, auxin (naphthalene-1-acetic acid) together with cytokinin (6-benzylaminopurine) induced a greater than 40-fold increase in a p34^cdc2-like protein, recoverable in the p13^suc1-binding fraction, that had high H1 histone kinase activity, but enzyme induced without cytokinin was inactive. In suspension-cultured N. plumbaginifolia Viv., cytokinin (kinetin) was stringently required only in late G2 phase of the cell division cycle (cdc) and cells lacking kinetin arrested in G2 phase with inactive p34^cdc2-like H1 histone kinase. Control of the Cdc2 kinase by inhibitory tyrosine phosphorylation was indicated by high phosphotyrosine in the inactive enzyme of arrested pith and suspension cells. Yeast cdc25 phosphatase, which is specific for removal of phosphate from tyrosine at the active site of p34^cdc2 enzyme, was expressed in bacteria and caused extensive in-vitro activation of p13^suc1-purified enzyme from pith and suspension cells cultured without cytokinin. Cytokinin stimulated the removal of phosphate, activation of the enzyme and rapid synchronous entry into mitosis. Therefore, plants can control cell division by tyrosine phosphorylation of Cdc2 but differ from somatic animal cells in coupling this mitotic control to hormonal signals.Abbreviations BAP 6-benzylaminopurine - BrdUrd 5-bromo-2-deoxyuridine - cdc cell division cycle - Cdc25 cdc phospho-protein phosphatase - CKI cyclin dependent kinase inhibitor - 2,4-D 2,4-dichlorophenoxyacetic acid - DAPI 4,6 diamidino-2-phenylindole - GST-cdc25 glutathione sulfur transferase-truncated cdc25 fusion - MS Murashige and Skoog (1962) - NAA naphthalene-1-acetic acid - p34^cdc2 34-kDa product of the cdc2 gene     

The plant cell cycle

The first aim of this paper is to review recent progress in identifying genes in plants homologous to cell division cycle (cdc) genes of fission yeast. In the latter, cdc genes are well-characterised. Arguably, most is known about cdc2 which encodes a 34 kDa protein kinase (p34^cdc2) that functions at the G2-M and G1-S transition points of the cell cycle. At G2-M, the p34^cdc2 protein kinase is regulated by a number of gene products that function in independent regulatory pathways. The cdc2 kinase is switched on by a phosphatase encoded by cdc25, and switched off by a protein kinase encoded by weel. p34 Must also bind with a cyclin protein to form maturation promoting factor before exhibiting protein kinase activity. In plants, homologues to p34^cdc2 have been identified in pea, wheat, Arabidopsis, alfalfa, maize and Chlamydomonas. They all exhibit the PSTAIRE motif, an absolutely conserved amino acid sequence in all functional homologues sequenced so far. As in animals, some plant species contain more than one cdc2 protein kinase gene. but in contrast to animals where one functions at G2-M and the other (CDK2 in humans and Egl in Xenopus) at G1-S, it is still unclear whether there are functional differences between the plant p34^cdc2 protein kinases. Again, whereas in animals cyclins are well characterised on the basis of sequence analysis, into class A, class B (G2-M) and CLN (G1 cyclins), cyclins isolated from several plant species cannot be so clearly characterised. The differences between plant and animal homologues to p34^cdc2 and cyclins raises the possibility that some of the regulatory controls of the plant genes may be different from those of their animal counterparts. The second aim of the paper is to review how planes of cell division and cell size are regulated at the molecular level. We focus on reports showing that p34^cdc2 binds to the preprophase band (ppb) in late G2 of the cell cycle. The binding of p34^cdc2 to ppbs may be important in regulating changes in directional growth but, more importantly, there is a requirement to understand what controls the positioning of ppbs. Thus, we highlight work resolving proteins such as the microtubule associated proteins (MAPs) and those mitogen activated protein kinases (MAP kinases), which act on, or bind to, mitotic microtubules. Plant homologues to MAP kinases have been identified in alfalfa. Finally, some consideration is given to cell size at division and how alterations in cell size can alter plant development. Transgenic tobacco plants expressing the fission yeast gene, cdc25, exhibited various perturbations of development and a reduced cell size at division. Hence, cdc25 affected the cell cycle (and as a consequence, cell size at division) and cdc25 expression was correlated with various alterations to development including precocious flowering and altered floral morphogenesis. Our view is that the cell cycle is a growth cycle in which a cell achieves an optimal size for division and that this size control has an important bearing on differentiation and development. Understanding how cell size is controlled, and how plant cdc genes are regulated, will be essential keys to ‘the cell cycle locks’, which when ‘opened’, will provide further clues about how the cell cycle is linked to plant development.     

