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EVOLUTIONARY HOMOLOGS (part 1/2)

Cdc25 in yeast

Fission yeast cells tolerate the total absence of the cdc25 mitotic inducer in two cases, either in cdc2-3w or in wee1 genetic backgrounds. In the cdc2-3w;cdc25delta double mutant, the rate-limiting step leading to mitosis is reaching a critical size. However, the size control of this mutant operates in late G2, which is different from wild-type (WT) cells. This fact suggests that in WT the rate-limiting molecular process during the G2 timer is the Tyr15 dephosphorylation of cdc2, for which the cdc25 phosphatase (together with its back-up, pyp3) is dependent. In the cdc2-3w;cdc25delta mutant, the population splits into different clusters, all lacking mitotic size control. This strain maintains size homeostasis by a novel method, which is random movement of the cells from one cluster to another in the successive generations. These cells should normally have a 'minimal cycle', a 'timer' with short G1 and G2 phases. However, very often the cells abort mitosis, possibly at an early event and return back to early G2, thus lengthening their cycles. The inability of these cells to start anaphase might be caused by the absence of the main mitotic regulators (wee1 and cdc25) and the improper regulation of their back-up copies (mik1 and pyp3, respectively) (Sveiczer, 1999).

Cdc25 in C. elegans

The precise control of cell division during development is pivotal for morphogenesis and the correct formation of tissues and organs. One important gene family involved in such control is the p21/p27/p57 class of negative cell cycle regulators. Loss of function of the C. elegans p27 homolog, cki-1, causes extra cell divisions in numerous tissues including the hypodermis, the vulva, and the intestine. This study seeks to better understand how cell divisions are controlled upstream or in parallel to cki-1 in specific organs during C. elegans development. By taking advantage of the invariant cell lineage of C. elegans, an intestinal-specific GFP reporter was used in a screen to identify mutants that undergo cell division abnormalities in the intestinal lineage. A mutant was isolated with twice the wild-type complement of intestinal cells, all of which arise during mid-embryogenesis. This mutant, called rr31, is a fully dominant, maternal-effect, gain-of-function mutation in the cdc-25.1 cell cycle phosphatase that sensitizes the intestinal lineage to an extra cell division. cdc-25.1 acts at the G1/S transition, since ectopic expression of CDC-25.1 caused entry into S phase in intestinal cells. In addition, the cdc-25.1(gf) (the gain of function mutation) requires cyclin E. The extra cell division defect is to be restricted to the E lineage and the E fate is necessary and sufficient to sensitize cells to this mutation. In Drosophila, CDC25/String proteolysis has been shown to be important for the proper coordination of gastrulation and ingression of the mesoderm anlage. A similar mechanism might be acting in the coordination of C. elegans endodermal divisions, whereby correct division timing, with specification and function, is essential for gastrulation and ensuing embryogenesis (Kostic, 2002).

In multicellular organisms, developmental programs 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 behavior is a probable step toward 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, a gain-of-function mutant allele was identified 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, modulation of the oncogenic behaviour of this mutant has been demonstrated. A reduction of the wild-type cdc-25.1 activity can cause a failure of proliferation of intestinal and other cell types. The fact 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 (Cucas, 2002).

Cdc25 phosphatases are key positive cell cycle regulators that coordinate cell divisions with growth and morphogenesis in many organisms. Intriguingly in C. elegans, two cdc-25.1(gf) mutations induce tissue-specific and temporally restricted hyperplasia in the embryonic intestinal lineage, despite stabilization of the mutant CDC-25.1 protein in every blastomere. This study investigated the molecular basis underlying the CDC-25.1(gf) stabilization and its associated tissue-specific phenotype. Both mutations were found affect a canonical β-TrCP phosphodegron motif, while the F-box protein LIN-23, the β-TrCP orthologue, is required for the timely degradation of CDC-25.1. Accordingly, depletion of lin-23 in wild-type embryos stabilizes CDC-25.1 and triggers intestinal hyperplasia, which is, at least in part, cdc-25.1 dependent. lin-23(RNAi) causes embryonic lethality owing to cell fate transformations that convert blastomeres to an intestinal fate, sensitizing them to increased levels of CDC-25.1. This characterization of a novel destabilizing cdc-25.1(lf) intragenic suppressor that acts independently of lin-23 indicates that additional cues impinge on different motifs of the CDC-25.1 phosphatase during early embryogenesis to control its stability and turnover, in order to ensure the timely divisions of intestinal cells and coordinate them with the formation of the developing gut (Hebeisen, 2008).

The early cell divisions of the C. elegans embryo are precisely controlled by gene products that are provided from the maternal germ line. The isolation of two gain-of-function alleles of CDC-25.1 that demonstrate a strict maternal effect indicates that this regulation can be perturbed, resulting in supernumerary cell divisions specifically within the E lineage. How these mutations give rise to the extra divisions is unclear, although CDC-25.1(rr31) is more stable than its wild-type counterpart. By developing a GFP-based transgenic assay to assess the dynamics of protein degradation during early embryogenesis, this study shows that the stabilization of CDC-25.1 is mediated by a point mutation within a conserved DSGX4S β-TrCP-like phosphodegron in both cdc-25.1(gf) mutants. This stabilizes the protein, resulting in the abnormal presence of CDC-25.1 during a short, yet crucial, window during early development, which is presumably the cause of the observed cell cycle defect (Hebeisen, 2008).

As a fundamental process of development, cell proliferation must be coordinated with other processes such as fate differentiation. Through statistical analysis of individual cell cycle lengths of the first 8 out of 10 rounds of embryonic cell division in C. elegans, synchronous and invariantly ordered divisions were identified that are tightly associated with fate differentiation. The results suggest a three-tier model for fate control of cell cycle pace: the primary control of cell cycle pace is established by lineage and the founder cell fate, then fine-tuned by tissue and organ differentiation within each lineage, then further modified by individualization of cells as they acquire unique morphological and physiological roles in the variant body plan. Then, attempts were made to identify the pace-setting mechanisms in different fates. The results suggest that ubiquitin-mediated degradation of CDC-25.1 is a rate-determining step for the E (gut) and P3 (muscle and germline) lineages but not others, even though CDC-25.1 and its apparent decay have been detected in all lineages. These results demonstrate the power of C. elegans embryogenesis as a model to dissect the interaction between differentiation and proliferation, and an effective approach combining genetic and statistical analysis at single-cell resolution (Bao, 2008).

Stat3/Cdc25a-dependent cell proliferation promotes embryonic axis extension during zebrafish gastrulation

Cell proliferation has generally been considered dispensable for anteroposterior extension of embryonic axis during vertebrate gastrulation. Signal transducer and activator of transcription 3 (Stat3) (see Drosophila Stat92E), a conserved controller of cell proliferation, survival and regeneration, is associated with human scoliosis, cancer and Hyper IgE Syndrome. Zebrafish Stat3 has been proposed to govern convergence and extension gastrulation movements in part by promoting Wnt/Planar Cell Polarity (PCP) signaling (see Drosophila wg), a conserved regulator of mediolaterally polarized cell behaviors. Using zebrafish stat3 null mutants and pharmacological tools, this study demonstrates that cell proliferation contributes to anteroposterior embryonic axis extension. Zebrafish embryos lacking maternal and zygotic Stat3 expression exhibit normal convergence movements and planar cell polarity signaling, but transient axis elongation defect due to insufficient number of cells resulting largely from reduced cell proliferation and increased apoptosis. Pharmacologic inhibition of cell proliferation during gastrulation phenocopies axis elongation defects. Stat3 regulates cell proliferation and axis extension in part via upregulation of Cdc25a (see Drosophila stg) expression during oogenesis. Accordingly, restoring Cdc25a expression in stat3 mutants partially suppresses cell proliferation and gastrulation defects. During later development, stat3 mutant zebrafish exhibit stunted growth, scoliosis, excessive inflammation, and fail to thrive, affording a genetic tool to study Stat3 function in vertebrate development, regeneration, and disease (Liu, 2017).

