twins: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

Gene name - twins

Synonyms - Protein phosphatase 2A at 85F (PP2A-85)

Cytological map position - 85F12-F13

Function - serine/threonine protein phosphatase

Keyword(s) - ras pathway, peripheral nervous system, wing, eye, cell cycle

Symbol - tws

FlyBase ID:FBgn0004889

Genetic map position - 3-[49]

Classification - regulatory subunit of PP2A

Cellular location - unknown



NCBI links: Precomputed BLAST | Entrez Gene
Recent literature
Merigliano, C., Marzio, A., Renda, F., Somma, M.P., Gatti, M. and Vernì, F. (2016). A role for the Twins protein phosphatase (PP2A-B55) in the maintenance of Drosophila genome integrity. Genetics [Epub ahead of print]. PubMed ID: 28040742
Summary:
The protein phosphatase 2A (PP2A) is a conserved heterotrimeric enzyme that is mutated in many types of cancer and acts as a tumor suppressor. In mammalian cells, PP2A inhibition results in DNA double strand breaks (DSBs) and chromosome aberrations (CABs). However, the mechanisms through which PP2A prevents DNA damage are still unclear. This study focuses on the role of the Drosophila twins (tws) gene in the maintenance of chromosome integrity; tws encodes the B regulatory subunit (B/B55) of PP2A. Mutations in tws cause high frequencies of CABs (0.5 CABs/cell) in Drosophila larval brain cells and lead to an abnormal persistence of γ-H2Av repair foci. However, mutations that disrupt the PP4 phosphatase activity impair foci dissolution but do not cause CABs, suggesting that a delayed foci regression is not clastogenic. Tws is required for activation of the G2/M DNA damage checkpoint, while PP4 is required for checkpoint recovery, a result that points to a conserved function of these phosphatases from flies to humans. Mutations in the ATM-coding gene tefu are strictly epistatic to tws mutations for the CAB phenotype, suggesting that failure to dephosphorylate an ATM substrate(s) impairs DNA DSBs repair. In addition, mutations in the Ku70 gene, which do not cause CABs, completely suppress CAB formation in tws Ku70 double mutants. These results suggest the hypothesis that an improperly phosphorylated Ku70 protein can lead to DNA damage and CABs.
BIOLOGICAL OVERVIEW

Before any discussion of Twins function, it will prove useful to have a look at its somewhat complicated enzymatic structure. Twins is the B-subunit (PR55) of a heterotrimeric protein phosphatase known as 2A (PP2A). The trimer is composed of three subunits -- the so-called A and B subunits, plus a catalytic subunit: mammalian PP2A then, is composed of a catalytic 36-kD subunit, a 65kD regulatory subunit (the A subunit or PR65) and the B-subunit. The dimeric core complex (catalytic and A-subunit) may be combined with one of a number of regulatory subunits (the B-subunits) ranging in size from 54 to 74kD. The activity and substrate specificity of the core complex is modulated by association with a B-subunit. In Drosophila, genes for the catalytic and PR65 (A) subunits of PP2A were cloned by sequence similarity to their mammalian counterparts (Mayer-Jaekel, 1992). Cloning of the B subunit PR55, coding for Twins protein, was accomplished through homology to the human homolog (Mayer-Jaekel, 1993 and Uemura, 1993).

Twins protein and the catalytic subunit of PP2A, the two proteins for which mutations have been isolated, have a multitude of functions, suggesting involvement in a number of pathways. Initially implicated as a regulator of mitotic progression (see Effects of mutation and Evolutionary homologs sections) , Twins has subsequently been implicated in three additional roles.

In the first of these, twins mutation causes a peripheral nervous system defect similar to that observed in numb and musashi mutations. How does this defect manifest itself? At this point, a quick look at the non-mutated phenotype is in order. Adult mechanosensory bristles normally consist of four cells: two cells of neural derivation (neuron and glial or thecogen cells), and two support cells ( a socket cell [tormogen] and a shaft cell [trichogen]). These four cells are derived from a single precursor called the sensory organ precursor (SOP). Mutations in twins (Shiomi, 1994), musashi (Nakamura, 1994) and numb result in an increase in the ratio of support cells, at the expense of neural cells.