Sensor and effector kinases in DNA damage checkpoint regulate capacity for homologous recombination repair of fission yeast in G2 phase

Although the G2/M DNA damage checkpoint is currently viewed as a set of coordinated cellular responses affecting both cell cycle progression and non-cell cycle targets, the relative contributions of the two target categories to DNA repair and cell survival after exposure to ionizing radiation have not been clearly addressed. We investigated how rad3 (ATR ortholog) or chk1/cds1 (CHK1/CHK2 orthologs) null mutations change the kinetics of double-strand break (DSB) repair in Schizosaccharomyces pombe cells under conditions of forced G2 arrest. After 200-Gy γ-ray irradiation, DSBs were repaired in rad3Δ cdc25-22 or chk1Δ cds1Δ cdc25-22 cells, almost as efficiently as in cdc25-22 cells at the restrictive temperature. In contrast, little repair was observed in the checkpoint-deficient cells up to 4h after higher-dose (500Gy) irradiation, whereas repair was still efficient in the control cdc25-22 cells. Immediate loss of viability appeared not be responsible for the repair defect after the higher dose, since both checkpoint-proficient and deficient cells with cdc25-22 allele synchronously resumed cycling with a similar time course when released to the permissive temperature 4h after irradiation. Recruitment of repair proteins Rad11 (Rpa1 ortholog), Rad22 (Rad52 ortholog), and Rhp54 (Rad54 ortholog) to the damage sites was not significantly impaired in the checkpoint-deficient cells, whereas their release was profoundly delayed. Our results suggest that sensor and effector kinases in the damage checkpoint machinery affect the efficiency of repair downstream of, or in parallel with the core repair reaction.     

Purification and biochemical analysis of catalytically active human cdc25C dual specificity phosphatase

We describe a reliable and efficient method for the purification of catalytically active and mutant inactive full-length forms of the human dual specificity phosphatase cdc25C from bacteria. The protocol involves isolating insoluble cdc25C protein in inclusion bodies, solubilization in guanidine HCL, and renaturation through rapid dilution into low salt buffer. After binding renatured proteins to an ion exchange resin, cdc25C elutes in two peaks at 350 and 450 mM NaCl. Analysis by gel exclusion chromatography and enzymatic assays reveals the highest phosphatase activity is associated with the 350 mM NaCl with little or no activity present in the 450 mM peak. Furthermore, active cdc25C has a native molecular mass of 220 kDa consistent with a potential tetrameric complex of the 55-kDa cdc25C protein. Assaying phosphatase activity against artificial substrates pNPP and 3-OMFP reveals a 220 kDa form of the phosphatase is active in a non-phosphorylated state. The protein effectively activates cdk1/cyclin B prokinase complexes in vitro in the absence of cdk1 kinase activity in an orthovanadate sensitive manner but is inactivated by A-kinase phosphorylation. In vitro phosphorylation of purified cdc25C by cdk1/cyclin B1, cdk2/cyclin A2 and cdk2/cyclin E shows that distinct TP/SP mitotic phosphorylation sites on cdc25C are differentially phosphorylated by these 3 cdk/cyclin complexes associated with different levels of cdc25C activation. Finally, we show that endogenous native cdc25C from human cells is present in high molecular weight complexes with other proteins and resolves mostly above 200-kDa. These data show that untagged cdc25C can be purified with a simple protocol as an active dual specificity phosphatase with a native molecular mass consistent with a homo-tetrameric configuration.     

人源Cdc25C蛋白的融合表达及纯化

目的:表达并纯化有活性的GST-Cdc25C融合蛋白,以用于Cdc25C功能研究。方法:利用RT-PCR克隆MCF-7细胞的cdc25c全长基因;在大肠杆菌中表达GST-Cdc25C融合蛋白;利用GSH交联的琼脂糖珠纯化GST-Cdc25C融合蛋白;通过体外磷酸酶活性分析检测GST-Cdc25C融合蛋白的磷酸酶活性。结果:克隆获得1465 bp的人源cdc25c全长基因,并克隆至pGEX-4T-1原核表达载体;在原核系统中可溶性表达了相对分子质量约87×103的GST-Cdc25C融合蛋白;通过亲和纯化获得的GST-Cdc25C融合蛋白具有较好的磷酸酶活性。结论:得到了有磷酸酶活性的GST-Cdc25C融合蛋白,可用于后续的Cdc25C功能研究。     

The Caenorhabditis elegans gene ncc-1 encodes a cdc2-related kinase required for M phase in meiotic and mitotic cell divisions, but not for S phase

We have identified six protein kinases that belong to the family of cdc2-related kinases in Caenorhabditis elegans. Results from RNA interference experiments indicate that at least one of these kinases is required for cell-cycle progression during meiosis and mitosis. This kinase, encoded by the ncc-1 gene, is closely related to human Cdk1/Cdc2, Cdk2 and Cdk3 and yeast CDC28/cdc2(+). We addressed whether ncc-1 acts to promote passage through a single transition or multiple transitions in the cell cycle, analogous to Cdks in vertebrates or yeasts, respectively. We isolated five recessive ncc-1 mutations in a genetic screen for mutants that resemble larval arrested ncc-1(RNAi) animals. Our results indicate that maternal ncc-1 product is sufficient for embryogenesis, and that zygotic expression is required for cell divisions during larval development. Cells that form the postembryonic lineages in wild-type animals do not enter mitosis in ncc-1 mutants, as indicated by lack of chromosome condensation and nuclear envelope breakdown. However, progression through G1 and S phase appears unaffected, as revealed by expression of ribonucleotide reductase, incorporation of BrdU and DNA quantitation. Our results indicate that C. elegans uses multiple Cdks to regulate cell-cycle transitions and that ncc-1 is the C. elegans ortholog of Cdk1/Cdc2 in other metazoans, required for M phase in meiotic as well as mitotic cell cycles.     