Coordination of cell proliferation and cell fate determination by CES-1 snail

The coordination of cell proliferation and cell fate determination is critical during development but the mechanisms through which this is accomplished are unclear. This study presents evidence that the Snail-related transcription factor CES-1 of Caenorhabditis elegans coordinates these processes in a specific cell lineage. CES-1 can cause loss of cell polarity in the NSM neuroblast. By repressing the transcription of the BH3-only gene egl-1, CES-1 can also suppress apoptosis in the daughters of the NSM neuroblasts. CES-1 also affects cell cycle progression in this lineage. Specifically, it was found that CES-1 can repress the transcription of the cdc-25.2 gene, which encodes a Cdc25-like phosphatase, thereby enhancing the block in NSM neuroblast division caused by the partial loss of cya-1, which encodes Cyclin A. The results indicate that CDC-25.2 and CYA-1 control specific cell divisions and that the over-expression of the ces-1 gene leads to incorrect regulation of this functional 'module'. Finally, evidence is provided that dnj-11 MIDA1 not only regulate CES-1 activity in the context of cell polarity and apoptosis but also in the context of cell cycle progression. In mammals, the over-expression of Snail-related genes has been implicated in tumorigenesis. These findings support the notion that the oncogenic potential of Snail-related transcription factors lies in their capability to, simultaneously, affect cell cycle progression, cell polarity and apoptosis and, hence, the coordination of cell proliferation and cell fate determination (Yan, 2013).

Function of the different mammalian Cdc25 isoforms

In mammalian cells the Cdc25 family of dual-specificity phosphatases has three distinct isoforms, termed A, B, and C, which are thought to play discrete roles in cell-cycle control. Xenopus Cdc25A exhibits developmental regulation and demonstrates a key role in embryonic cell-cycle control. Northern and Western blot analyses show that Cdc25A is absent in oocytes, and synthesis begins within 30 min after fertilization. The protein product is localized in the nucleus during interphase and accumulates continuously until the midblastula transition (MBT), after which it is degraded. Upon injection into newly fertilized eggs, wild-type Cdc25A shortens the cell cycle and accelerates the timing of cleavage, whereas embryos injected with phosphatase-dead Cdc25A display a dose-dependent increase in the length of the cell cycle and a slower rate of cleavage. In contrast, injection of the phosphatase-dead Cdc25C isoform has no effect. Western blotting with an antibody specific for phosphorylated tyr15 in Cdc2/Cdk2 reveals a cycle of phosphorylation/dephosphorylation in each cell cycle in control embryos, and in embryos injected with phosphatase-dead Cdc25A there is a twofold increase in the level of phospho-tyrosine in Cdc2/Cdk2. Consistent with this, the levels of cyclin B/Cdc2 and cyclin E/Cdk2 histone H1 kinase activity are both reduced by approximately 50% after phosphatase-dead Cdc25A injection. The phosphatase-dead Cdc25A can be recovered in a complex with both Cdks, suggesting that it acts in a dominant-negative fashion. These results indicate that periodic phosphorylation of Cdc2/Cdk2 on tyr15 occurs in each pre-MBT cell cycle, and dephosphorylation of Cdc2/Cdk2 by Cdc25A controls at least in part the length of the cell cycle and the timing of cleavage in pre-MBT embryos. The disappearance of Cdc25A after the MBT may underlie in part the lengthening of the cell cycle at that time (Kim, 1999).

Time-lapse fluorescence microscopy has been used to study the properties of the Cdc25B and Cdc25C phosphatases that have both been implicated as initiators of mitosis in human cells. To differentiate between the functions of the two proteins, expression constructs encoding Cdc25B or Cdc25C or their GFP-chimeras have been microinjected into synchronized tissue culture cells. This assay allows the proteins to be expressed at defined points in the cell cycle. The microinjected cells were followed by time-lapse microscopy, in the presence or absence of DNA synthesis inhibitors, and whether they enter mitosis prematurely or at the correct time was assayed. Overexpressing Cdc25B alone rapidly causes S phase and G2 phase cells to enter mitosis, whether or not DNA replication is complete, whereas overexpressing Cdc25C does not cause premature mitosis. Overexpressing Cdc25C together with cyclin B1 does shorten the G2 phase and can override the unreplicated DNA checkpoint, but much less efficiently than overexpressing Cdc25B. These results suggest that Cdc25B and Cdc25C do not respond identically to the same cell cycle checkpoints. This difference may be related to the differential localization of the proteins; Cdc25C is nuclear throughout interphase, whereas Cdc25B is nuclear in the G1 phase and cytoplasmic in the S and G2 phases. The change in subcellular localization of Cdc25B is due to nuclear export and this is dependent on cyclin B1. The data suggest that although both Cdc25B and Cdc25C can promote mitosis, they are likely to have distinct roles in controlling the initiation of mitosis (Karlsson, 1999).

Activation of cdc25

The auto-catalytic activation of the cyclin-dependent kinase Cdc2 or MPF (M-phase promoting factor) is an irreversible process responsible for the entry into M phase. In Xenopus oocyte, a positive feed-back loop between Cdc2 kinase and its activating phosphatase Cdc25 allows the abrupt activation of MPF and the entry into the first meiotic division. The Cdc2/Cdc25 feed-back loop was studied using cell-free systems derived from Xenopus prophase-arrested oocyte. The findings support the following two-step model for MPF amplification: during the first step, Cdc25 acquires a basal catalytic activity resulting in a linear activation of Cdc2 kinase. In turn, Cdc2 partially phosphorylates Cdc25 but no amplification takes place; under this condition Plx1 kinase and its activating kinase Plkk1 are activated. However, their activity is not required for the partial phosphorylation of Cdc25. This first step occurs independent of PP2A or Suc1/Cks-dependent Cdc25/Cdc2 association. On the contrary, the second step involves the full phosphorylation and activation of Cdc25 and the initiation of the amplification loop. It depends both on PP2A inhibition and Plx1 kinase activity. Suc1-dependent Cdc25/Cdc2 interaction is required for this process (Karaiskou, 1999).

The Ras and Raf1 proto-oncogenes transduce extracellular signals that promote cell growth. Cdc25 phosphatases activate the cell division cycle by dephosphorylation of critical threonine and tyrosine residues within the cyclin-dependent kinases. Cdc25 phosphatase associates with Raf1 in somatic mammalian cells and in meiotic frog oocytes. Furthermore, cdc25 phosphatase can be activated in vitro in a Raf1-dependent manner. Activation of the cell cycle by the Ras/Raf1 pathways might be mediated in part by cdc25 (Galaktionov, 1995).

Human Cdc25 is a target of c-myc (Drosophila homolog: dmyc). There are three known CDK-activating phosphatases in humans. cdc25A is expressed early in G1 following serum stimulation of quiescent fibroblasts. cdc25B is expressed closer to the G1/S transition, and cdc25C is activated in G2. cdc25A and cdc25B cooperate with Ha-ras in the oncogenic transformation of primary rodent fibroblasts. The Myc/Max heterodimer binds to elements in the cdc25A gene and activates transcripion. Like myc, cdc25A, itself a proto-oncogene, can induce apoptosis in cells depleted of growth factor, and myc-induced apoptosis also requires cdc25A. cdc25B is also a target of Myc, although at a later time and to a lesser extent than cdc25A. Thus it is possible that cdc25A and cdc25B might act cooperatively in Myc-driven cell-cycle activation and/or apoptosis (Galaktionov, 1996).

Cdc2 kinase is capable of phosphorylating and activating cdc25, suggesting the existence of a positive feedback loop. Kinases other than cdc2 that can phosphorylate and activate cdc25. Cdc25 is phosphorylated and activated by Cyclin A/cdk2 and Cyclin E/cdk2 in vitro. However, in interphase Xenopus egg another kinase other than cdc2 and cdk2 may initially activate cdc25 in vivo suggesting that this kinase may also phosphorylate M-phase substrates even in the absence of cdc2 kinase (Izumi, 1995).

Cdc2, the cyclin-dependent kinase that controls mitosis (See Cyclin A and Cyclin B) is negatively regulated by phosphorylation on its threonine-14 and tyrosine-15 residues. Cdc25 (Drosophila homolog: String), the phosphatase that dephosphorylates both of these residues, undergoes activation and phosphorylation by multiple kinases at mitosis. Plx1, a kinase that associates with and phosphorylates the amino-terminal domain of Cdc25, was purified from Xenopus. Cloning reveals the Plx1 is related to the Polo family of protein kinases (See Drosophila Polo). Recombinant Plx1 phosphorylates Cdc25 and stimulates its activity in a purified system. It is likely that Plx1 participates in the control of mitotic progression by targeting Cdc25 (Kumagai, 1996).