One additional digression will clarify Twins's involvement in such defects, with respect to numb, taking us by way of tramtrack: mutation of tramtrack results in the the transformation of socket and shaft cells to neuron and glial cells. Ectopic expression of ttk has just the opposite effect: the transformation of neuron and glial cells into socket and shaft cells. Numb protein is asymmetrically distributed to neural precursor cells in the first division of the SOP, and Numb targets tramtrack, which then acts as a repressor of support cell fate in neural and glial progeny (Rhyu, 1994 and Guo, 1995). Interestingly, an N-terminal domain of Numb protein consists of residues predictive of a phosphotyrosine binding domain (PTB domain) (Uemura, 1989 and Zhong, 1996). It is possible that Twins protein regulates a Numb phosphoprotein target, and in this manner is involved in the peripheral nervous system defect.

A second function for Twins is found in wing morphogenesis. Mutation of twins causes a pattern duplication in Drosophila imaginal discs. Inactivation of twins induces the formation of extra wing blade anlagen in the posterior compartment. The duplication is mirror symmetrical, and the line of symmetry does not correspond to any of the known compartment borders (Uemura, 1993).

A third function for Twins has to do with eye morphogenesis. Mutations in the catalytic subunit of PP2P interfere with Ras pathway function in eye morphogenesis. PP2P mutation exacerbates defects caused by unregulated Ras function and diminishes defects cause by unregulated Raf function. Ras and Raf are both components of the EGF-receptor signaling pathway required for determination of photoreceptor fate in the compound eye (Wassarman, 1996).

These four defects, associated with mutant PP2A (defective mitosis, defective cell fate determination in the PNS, alteration of patterning in the wing, and interferance in Ras pathway function in eye morphogenesis) do not seem to share any common denominator, other than the involvement of PP2A. It is likely that PP2A substrates are different for each of these defects.

One of the most exciting aspects of PP2A research is PP2A involvement in differentiation. In terms of understanding developmental roles, Drosophila is the organism of choice, but in terms of an understanding of biochemical pathways, a multitude of other organisms are proving useful. Organisms as diverse as yeast, Acetabularia, Xenopus and mammals reveal an intimate interaction between PP2A in the cytoskeleton and cyclinB/cdc2. Different PP2A subunits in mammals react differently with respect to subcellular location, suggesting cell type specific functions (McCright, 1996). Other work shows that PP2A is involved in positively and negatively regulating response to neuropeptides with different responses depending on receptor subtype (Huang, 1996). Finally, PP2A plays a key role in the differentiation-dependent expression of the neurofilament gene, suggesting an involvement in neural cell fate (Sasahara, 1996). It is clear that PP2A takes on many developmental roles, and only the combination of investigative genetics and the resources of developmental biology coupled with biochemical techniques will reveal the complexities of PP2A's functions.

PP2A and GSK-3β act antagonistically to regulate active zone development
The synapse is composed of an active zone apposed to a postsynaptic cluster of neurotransmitter receptors. Each Drosophila neuromuscular junction comprises hundreds of such individual release sites apposed to clusters of glutamate receptors. This study shows that protein phosphatase 2A (PP2A) is required for the development of structurally normal active zones opposite glutamate receptors. When PP2A is inhibited presynaptically, many glutamate receptor clusters are unapposed to Bruchpilot (Brp), an active zone protein required for normal transmitter release. These unapposed receptors are not due to presynaptic retraction of synaptic boutons, since other presynaptic components are still apposed to the entire postsynaptic specialization. Instead, these data suggest that Brp localization is regulated at the level of individual release sites. Live imaging of glutamate receptors demonstrates that this disruption to active zone development is accompanied by abnormal postsynaptic development, with decreased formation of glutamate receptor clusters. Remarkably, inhibition of the serine-threonine kinase GSK-3beta completely suppresses the active zone defect, as well as other synaptic morphology phenotypes associated with inhibition of PP2A. These data suggest that PP2A and GSK-3beta function antagonistically to control active zone development, providing a potential mechanism for regulating synaptic efficacy at a single release site (Viquez, 2009).

This study demonstrates that the serine-threonine phosphatase PP2A is required in the presynaptic neuron for normal development and maturation of presynaptic release sites. This action of PP2A is opposed by the serine-threonine kinase GSK-3β, suggesting that this phosphatase/kinase pair co-regulate the phosphorylation state and activity of proteins that are required for proper synaptic development (Viquez, 2009).