Evolution of the PP2C Family in Caenorhabditis: Rapid Divergence of the Sex-Determining Protein FEM-2

To investigate the causes and functional significance of rapid sex-determining protein evolution we compared three Caenorhabditis elegans genes encoding members of the protein phosphatase 2C (PP2C) family with their orthologs from another Caenorhabditis species (strain CB5161). One of the genes encodes FEM-2, a sex-determining protein, while the others have no known sex-determining role. FEM-2's PP2C domain was found to be more diverged than the other PP2C domains, supporting the notion that sex-determining proteins are subjected to selective pressures that allow for or cause rapid divergence. Comparison of the positions of amino acid substitutions in FEM-2 with a solved three-dimensional structure suggests that the catalytic face of the protein is highly conserved among C. elegans, CB5161, and another closely related species C. briggsae. However, the non-conserved regions of FEM-2 cannot be said to lack functional importance, since fem-2 transgenes from the other species were unable to rescue the germ-line defect caused by a C. elegans fem-2 mutation. To test whether fem-2 functions as a sex-determining gene in the other Caenorhabditis species we used RNA-mediated interference (RNAi). fem-2 (RNAi) in C. elegans and C. briggsae caused germ-line feminization, but had no noticeable effect in CB5161. Thus the function of fem-2 in CB5161 remains uncertain. Received: 11 April 2001 / Accepted: 6 August 2001     

Cdc25A is a novel phosphatase functioning early in the cell cycle.

The cdc25+ tyrosine phosphatase is a key mitotic inducer of the fission yeast Schizosaccharomyces pombe, controlling the timing of the initiation of mitosis. Mammals contain at least three cdc25+ homologues called cdc25A, cdc25B and cdc25C. In this study we investigate the biological function of cdc25A. Although very potent in rescuing the S.pombe cdc25 mutant, cdc25A is less structurally related to the S.pombe enzyme. Northern and Western blotting detection reveals that unlike cdc25B, cdc25C and cdc2, cdc25A is predominantly expressed in late G1. Moreover, immunodepletion of cdc25A in rat cells by microinjection of a specific antibody effectively blocks their cell cycle progression from G1 into the S phase, as determined by laser scanning single cell cytometry. These results indicate that cdc25A is not a mitotic regulator but a novel phosphatase that plays a crucial role in the start of the cell cycle. In view of its strong ability to activate cdc2 kinase and its specific expression in late G1, cdc2-related kinases functioning early in the cell cycle may be targets for this phosphatase.     

The disappearance of cyclins A and B and the increase in activity of the G(2)/M-phase cellular kinase cdc2 in herpes simplex virus 1-infected cells require expression of the alpha22/U(S)1.5 and U(L)13 viral genes

In uninfected cells the G₂/M transition is regulated by cyclin kinase complex containing cdc2 and, initially, cyclin A, followed by cyclin B. cdc2 is downregulated through phosphorylation by wee-1 and myt-1 and upregulated by cdc-25C phosphatase. We have examined the accumulation and activities of these proteins in cells infected with wild type and mutants of herpes simplex virus 1. The results were as follows. (i) Cyclin A and B levels were reduced beginning 4 h after infection and were undetectable at 12 to 16 h after infection. (ii) cdc2 protein also decreased in amount but was detectable at all times after infection. In addition, a fraction of cdc2 protein from infected cells exhibited altered electrophoretic mobility in denaturing gels. (iii) The levels of cdk7 or myt-1 proteins remained relatively constant throughout infection, whereas the level of wee-1 was significantly decreased. (iv) cdc-25C formed novel bands characterized by slower electrophoretic mobility that disappeared after treatment with phosphatase. In addition, one phosphatase-sensitive band reacted with MPM-2 antibody that recognizes a phosphoepitope phosphorylated exclusively in M phase. (v) cdc2 accumulating in infected cells exhibited kinase activity. The activity of cdc2 was higher in infected cell lysates than those of corresponding proteins present in lysates of mock-infected cells even though cyclins A and B were not detectable in lysates of infected cells. (vi) The decrease in the levels of cyclins A and B, the increase in activity of cdc2, and the hyperphosphorylation of cdc-25C were mediated by U_L13 and α22/U_S1.5 gene products. In light of its normal functions, the activated cdc2 kinase may play a role in the changes in the morphology of the infected cell. These results are consistent with the accruing evidence that herpes simplex virus scavenges the cell for useful cell cycle proteins and subverts them for its own use.