Entry into mitosis depends on activation of the dual-specificity phosphatase Cdc25C, which dephosphorylates and activates the cyclin B-Cdc2 complex. Previous work has shown that the Xenopus polo-like kinase Plx1 can phosphorylate and activate Cdc25C in vitro. Plx1 is activated in vivo during oocyte maturation with the same kinetics as Cdc25C. Microinjection of wild-type Plx1 into Xenopus oocytes accelerates the rate of activation of Cdc25C and cyclin B-Cdc2. Conversely, microinjection of either an antibody against Plx1 or kinase-dead Plx1 significantly inhibits the activation of Cdc25C and cyclin B-Cdc2. This effect can be reversed by injection of active Cdc25C, indicating that Plx1 is upstream of Cdc25C. However, injection of Cdc25C, which directly activates cyclin B-Cdc2, also causes activation of Plx1, suggesting that a positive feedback loop exists in the Plx1 activation pathway. Other experiments show that injection of Plx1 antibody into early embryos, which do not require Cdc25C for the activation of cyclin B-Cdc2, results in an arrest of cleavage that is associated with monopolar spindles. These results demonstrate that in Xenopus laevis, Plx1 plays important roles both in the activation of Cdc25C at the initiation of mitosis and in spindle assembly at late stages of mitosis (Qian, 1998).

The results reported here establish that the activity of Plx1 is cell cycle regulated and involved in the G2/M transition during Xenopus oocyte maturation. Thus Plx1 plays two roles in mitosis, regulating entry into mitosis and exit from mitosis. The inhibitory effects of Plx1 antibody injection reported here, as well as the effects of wild-type and kinase-dead Plx1 on progesterone-induced Cdc25C activation, demonstrate that Plx1 functions in the Cdc25C activation pathway. Several lines of evidence support the hypothesis that these effects occur due to direct phosphorylation of Cdc25C by Plx1. (1) Cdc25C is a substrate for Plx1 in vitro. (2) Activation of Plx1 correlates exactly with activation of Cdc25C during both meiosis I and meiosis II and Plx1 antibody or kinase-dead Plx1 significantly delays the phosphorylation and activation of Cdc25C. (3) Cdc25C itself can overcome the inhibitory effect of Plx1 antibody. These results support the hypothesis that Plx1 could be a trigger kinase, also termed kinase X, that initiates the positive feedback loop between Cdc25C and cyclin B-Cdc2. The existence of a trigger kinase had been predicted from studies in which initial activation of Cdc25C occurred prior to or in the absence of cyclin B-Cdc2. The results shown here indicate that Plx1 may be a trigger kinase, but they do not exclude the possibility that other kinases also function as trigger kinases. Since injection of Plx1 antibody almost completely inhibits Plx1 activity yet does not completely block Cdc25C activation, it is likely that, in addition to Plx1 and cyclin B-Cdc2, other protein kinases also contribute to Cdc25C activation (Qian, 1998).

During mitosis the Xenopus polo-like kinase 1 (Plx1) plays key roles in the activation of Cdc25C, in spindle assembly, and in cyclin B degradation. Previous work has shown that the activation of Plx1 requires phosphorylation on serine and threonine residues. In the present work, it is demonstrated that replacement of Ser-128 or Thr-201 with a negatively charged aspartic acid residue (S128D or T201D) elevates Plx1 activity severalfold and that replacement of both Ser-128 and Thr-201 with Asp residues (S128D/T201D) increases Plx1 activity approximately 40-fold. Microinjection of mRNA encoding S128D/T201D Plx1 into Xenopus oocytes directly induces the activation of both Cdc25C and cyclin B-Cdc2. In egg extracts T201D Plx1 delays the timing of deactivation of Cdc25C during exit from M phase and accelerates Cdc25C activation during entry into M phase. This supports the concept that Plx1 is a 'trigger' kinase for the activation of Cdc25C during the G(2)/M transition. In addition, during anaphase T201D Plx1 reduces preferentially the degradation of cyclin B2 and delayed the reduction in Cdc2 histone H1 kinase activity. In early embryos S128D/T201D Plx1 resulted in arrest of cleavage and formation of multiple interphase nuclei. Consistent with these results, Plx1 has been found to be localized on centrosomes at prophase, on spindles at metaphase, and at the midbody during cytokinesis. These results demonstrate that in Xenopus laevis activation of Plx1 is sufficient for the activation of Cdc25C at the initiation of mitosis and that inactivation of Plx1 is required for complete degradation of cyclin B2 after anaphase and completion of cytokinesis (Qian, 1999).

In the Xenopus oocyte system mitogen treatment triggers the G2/M transition by transiently inhibiting the cAMP-dependent protein kinase (PKA); subsequently, other signal transduction pathways are activated, including the mitogen-activated protein kinase (MAPK) and polo-like kinase pathways. To study the interactions between these pathways, a cell-free oocyte extract was used that carries out the signaling events of oocyte maturation after addition of PKI, the heat-stable inhibitor of PKA. PKI stimulates the synthesis of Mos and activation of both the MAPK pathway and the Plx1/Cdc25C/cyclin B-Cdc2 pathway. Activation of the MAPK pathway alone by glutathione S-transferase (GST)-Mos does not lead to activation of Plx1 or cyclin B-Cdc2. Inhibition of the MAPK pathway in the extract by the MEK1 inhibitor U0126 delays, but does not prevent, activation of the Plx1 pathway, and inhibition of Mos synthesis by cycloheximide has a similar effect, suggesting that MAPK activation is the only relevant function of Mos. Immunodepletion of Plx1 completely inhibits activation of Cdc25C and cyclin B-Cdc2 by PKI, indicating that Plx1 is necessary for Cdc25C activation. In extracts containing fully activated Plx1 and Cdc25C, inhibition of cyclin B-Cdc2 by p21Cip1 has no significant effect on either the phosphorylation of Cdc25C or the activity of Plx1. These results demonstrate that maintenance of Plx1 and Cdc25C activity during mitosis does not require cyclin B-Cdc2 activity. It is evident that Plx1 is an essential trigger kinase for Cdc25C activation at the G2/M transition. No other kinase appears to be able to substitute for this function of Plx1 in G2, although, once activated, cyclin B-Cdc2 is capable of activating Cdc25C in a positive feedback loop (Qian, 2001).

Pin1 is an essential protein that can peptidyl-prolyl-isomerize small phosphopeptides. Pin1 is a small (18 kDa), abundant (0.5 ┬ÁM), ubiquitously expressed protein that is essential for cell cycle progression in yeast and in mammalian cells. Although Pin1 homologs are not essential in Drosophila (see Dodo; Maleszka, 1996). Loss of Pin1 in S. cerevisiae or inhibition of Pin1 expression by antisense RNA in HeLa cells causes mitotic arrest. Overexpression of Pin1 leads to G2 arrest through a failure to activate the Cdc2 mitotic kinase. Pin1 contains two domains: an N-terminal WW domain, which specifically binds phosphorylated peptides containing the sequence P-Ser/P-Thr-Pro, and a C-terminal peptidyl-prolyl isomerase (PPIase) domain. The PPIase activity is sensitive to phosphorylation of the substrate, increasing 100-fold for peptides phosphorylated on the serine adjacent to the isomerized proline (Stukenberg, 2001 and references therein).

Pin1 interacts with a number of important regulatory proteins in a phosphorylation-dependent manner, such as the C-terminal domain of RNA polymerase II, as well as several regulators of the G2/M transition including Cdc25c, Wee1, Myt1, and Polo kinase. However, Pin 1 does not have an exceptionally strong preference for any one substrate. Using affinity chromatography and protein blots, it has been shown that Pin1 has measurable affinity for dozens or perhaps hundreds of mitotically phosphorylated proteins. Many of these same proteins are recognized by a monoclonal antibody called MPM-2 that had long been used to identify mitotic cells. It has been suggested that Pin1 regulates entry into mitosis by catalyzing the cis/trans-isomerization of prolines on critical protein substrates in response to phosphorylation. Pin1 is shown in this study to catalytically generate a conformational change on the mitotic phosphatase Cdc25, as assayed by limited protease digestion, differential reactivity to a phosphoserine-proline-directed monoclonal antibody (MPM-2), and by changes in Cdc25 enzymatic activity. Pin1 catalytically modifies the conformation of Cdc25 at stoichiometries less than 0.0005, and mutants of Pin1 in the prolyl isomerase domain are not active. It is suggested that, although difficult to detect, phosphorylation-dependent conformational changes mediated by prolyl isomerization may play an important regulatory role in the cell cycle (Stukenberg, 2001).