At the Drosophila NMJ, the synaptic terminal of a motoneuron is a branched chain of synaptic boutons whose gross structure is strongly influenced by the cytoskeleton. Within each synaptic terminal, there are hundreds of individual synapses, neurotransmitter release sites with an active zone directly apposed to a cluster of postsynaptic glutamate receptors. Most studies in Drosophila have focused on genes controlling synaptic terminal development. However with the recent development of antibodies to the active zone component Bruchpilot and the essential glutamate receptor DGluRIII, a genetic analysis of active zone and postsynaptic density development is now feasible. Previous studies have demonstrated that PP2A acts in the motoneuron to control synaptic terminal morphology likely via regulation of microtubules. This study demonstrate that PP2A is also essential for the proper development of the individual synaptic unit, the active zone and glutamate receptor dyad (Viquez, 2009).

Presynaptic inhibition of PP2A impairs synaptic transmission, leading to a large decrease in quantal content. While investigating potential morphological explanations for defective transmitter release, it was observed that many glutamate receptor clusters are unapposed to the active zone protein Bruchpilot. This is not due to retraction of the presynaptic terminal, since apposed and unapposed GluR clusters are intermingled throughout the terminal in a salt and pepper pattern, and presynaptic structures such as synaptic vesicles are still apposed to the entire extent of the postsynaptic specialization. Instead, there is a defect at the level of the individual synapse. These GluR clusters may be unapposed to active zones, or may be apposed to abnormal active zones lacking Bruchpilot. Two lines of evidence suggest that these GluR clusters may be unapposed to active zones. First, Bruchpilot is required for the localization of T-bars to the active zone, so if many active zones are missing Bruchpilot, then there should be a decrease in the proportion of active zones with T-bars. However when PP2A is inhibited no change was seen in the proportion of active zones with T-bars. Second, with PP2A inhibition the number of Brp puncta is down, as is the density of active zones as defined by ultrastructural analysis. This suggests that there is not a large pool of active zones without Brp. Both of these findings suggest that there are fewer active zones, and that those active zones that form do contain Brp. If this is so, then why are GluR clusters present that are unapposed to active zones? This could be due either to a problem with synapse formation/maturation or maintenance. While it is not known which is the case, the model that there is a defect in the formation or maturation is preferred for the following reasons. First, unapposed receptors are more prevalent in the distal regions of the NMJ where new synapses tend to be added. Second, the unapposed receptors form quite small clusters, while newly forming GluR clusters in wild type are also quite small. Finally, live imaging reveals that fewer GluR clusters form late in larval development, demonstrating a defect in synapse formation (Viquez, 2009).

A model is proposed in which PP2A activity is required for the maturation phase of synapse development. In this view, at a wild type synapse a signal would initiate synapse formation, leading to postsynaptic clustering of glutamate receptors as well as transsynaptic interactions that form the tightly apposed pre- and postsynaptic membranes as seen in electron micrographs. Later, additional active zone components such as Brp would be recruited to the active zone, a process known to occur after GluR clustering. With PP2A inhibition, this unknown signal would still initiate synapse formation and induce GluR clusters. However, at some fraction of nascent synapses the maturation process would fail. The GluR clusters could be trapped in their small, immature state or lost, while the transsynaptic process leading to the tight apposition of pre- and postsynaptic membranes would also fail and Brp would not be recruited. The alternate model that synaptic maintenance is disrupted, and that unapposed GluR clusters are the remains of synapses at which the presynaptic terminal has been lost, cannot, however, be ruled out. Regardless of the precise mechanism, these data demonstrate that PP2A is required to ensure the correct apposition of structurally normal active zones and glutamate receptors at the synapse (Viquez, 2009).

PP2A is one of the major serine/threonine phosphatases in the cell, so inhibiting its function likely leads to hyperphosphorylation of many proteins. Hence, phenotypes could be due to the pleiotropic effects of misregulating numerous pathways. The data, however, argue for a good deal of specificity in the function of PP2A for the synaptic morphology phenotypes assayed. Inhibiting PP2A in the neuron leads to misapposed GluR clusters, a disrupted synaptic cytoskeleton, and an altered bouton morphology. Each of these phenotypes is suppressed when GSK-3β is inhibited. This suggests that these synaptic phenotypes are due to the misregulation of a pathway that is antagonistically regulated by PP2A and GSK-3β. Opposite phenotypes are not seen, however, when PP2A is overexpressed, suggesting that hyperphosphorylation affects this pathway more than hypophosphorylation. While genetic studies cannot prove that this phosphatase/kinase pair act directly on the same substrate, the simplest interpretation of the data is that PP2A and GSK-3β co-regulate the phosphorylation state and activity of a protein or proteins that are required for the proper development of active zones and the synaptic cytoskeleton. While these PP2A phenotypes are all suppressed by inhibition of GSK-3β, there is no suppression of the accumulation of synaptic material in the axon, a phenotype consistent with defects in axonal transport. Decreased transport of active zone material such as Brp is a plausible mechanism for the active zone defects in this mutant. However, the failure of GSK-3β inhibition to suppress the axonal transport phenotype demonstrates that the active zone maturation and axon transport phenotypes are genetically separable. Hence, the accumulation of Brp in the axon cannot be responsible for the defects in synaptic maturation (Viquez, 2009).