During oogenesis, the Xenopus oocyte is blocked in prophase of meiosis I. It becomes competent to resume meiosis in response to progesterone at the end of its growing period (stage VI of oogenesis). Stage IV oocytes contain a store of inactive pre-MPF (Tyr15-phosphorylated Cdc2 bound to cyclin B2); the Cdc25 phosphatase that catalyzes Tyr15 dephosphorylation of Cdc2 is also present. However, the positive feedback loop that allows MPF autoamplification is not functional at this stage of oocyte growth. When cyclin B is overexpressed in stage IV oocytes, MPF autoamplification does not occur and the newly formed cyclin B-Cdc2 complexes are inactivated by Tyr15 phosphorylation, indicating that Myt1 kinase remains active and that Cdc25 is prevented from being activated. Plx1 kinase (or polo-like kinase), which is required for Cdc25 activation and MPF autoamplification in full grown oocytes is not expressed at the protein level in small stage IV oocytes. In order to determine if Plx1 could be the missing regulator that prevents MPF autoamplification, polo kinase was overexpressed in stage IV oocytes. Under these conditions, the MPF-positive feedback loop was restored. Moreover, acquisition of autoamplification competence does not require the Mos/MAPK pathway (Karaiskou, 2004).

Thus, Plx1 protein, crucial for the function of the auto-amplification feedback loop in full-grown oocytes is not expressed in small oocytes. Both Cdc25 and Myt1 are direct substrates of Plk1 during M phase. The results indicate that overexpression of Plk1 in stage IV oocytes authorizes cyclin B1 to form active complexes with Cdc2. This observation shows that in oocytes, Plk1 participates in the formation of an active MPF trigger by downregulating Myt1. Moreover, it indicates that progesterone unresponsiveness of small oocytes depends on a stable activity of Myt1 kinase, because of Plx1 absence. Although Plk1 expression prevents Tyr15 phosphorylation of Cdc2 after cyclin B overexpression, Cdc25 is not fully activated. This shows that full activation of Cdc25 requires a further regulatory mechanism. Indeed, Xenopus Cdc25 can be negatively regulated through Ser287 phosphorylation by several kinases, including Chk1 and PKA. Cdc25C, which is phosphorylated on Ser287 in Xenopus prophase oocytes, is dephosphorylated by type 1 phosphatase (PP1) at GVBD. Since the PP1 inhibitor I prevents meiotic maturation, PP1 could participate in the regulation of the MPF autoamplification loop by catalyzing the removal of the inhibitory Ser287 phosphate, and could therefore be involved in the regulation of Cdc25 during oogenesis (Karaiskou, 2004).

In competent oocytes, Plx1 action on Cdc25 is antagonized by an okadaic acid-sensitive phosphatase, involving PP2A activity. This explains why the auto-amplification mechanism can be artificially activated by okadaic acid. However, okadaic acid is unable to promote Cdc2 activation in small incompetent oocytes, showing that the loop implying Cdc2, Cdc25, Plx1 and PP2A is not functional in growing oocytes. The most probable explanation for this defect is the absence of Plx1 in stage IV oocytes. Indeed, it has been shown, both in vivo and in vitro, that expression of Plk1 is sufficient to restore the activation of MPF in response to okadaic acid in incompetent oocytes. Plx1 is therefore the missing factor explaining why the auto-amplification of MPF is defective in small oocytes (Karaiskou, 2004).

Altogether, these experiments show that the incompetence of small oocytes to resume meiosis is ensured by the absence of Plx1 resulting in a double negative control on MPF activation. (1) The formation of active complexes between Cdc2 and newly synthesized cyclins is prevented by a sustained activity of Myt1 that escapes downregulation by Plx1. (2) Cdc25 activation that is normally achieved through a feedback loop involving Plx1 is also prevented. Further investigation will be necessary to discover (1) how Plx1 expression is controlled by cell size at the end of oogenesis; (2) how PP2A controls Cdc25 activity in small oocytes, and (3) how the initial steps of the progesterone transduction pathway connect to MPF regulators, allowing the female germ cell to resume meiosis when oocyte growth is completed (Karaiskou, 2004).

Regulation of Cdc25C by ERK-MAP kinases during the G2/M transition

Induction of G2/M phase transition in mitotic and meiotic cell cycles requires activation by phosphorylation of the protein phosphatase Cdc25. Although Cdc2/cyclin B and polo-like kinase (PLK) can phosphorylate and activate Cdc25 in vitro, phosphorylation by these two kinases is insufficient to account for Cdc25 activation during M phase induction. This study demonstrates that p42 MAP kinase (MAPK), the Xenopus ortholog of ERK2, is a major Cdc25 phosphorylating kinase in extracts of M phase-arrested Xenopus eggs. In Xenopus oocytes, p42 MAPK interacts with hypophosphorylated Cdc25 before meiotic induction. During meiotic induction, p42 MAPK phosphorylates Cdc25 at T48, T138, and S205, increasing Cdc25's phosphatase activity. In a mammalian cell line, ERK1/2 interacts with Cdc25C in interphase and phosphorylates Cdc25C at T48 in mitosis. Inhibition of ERK activation partially inhibits T48 phosphorylation, Cdc25C activation, and mitotic induction. These findings demonstrate that ERK-MAP kinases are directly involved in activating Cdc25 during the G2/M transition (Wang, 2007).

Having established the role of ERK-MAP kinases in the Cdc25C-activation system, the next question is whether MAPK is the only previously unrecognized kinase involved in Cdc25C activation during M phase induction. Although phosphorylation of Cdc25 by p42 MAPK accounts for Mos-induced Cdc2 activation in Xenopus oocyte extracts, this finding does not explain how the phosphatase inhibitor OA, which can bypass the requirement for progesterone to induce Xenopus oocyte maturation, induces Cdc2 activation without activation of p42 MAPK. In some of the previous studies, inhibition of MAPK activation delays, but does not block, progesterone-induced Xenopus oocyte maturation. In contrast to the natural stimulus progesterone, forced activation of MAPK does not always induce Cdc2 activation in Xenopus oocytes or cell-free systems. When it does, there is a significant delay between robust MAPK activation and Cdc2 dephosphorylation. These multiple unexplained observations indicate that progesterone stimulation of Xenopus oocytes activates at least one additional kinase that is involved in Cdc25 activation. While sufficient activation of p42 MAPK is probably able to initiate and amplify Cdc25 activation in cooperation with Cdc2 kinase and Plx1, composite roles of p42 MAPK and this additional kinase may cause quicker and more robust activation of Cdc25 and Cdc2/cyclin B during Xenopus oocyte maturation. In agreement with this hypothesis, gel filtration of MEE resulted in recovery of a much broader peak of Cdc25-phosphorylating activity than can be accounted for by p42 MAPK and/or Cdc2 activities. Depletion of MAPK from QE1, which contained most of the unaccounted-for Cdc25 phosphorylating activity in Xenopus egg extracts, removed only 50% of the Cdc25-phosphorylating activity. The remaining activity can be stabilized by OA and thiophosphorylation and is almost certainly due to a kinase complex of >200 kDa that does not contain Cdc2, Plx1, or p42 MAPK. Thus, it is hypothesized that this yet-to-be-identified Cdc25-phosphorylating activity represents the additional kinase or one of the additional kinases involved in Cdc25 activation during Xenopus oocyte maturation. The presence of alternative regulators in Cdc25 activation may explain why studies by different investigators have yielded differing results with respect to the requirement of MAPK for Cdc2 activation during Xenopus oocyte maturation (Wang, 2007).

Transcriptional regulation of Cdc25

The S/G2-specific transcription of the human cdc25C gene is due to the periodic occupation of a repressor element ('cell cycle-dependent element' or CDE) located in the region of the basal promoter. Protein binding to the major groove of the CDE in G0 and G1 results in a phase-specific repression of activated transcription. CDE-mediated repression is also the major principle underlying the periodic transcription of the human cyclin A and cdc2 genes. A single point mutation within the CDE results in a 10- to 20-fold deregulation in G0 and an almost complete loss of cell cycle regulation for all three genes. The cdc25C, cyclin A and cdc2 genes also share an identical 5 bp region ('cell cycle genes homology region' or CHR) starting at an identical position, six nucleotides 3' to the CDE. Strikingly, mutation of the CHR region in each of the three promoters produces the same phenotype as the mutation of the CDE, i.e. a dramatic deregulation in G0. In agreement with these results, in vivo DMS footprinting shows the periodic occupation of the cyclin A CDE in the major groove and of the CHR in the minor groove. All three genes bear conspicuous similarities in their upstream activating sequences (UAS). This applies in particular to the presence of NF-Y and Sp1 binding sites which, in the cdc25C gene, have been shown to be the targets of repression through the CDE (Zwicker, 1995).

The cdc25C , cyclin A and cdc2 genes are regulated during the cell cycle through two contiguous repressor binding sites, the CDE and CHR, located in the region of transcription initiation and interacting with a factor termed CDF-1. The target of this repression seems to be transcriptional activation of these promoters by transcription factors bound upstream. The majority of these factors fall into the class of glutamine-rich activators, suggesting that CDF-1-mediated repression might be activation domain specific. Chimeric promoter constructs have been used to demonstrate that the cdc25C UAS, but not the core promoter, is crucial for repression. Only specific transcription factors and activation domains are responsive to CDE-CHR-mediated cell cycle regulation. These observations clearly indicate that CDF-1 interferes with activation of transcription by a specific subset of transactivators. The repressible activation domains belong to the same class of glutamine-rich activators, pointing to specific interactions of CDF-1 with components of the transcription machinery. In agreement with this conclusion it has been found that a simple inversion of the CDE-CHR module completely abrogates cell cycle-regulated repression (Zwicker, 1997).

The cdc25C , cdc2 and cyclin A promoters are controlled by transcriptional repression through two contiguous protein binding sites, termed the CDE and CHR. In the present study CDF-1 has been identified as the factor that interacts with the cdc25C CDE-CHR module. CDF-1 binds to the CDE in the major groove and to the CHR in the minor grove in a cooperative fashion in vitro, in a manner similar to that seen by genomic footprinting. In agreement with in vivo binding data and its putative function as a periodic repressor, DNA binding by CDF-1 in nuclear extracts is down-regulated during cell cycle progression. CDF-1 also binds avidly to the CDE-CHR modules of the cdc2 and cyclin A promoters, but not to the E2F site in the B-myb promoter (see Drosophila Myb oncogene-like). Conversely, E2F complexes do not recognize the cdc25C CDE-CHR and CDF-1 is immunologically unrelated to all known E2F and DP family members. This indicates that E2F- and CDF-mediated repression is controlled by different factors acting at different stages during the cell cycle. While E2F-mediated repression seems to be associated with genes such as B-myb that are up-regulated early (around mid G1), CDE-CHR-controlled genes (such as cdc25C, cdc2 and cyclin A), become derepressed later. The fractionation of native nuclear extracts on glycerol gradients leads to separation of CDF-1 from both E2F complexes and pocket proteins of the pRb family. This emphasizes the conclusion that CDF-1 is not an E2F family member and points to profound differences in the cell cycle regulation of CDF-1 and E2F (Liu, 1997).

The activity of the cyclin-dependent kinases (CDKs) that control cell growth and division can be negatively regulated, either by tyrosine phosphorylation or by the binding of various CDK inhibitors. Whereas regulation by tyrosine phosphorylation is well documented in CDKs that function during mitosis, little is known about its role in the regulation of CDKs that act in the G1 phase of the cell cycle. In contrast, much evidence has accumulated on the regulation of G1 CDKs by CDK inhibitors. The cytokine TGF-beta inhibits growth by causing cell-cycle arrest as a result of increasing the concentration of the Cdk4/6 inhibitor p15(INK4B/MTS2). TGF-beta can also cause the inhibition of Cdk4 and Cdk6 by increasing their level of tyrosine phosphorylation. Tyrosine phosphorylation and inactivation of Cdk4/6 in a human mammary epithelial cell line are shown to result from the ability of TGF-beta to repress expression of the CDK tyrosine phosphatase Cdc25A. Repression of Cdc25A and induction of p15 are independent effects mediating the inhibition of Cdk4/6 by TFG-beta (Iavarone, 1997).

Dephosphorylation of cdc2 (cyclin dependent kinase)

Tyrosine phosphorylation of cdc2 occurs on Tyr15, a residue located within the ATP binding site of the protein: this is required for maintaining inactivity in the cdc2-cyclin B complex until DNA replication is completed. At the end of G2, the activation of the cdc25 phosphatase (String in Drosophila) causes cdc2 dephosphorylation and the activation of the histone H1 kinase activity (Krek, 1991).

Interaction of Cdc25 with cyclin/cdk dimers

In human and murine cells, there are three known Cdc25 proteins: Cdc25A, Cdc25B, and Cdc25C. The three phosphatases share approximately 40 to 50% homology. Cdc25C and Cdc25A function at G2-M and G1-S transitions during the human cell cycle, respectively. Cdc25A associates with, dephosphorylates, and activates the cell cycle kinase cyclin E-cdk2 (See Drosophila Cyclin E). p21CIP1 and p27 are cyclin-dependent kinase (cdk) inhibitors (See Drosophila Dacapo) induced by growth-suppressive signals (such as p53) and transforming growth factor beta (TGF-beta). A cyclin binding motif has been identified near the N terminus of Cdc25A that is similar to the cyclin binding Cy (or RR LFG) motif of the p21CIP1 family of cdk inhibitors and separate from the catalytic domain. Mutations in this motif disrupt the association of Cdc25A with cyclin E- or cyclin A-cdk2 in vitro and in vivo and selectively interfere with the dephosphorylation of cyclin E-cdk2. A peptide based on the Cy motif of p21 competitively disrupts the association of Cdc25A with cyclin-cdks and inhibits the dephosphorylation of the kinase. p21 inhibits Cdc25A-cyclin-cdk2 association and the dephosphorylation of cdk2. Conversely, Cdc25A, which is itself an oncogene up-regulated by the Myc oncogene, associates with cyclin-cdk and protects it from inhibition by p21. Cdc25A also protects DNA replication in Xenopus egg extracts from inhibition by p21. These results describe a mechanism by which the Myc- or Cdc25A-induced oncogenic and p53- or TGF-beta-induced growth-suppressive pathways counterbalance each other by competing for cyclin-cdks (Saha, 1997).

Cdc25 regulates entry into mitosis by regulating the activation of cyclin B/cdc2. In humans, at least two cdc25 isoforms have roles in controlling the G2/M transition. Two cdc25B splice variants, cdc25B2 and cdc25B3, are capable of activating cyclin A/cdk2 and cyclin B/cdc2, but the mitotic hyperphosphorylation of these proteins increases their activity toward only cyclin B1/cdc2. Cdc25C has only very low activity in its unphosphorylated form; following hyperphosphorylation it will efficiently catalyze the activation of only cyclin B/cdc2. This is reflected by the in vivo activity of the immunoprecipitated cdc25B and cdc25C from interphase and mitotic HeLa cells. The increased activity of the hyperphosphorylated cdc25s toward cyclin B1/cdc2 is in large part due to increased binding of this substrate. The substrate specificity, activities, and timing of the hyperphosphorylation of cdc25B and cdc25C during G2 and M suggest that these two mitotic cdc25 isoforms are activated by different kinases and perform different functions during progression through G2 into mitosis (Gabrielli, 1997a).

DNA replication in higher eukaryotes requires activation of a Cdk2 kinase by Cdc25A, a labile phosphatase subject to further destabilization upon genotoxic stress. A distinct, markedly stable form of Cdc25A, is described that plays a previously unrecognized role in mitosis. Mitotic stabilization of Cdc25A reflects its phosphorylation on Ser17 and Ser115 by cyclin B-Cdk1, modifications required to uncouple Cdc25A from its ubiquitin-proteasome-mediated turnover. Cdc25A binds and activates cyclin B-Cdk1, accelerates cell division when overexpressed, and its downregulation by RNA interference (RNAi) delays mitotic entry. DNA damage-induced G2 arrest, in contrast, is accompanied by proteasome-dependent destruction of Cdc25A, and ectopic Cdc25A abrogates the G2 checkpoint. Thus, phosphorylation-mediated switches among three differentially stable forms ensure distinct thresholds, and thereby distinct roles for Cdc25A in multiple cell cycle transitions and checkpoints (Mailand, 2002).

Arguably the most unexpected finding of this study was that entry into mitosis is accompanied by an abrupt switch, which converts Cdc25A from a labile to a stable protein. Hence, while acceleration of the basal Cdc25A protein turnover participates in response to genotoxic stress or stalled replication in interphase, uncoupling of Cdc25A from the ubiquitin-proteasome-dependent degradation plays an important role once the cells become committed to undergo cell division. Whereas destruction of the phosphatase guards against premature mitosis of cells with an incompletely replicated and/or damaged genome, stabilization of Cdc25A ensures that even in the absence of de novo mRNA synthesis, Cdc25A is abundant and active during initial stages of mitotis. Both reduction and extension of the Cdc25A protein half-life rely on phosphorylations within its N-terminal regulatory region. It has been shown that in S-phase cells, phosphorylation of Ser123 by the ATM-Chk2 kinase cascade (see loki) is required to induce degradation of Cdc25A in response to ionizing radiation. Such a mechanism may also operate in the DNA damage-induced G2 checkpoint. The mitotic stabilization of Cdc25A also reflects site-specific phosphorylation. Both Ser17 and Ser115 fulfill the minimum requirements for a CDK consensus site, and phosphorylation of Ser17 and Ser115 is mediated by cyclin B-Cdk1 in vitro, and is detectable in vivo only after entry into mitosis when the cyclin B-Cdk1 complex becomes physiologically active. Although the integrity of Ser17 and Ser115 appears crucial for Cdc25A stabilization, the data do not exclude the potential involvement of other residues. Thus, while concomitant mutation of Ser17 and Ser115 does confer marked instability in mitosis, the protein turnover of such a Cdc25A(2A) mutant could still be accelerated moderately by roscovitine, and the Ser17/Ser115-deficient protein still undergoes a partial mitosis-associated electrophoretic mobility shift. This indicates that cyclin B-Cdk1 might phosphorylate more residues than those identified by mass spectrometry analysis. Moreover, the fact that the endogenous Cdc25A protein remains partly shifted in roscovitine-treated mitotic cells suggests that other kinases (such as Plk1), specifically activated at the G2/M transition, assist in inducing and/or maintaining the stability of Cdc25A (Mailand, 2002).

Inactivation and degradation of cdc25

The level of the mitotic activating tyrosine phosphatase cdc25 is regulated by both transcriptional and post-transcriptional mechanisms in the fission yeast S. pombe. Cdc25 is ubiquitinated prior to destruction and the gene pub1 regulates this event. Disruption of pub1 elevates the level of cdc25 protein in vivo, rendering cells relatively resistant to the cdc25-opposing tyrosine kinases wee1 and mik1. In addition, loss of wee1 activity in a pub1-disruption background results in a lethal premature entry into mitosis that can be rescued by loss of cdc25 function (Nefsky, 1996).

Phosphorylation of mitotic proteins on the Ser/Thr-Pro motifs has been shown to play an important role in regulating mitotic progression. Pin1 is a novel essential peptidyl-prolyl isomerase (PPIase) that inhibits entry into mitosis and is also required for proper progression through mitosis, but its substrate(s) and function(s) remain to be determined. In both human cells and Xenopus extracts, Pin1 interacts directly with a subset of mitotic phosphoproteins on phosphorylated Ser/Thr-Pro motifs in a phosphorylation-dependent and mitosis-specific manner. Many of these Pin1-binding proteins are also recognized by the monoclonal antibody MPM-2, and they include the important mitotic regulators Cdc25, Myt1, Wee1, Plk1, and Cdc27. The importance of this Pin1 interaction was tested by constructing two Pin1 active site point mutants that fail to bind a phosphorylated Ser/Thr-Pro motif in mitotic phosphoproteins. Wild-type, but not mutant, Pin1 inhibits both mitotic division in Xenopus embryos and entry into mitosis in Xenopus extracts. The interaction between Pin1 and Cdc25 was examined in detail. Pin1 not only binds the mitotic form of Cdc25 on the phosphorylation sites important for its activity in vitro and in vivo, but it also inhibits its activity, offering one explanation for the ability of Pin1 to inhibit mitotic entry. Pin1 is a phosphorylation-dependent PPIase that can recognize specifically the phosphorylated Ser/Thr-Pro bonds present in mitotic phosphoproteins. Thus, Pin1 likely acts as a general regulator of mitotic proteins that have been phosphorylated by Cdc2 and other mitotic kinases (Shen, 1998).

The cis/trans peptidyl-prolyl isomerase, Pin1, is a regulator of mitosis that is well conserved from yeast to man. Depletion of Pin1-binding proteins from Xenopus egg extracts results in hyperphosphorylation and inactivation of the key mitotic regulator, Cdc2/cyclin B. This phenotype is a consequence of Pin1 interaction with critical upstream regulators of Cdc2/cyclin B, including the Cdc2-directed phosphatase, Cdc25, and its known regulator, Plx1. Although Pin1 could interact with Plx1 during interphase and mitosis, only the phosphorylated, mitotically active form of Cdc25 is able to bind Pin1, an event that has been recapitulated using in vitro phosphorylated Cdc25. Taken together, these data suggest that Pin1 may modulate cell cycle control through interaction with Cdc25 and its activator, Plx1 (Crenshaw, 1998).

The peptidyl-prolyl isomerase Pin1 has been implicated in regulating cell cycle progression. Pin1 has been found to be required for the DNA replication checkpoint in Xenopus laevis. Egg extracts depleted of Pin1 inappropriately transit from the G2 to the M phase of the cell cycle in the presence of the DNA replication inhibitor aphidicolin. This defect in replication checkpoint function is reversed after the addition of recombinant wild-type Pin1, but not an isomerase-inactive mutant, to the depleted extract. Premature mitotic entry in the absence of Pin1 is accompanied by hyperphosphorylation of Cdc25, activation of Cdc2/cyclin B, and generation of epitopes recognized by the mitotic phosphoprotein antibody, MPM-2. Therefore, Pin1 appears to be required for the checkpoint delaying the onset of mitosis in response to incomplete replication (Winkler, 2000).

The reversible protein phosphorylation on Ser/Thr-Pro motifs for the control of various cellular processes, including cell division is governed by Pro-directed protein kinases and phosphatases, and is a major regulatory mechanism in organisms as diverse as yeast and mammals. For example, activation of the Pro-directed kinase Cdc2 at the G2/M transition leads to the phosphorylation of a number of conserved proteins. This phosphorylation occurs mainly on Ser/Thr-Pro motifs. Many of these pSer/Thr-Pro motifs are also known as MPM-2 epitopes because the phospho-specific monoclonal antibody MPM-2 recognizes them. MPM-2 antigens include proteins such as Cdc25, Myt1, and tau. Phosphorylation on these MPM-2 epitopes is believed to alter the conformation and function of proteins to trigger the events of mitosis. Conversely, dephosphorylation of these MPM-2 epitopes by phosphatases, notably PP2A, also plays an essential role in the regulation of Cdc2 activation and mitosis. However, little is known about conformational changes induced by phosphorylation or whether the conformation affects protein dephosphorylation (Zhao, 2000 and references therein).

Interestingly, the peptidyl-prolyl bond is important for determining protein structure because it exists in two completely distinct but slowly interconverting cis and trans conformations and can introduce kinks into a peptide chain. These two conformations can be detected by proteases, the only enzymes known to cleave the trans isomer exclusively. Furthermore, slow cis/trans isomerization is frequently rate limiting, as in the case of protein refolding. The prolyl isomerization is catalyzed by peptidyl-prolyl cis/trans isomerases (PPIases). The cyclophilins and the FKBPs (FK506 binding proteins) are two major families of conventional PPIases that have been well characterized because of their importance as targets of clinically relevant immunosuppressive drugs. However, all 12 known cyclophilin and FKBP genes in budding yeast can be disrupted altogether with no phenotype. Therefore, evidence for the biological importance of the PPIase activity has been elusive (Zhao, 2000 and references therein).

The only PPIases that seem to be essential for cell survival, at least in budding yeast and HeLa cells, are Pin1 and Pin1-like PPIases, which have been isolated in all eukaryotic cells examined (see the Drosophila Pin1 homolog Dodo. These Pin1-like PPIases belong to a new family of PPIases, with the bacterial parvulin being the prototype. In contrast to other parvulin-like PPIases, Pin1-like PPIases have a unique active-site structure, substrate specificity, and cellular function. They specifically isomerize only phosphroylated Ser/Thr-Pro bonds. Significantly, phosphorylation on Ser/Thr-Pro motifs restrains the already slow cis/trans prolyl isomerization. Moreover, phosphorylation renders pSer/Thr-Pro bonds resistant to the catalytic action of other PPIases. Therefore, phosphorylation on Ser/Thr-Pro leads to a unique structural feature and specifically requires Pin1-like PPIases to catalyze prolyl isomerization (Zhao, 2000 and references therein).

Pin1 also contains a WW domain that binds to specific pSer/Thr-Pro motifs present in a subset of phosphoproteins. MPM-2 recognizes many of these specific pSer/Thr-Pro motifs. Pin1 regulates the function of these proteins. For example, Pin1 inhibits the Cdc25C activity and restores the tau function. Pin1 is essential for mitosis, and its depletion or mutation induces premature mitotic entry and mitotic arrest accompanied by high levels of MPM-2 epitopes. In Alzheimer's disease neurons, Pin1 is sequestered in the tangles, and soluble Pin1 becomes depleted. Interestingly, these neurons also contain an elevated level of MPM-2 epitopes, including hyperphosphorylated tau. Although these results suggest that Pin1 may be required for dephosphorylation of some MPM-2 epitopes by phosphatases, the role of Pin1 in modulating dephosphorylation has not been described (Zhao, 2000 and references therein).

This study shows that Pin1-dependent prolyl isomerization regulates dephosphorylation of Cdc25C and Tau proteins. The major Pro-directed phosphatase PP2A is conformation-specific and effectively dephosphorylates only the trans pSer/Thr-Pro isomer. Furthermore, Pin1 catalyzes prolyl isomerization of specific pSer/Thr-Pro motifs both in Cdc25C and tau to facilitate their dephosphorylation by PP2A. Moreover, Pin1 and PP2A show reciprocal genetic interactions, and prolyl isomerase activity of Pin1 is essential for cell division in vivo. Thus, phosphorylation-specific prolyl isomerization catalyzed by Pin1 is a novel mechanism essential for regulating dephosphorylation of certain pSer/Thr-Pro motifs (Zhou, 2000).

The peptidyl-prolyl cis/trans isomerases (PPIases) activity is believed to allow spatial and temporal control of polypeptides by permitting relaxation of local energetically unfavorable Xaa-Pro conformational states. Although all cyclophilins and FKBPs can be deleted in yeast without significant growth effect, Pin1 is the PPIase gene essential for cell division in yeast. However, since Pin1 also contains a pSer/Thr-Pro binding WW domain that is essential for cell survival, an important question has been whether Pin1 functions simply as a phosphoprotein binding protein, like 14-3-3 proteins, or as a prolyl isomerase. It is shown here that although the WW domain is normally required for targeting Pin1 to its substrates, the PPIase domain is both necessary and sufficient to support cell growth. Extensive mutagenesis analysis has confirmed the essential role of the Pin1 PPIase activity in vivo (Zhou, 2000).

Why would the cell need the Pin1 enzymatic activity? The answer likely lies in the extraordinary substrate specificity of Pin1 toward pSer/Thr-Pro motifs and the unique structural feature and critical regulatory role of these motifs. Out of about 105 Ser/Thr-Pro bonds in sequence databases, only ~1% Ser/Thr-Pro bonds can be found in the protein structure database. The propensity of cis Ser/Thr-Pro bonds is in the range of 7%-25% and depends on the sequence identity criterion used to exclude closely related proteins (<25%) or same proteins (<95%), as described. Since phosphorylation on Ser/Thr-Pro motifs in peptides does not greatly affect the cis/trans prolyl bond ratio, the probability of pSer/Thr-Pro bonds in the cis conformation is estimated to be 10%-20%. This large number of cis pSer/Thr-Pro motifs suggests that their functional importance and dephosphorylation have to be considered. Therefore, some of the pSer/Thr-Pro bonds in proteins are likely to be accumulated in the cis form until Pin1, which is directed to the substrate by its WW domain, can convert the cis into the trans conformation. Significantly, most pSer/Thr-Pro motifs that are Pin1 targets are located in the regulatory domains and have crucial regulatory roles. For example, the Pin1 binding sites in Cdc25C (pThr48-Pro and pThr67-Pro) and in tau (Thr231-Pro) are critical in regulating their biological function to trigger mitotic entry and to promote microtubule assembly, respectively. The results have shown that Pin1 can isomerize these specific pSer/Thr-Pro motifs in tau and Cdc25C. Together with the fact that Pin1 can directly restore the biological function of phosphorylated tau and inhibit phosphorylated Cdc25C, these results indicate that prolyl isomerization of these specific pSer/Thr-Pro motifs is an important mechanism for regulating the protein function (Zhou, 2000 and references therein).

The biological importance of phosphorylation-specific prolyl isomerization becomes even more obvious because of the conformational specificity of the phosphatase. PP2A effectively dephosphorylates only the trans pSer/Thr-Pro isomer. Furthermore, Pin1 can isomerize specific pSer/Thr-Pro peptide bonds both in proteins and peptides and thereby facilitate their dephosphorylation by PP2A. Moreover, Pin1 and PP2A show reciprocal genetic interactions. These results provide compelling biochemical and genetic evidence for a novel role of phosphorylation-specific prolyl isomerization in regulating dephosphorylation of certain pSer/Thr-Pro motifs. It has been well established that reversible mitotic protein phosphorylation on pSer/Thr-Pro-containing MPM-2 epitopes plays an essential role in controlling the timing of mitotic progression. The findings that Pin1 modulates dephosphorylation of certain pSer/Thr-Pro motifs provide an explanation of why overexpression of Pin1 inhibits entry into mitosis, whereas mutation or depletion of Pin1 triggers premature mitotic entry accompanied by precocious hyperphosphorylation and activation of Cdc25C, as well as generation of MPM-2 epitopes. Furthermore, since some substrates for Pin1 and PP2A, such as Cdc25C, have been shown to be also involved in the G2/M checkpoint, these results may explain why depletion of Pin1 disrupts the replication checkpoint in Xenopus extracts. Therefore, Pin1-catalyzed phosphorylation-specific prolyl isomerization plays a crucial role in regulating protein dephosphorylation during cell division (Zhou, 2000 and references therein).

Given the importance of Pro-directed phosphorylation in diverse cell functions, this new mechanism is likely important in other processes and/or human diseases. For example, the findings that Pin1 facilitates dephosphorylation of tau suggest that depletion of soluble Pin1 may contribute to increased phosphorylation of tau and other MPM-2 antigens in Alzheimer's disease. Together with the fact that Pin1 can restore the biological function of phosphorylated tau, these results further support an important role of Pin1 in the pathogenesis of Alzheimer's disease. Therefore, by regulating the conformation of the pSer/Thr-Pro motifs, Pin1 can control the function of phosphoproteins directly or indirectly via modulating their dephosphorylation. Interestingly, both effects lead to the same consequences for Cdc25 and tau. Thus, phosphorylation-specific prolyl isomerization is an essential postphosphorylation regulatory mechanism that would allow the cell to turn the function of phosphorylated proteins on or off with high efficiency and precise timing during dynamic cellular processes (Zhou, 2000 and references therein).

The Cdc25 dual-specificity phosphatases control progression through the eukaryotic cell division cycle by activating cyclin-dependent kinases. Cdc25 A regulates entry into S-phase by dephosphorylating Cdk2, it cooperates with activated oncogenes in inducing transformation and is overexpressed in several human tumors. DNA damage or DNA replication blocks induce phosphorylation of Cdc25 A and its subsequent degradation via the ubiquitin-proteasome pathway. The regulation of Cdc25 A in the cell cycle was investigated. Cdc25 A degradation during mitotic exit and in early G1 is was found to be mediated by the anaphase-promoting complex or cyclosome (APC/C)Cdh1 ligase, and a KEN-box motif in the N-terminus of the protein was found to be required for its targeted degradation. Interestingly, the KEN-box mutated protein remains unstable in interphase and upon ionizing radiation exposure. Moreover, SCF (Skp1/Cullin/F-box) inactivation using an interfering Cul1 mutant accumulates and stabilizes Cdc25 A. The presence of Cul1 and Skp1 in Cdc25 A immunocomplexes suggests a direct involvement of SCF in Cdc25 A degradation during interphase. It is proposed that a dual mechanism of regulated degradation allows for fine tuning of Cdc25 A abundance in response to cell environment (Donzelli, 2002).

In the absence of HCLK2 degradation of the checkpoint phosphatase Cdc25A is compromised following replication stress as a result of accelerated Chk1 degradation

The human homologue of the C. elegans biological clock protein CLK-2 (HCLK2) associates with the S-phase checkpoint components ATR, ATRIP, claspin and Chk1. Consistent with a critical role in the S-phase checkpoint, HCLK2-depleted cells accumulate spontaneous DNA damage in S-phase, exhibit radio-resistant DNA synthesis, are impaired for damage-induced monoubiquitination of FANCD2 and fail to recruit FANCD2 and Rad51 (critical components of the Fanconi anaemia and homologous recombination pathways, respectively) to sites of replication stress. Although Thr 68 phosphorylation of the checkpoint effector kinase Chk2 remains intact in the absence of HCLK2, claspin phosphorylation and degradation of the checkpoint phosphatase Cdc25A are compromised following replication stress as a result of accelerated Chk1 degradation. ATR phosphorylation is known to both activate Chk1 and target it for proteolytic degradation, and depleting ATR or mutation of Chk1 at Ser 345 restored Chk1 protein levels in HCLK2-depleted cells. It is concluded that HCLK2 promotes activation of the S-phase checkpoint and downstream repair responses by preventing unscheduled Chk1 degradation by the proteasome (Collis, 2007).

Cdc25 and embryonic development

The CDC25 dual-specificity phosphatase family has been shown to play a key role in cell cycle regulation. The phosphatase activity of CDC25 drives the cell cycle by removing inhibitory phosphates from cyclin-dependent kinase/cyclin complexes. Although the regulation of CDC25 phosphatase activity has been elucidated both biochemically and genetically in other systems, the role of this enzyme during development is not well understood. To examine the expression pattern and function of CDC25 in C. elegans, a cdc25 homolog, cdc-25.1, was characterized during early embryonic development. The CDC-25.1 protein localizes to oocytes, embryonic nuclei, and embryonic cortical membranes. When the expression of CDC-25.1 is disrupted by RNA-mediated interference, the anterior cortical membrane of fertilized eggs becomes very fluid during meiosis and subsequent mitotic cell cycles. Mispositioning of the meiotic spindle, defects in polar body extrusion and chromosome segregation, and abnormal cleavage furrows are also observed. It is concluded that CDC-25.1 is required for a very early developmental process -- the proper completion of meiosis prior to embryogenesis (Ashcroft, 1999).

Trochoblasts are the first cells to differentiate during the development of spiralian embryos. Differentiation is accompanied by a cell division arrest. In embryos of the limpet Patella vulgata, the participation of cell cycle-regulating factors in trochoblast arrest was analysed as a first step to unravel its cause. The cell cycle phase in which the trochoblasts are arrested was determined by analysing the subcellular locations of mitotic cyclins. The results show that the trochoblasts are most likely arrested in the G2 phase. This was supported by measurement of the DNA content in trochoblast nuclei after the last division. Trochoblasts complete their final division at the sixth mitotic cycle. This mitotic cycle resembles the first postblastoderm cell cycle of Drosophila, in which mitotic activity is controlled by expression of the string gene. Since failure of string expression results in cell cycle arrest in the G2 phase, negative regulation of a Patella string homolog (stringlike) could be responsible for trochoblast arrest. Although Stl messengers disappear from trochoblasts during their final division, expression is observed again 20 min later. Messengers remain present in all trochoblasts at low levels during further development. Thus, expression of the stringlike gene allows the cell cycle arrest of these cells, whereas in Drosophila cells arrested in division lack String messengers (van der Kooij, 1998).

Major developmental events in early Xenopus embryogenesis coincide with changes in the length and composition of the cell cycle. These changes are mediated in part through the regulation of CyclinB/Cdc2 and they occur at the first mitotic cell cycle, the mid-blastula transition (MBT) and at gastrulation. The contribution has been investigated of maternal Wee1, a kinase inhibitor of CyclinB/Cdc2, to these crucial developmental transitions. By depleting Wee1 protein levels using antisense morpholino oligonucleotides, it is shown that Wee1 regulates M-phase entry and Cdc2 tyrosine phosphorylation in early gastrula embryos. Moreover, Wee1 is required for key morphogenetic movements involved in gastrulation, but is not needed for the induction of zygotic transcription. In addition, Wee1 is positively regulated by tyrosine autophosphorylation in early gastrula embryos and this upregulation of Wee1 activity is required for normal gastrulation. Overexpression of Cdc25C, a phosphatase that activates the CyclinB/Cdc2 complex, induces gastrulation defects that can be rescued by Wee1, providing additional evidence that cell cycle inhibition is crucial for the gastrulation process. Together, these findings further elucidate the developmental function of Wee1 and demonstrate the importance of cell cycle regulation in vertebrate morphogenesis (Murakami, 2004).

Modulation of the cell cycle appears to play an important role in both Xenopus and Drosophila embryogenesis. Prior to gastrulation, both organisms undergo a burst of rapid cell divisions followed by a gradual expansion of the cell cycle. Zygotic cell cycle components are synthesized after the MBT and previous studies have indicated that zygotic proteins do play a role in regulating Cdc2 activity during gastrulation. In Drosophila, cell cycle inhibition is observed at the ventral furrow, a region somewhat analogous to the Xenopus blastopore, and this inhibition is achieved by the removal of a zygotic activator of Cdc2. Specifically, the spatially restricted expression of the Tribbles protein results in the degradation of the String/Cdc25C phosphatase in cells surrounding the ventral furrow. In Xenopus, the zone of non-mitotic cells in the mid-late gastrula is identical to the area of zygotic Wee1B/Wee2 RNA expression, suggesting that zygotic expression of a Cdc2 inhibitor, Wee1B/Wee2, might play an analogous role in frog embryogenesis. Interestingly, the expansion of the cell cycle after the MBT (and during gastrulation) is regulated by zygotic components in Drosophila, but is regulated by maternally derived components in Xenopus. In Xenopus, the maternally regulated program of cell cycle expansion has been implicated in the onset of zygotic transcription, cytoplasmic blebbing and pseudopod formation (at the MBT), but has not been previously implicated in the coordinated tissue morphogenesis that takes place during gastrulation. This study demonstrates that the maternal Wee1 protein contributes to the cell cycle downregulation that occurs during Xenopus gastrulation. The findings also indicate that the maternally directed program of cell cycle control, rather than simply facilitating the transcription of zygotic components, plays a direct role in morphogenesis (Murakami, 2004).

The requirement of cell cycle regulation for the coordinated cell movements of gastrulation is another shared feature of Drosophila and Xenopus embryogenesis. In flies, the Tribbles-mediated degradation of Cdc25C permits the invagination of mesodermal cells at the ventral furrow, one of the earliest events of gastrulation. Similarly, in this study, cell cycle inhibition mediated by Wee1 was found to be important for epiboly, involution and convergent-extension, all of which are major morphogenetic processes that contribute to normal Xenopus gastrulation. Thus, although flies and frogs may use different molecular components to regulate the embryonic cell cycle, it appears that in both organisms the inhibition of cell division is essential for the complex morphogenetic movements required for gastrulation (Murakami, 2004).

Wee1 is upregulated by tyrosine autophosphorylation following the MBT and at gastrulation. This upregulation appears to be required for Wee1 function in early gastrula embryos given that neither kinase-inactive Wee1 or a Wee1 protein containing mutations in the tyrosine phosphorylation sites are able to rescue the defects produced by MO-Wee1-depletion. These findings are consistent with previous observations that upregulation of Wee1 activity by tyrosine autophosphorylation is critical for Wee1 function in the first mitotic cell cycle. Taken together, these studies indicate that the maternal Wee1 protein functions at distinct developmental points to coordinate cell cycle progression with events that control the organization of the embryonic body plan. Moreover, this work contributes to a growing body of evidence that cell cycle regulation is likely to be crucial for a wide variety of morphogenetic processes. Wee1 is a primary cell cycle target of the budding morphogenesis checkpoint in S. cerevisiae, and in mammalian cells, there is evidence that inhibition of cell proliferation is necessary for cell migration. Collectively, these studies suggest that 'morphogenesis' checkpoints, which coordinate cell shape changes and movement with cell proliferation, will be crucial for normal development and organogenesis, and may also play an important role in the balance between deregulated cell proliferation and metastasis (Murakami, 2004).

Cdc25 and checkpoint controls

Continued: String: Evolutionary Homologs part 2/2


string: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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