The identity of the pathway regulated by PP2A and GSK-3β is not known. One candidate substrate is APC2, which binds to and stabilizes the plus end of microtubules and which is a characterized substrate of both PP2A and GSK-3β. In hippocampal cells phosphorylation of APC by GSK-3β inhibits APC function and so disrupts microtubule stability and axon outgrowth. It was shown that loss of APC2 dominantly enhances the PP2A phenotype, which is consistent with the model from hippocampal cells that phosphorylating APC decreases its function. However, if APC2 were the key substrate, then it would be predicted that homozygous APC2 mutants, where all APC2 function is lost, should replicate the PP2A phenotype. However a synaptic apposition phenotype is not seen in recessive mutants for APC2 or in APC1/APC2 double mutants. Instead, the enhancement of the PP2A phenotype by the loss of APC2 suggests that APC2 promotes PP2A function, possibly in its role as a scaffolding molecule. Wnt signaling is candidate pathway for mediating these synaptic phenotypes because wnt signaling is required for normal Drosophila NMJ development and because GSK-3β and PP2A regulate the phosphorylation state of β-catenin in canonical wnt signaling. Inhibition of PP2A would be predicted to lead to hyperphosphorylation and destruction of β-catenin, thereby blocking wnt signaling. However it is unlikely that the PP2A synaptic phenotype is due to loss of canonical wnt signaling. First, this study found that expression of a constitutively active β-catenin does not suppress the PP2A synaptic phenotype but instead has a slight tendency to enhance the cytoskeletal defect. Second, APC functions as part of the destruction complex that leads to degradation of β-Catenin and block of wnt signaling, however APC mutants enhance rather than suppress the PP2A phenotype. These results are inconsistent with the model that the phenotype is due to decreased canonical wnt signaling through β-Catenin. However, the data are consistent with a role for β-catenin-independent wnt signaling. A third candidate substrate is Futsch, since it can be phosphorylated by GSK-3β and the effect of reduction of PP2A activity on Futsch structure is suppressed by reduction in GSK-3β levels. Continued genetic analysis may lead to the identification of the relevant substrate(s) that are antagonistically regulated by PP2A and GSK-3β to control synaptic development (Viquez, 2009).

There are interesting parallels between the function of PP2A and GSK-3β in the developing Drosophila neuromuscular system and in the pathogenesis of neurodegenerative diseases such as Alzheimer's. In Drosophila, PP2A antagonizes GSK-3β function to stabilize the synaptic cytoskeleton and promote synapse formation. In models of Alzheimer's Disease, PP2A and GSK-3β also act antagonistically, for example in regulating the phosphorylation state of tau. In addition, disruptions to the axonal cytoskeleton and synapse loss are early events in Alzheimer's pathogenesis. Characterizing the function of PP2A/GSK-3β in regulating cytoskeletal and synaptic integrity during development may provide insights into their role in regulating cytoskeletal and synaptic integrity during disease (Viquez, 2009).

Greatwall-phosphorylated Endosulfine is both an inhibitor and a substrate of PP2A-B55 heterotrimers

During M phase, Endosulfine (Endos) family proteins, that serve as protein phosphatase inhibitors, are phosphorylated by Greatwall kinase (Gwl), and the resultant pEndos inhibits the phosphatase PP2A-B55 (Twins), which would otherwise prematurely reverse many CDK-driven phosphorylations. This study shows that PP2A-B55 is the enzyme responsible for dephosphorylating pEndos during M phase exit. The kinetic parameters for PP2A-B55's action on pEndos are orders of magnitude lower than those for CDK-phosphorylated substrates, suggesting a simple model for PP2A-B55 regulation that is called inhibition by unfair competition. As the name suggests, during M phase PP2A-B55's attention is diverted to pEndos, which binds much more avidly and is dephosphorylated more slowly than other substrates. When Gwl is inactivated during the M phase-to-interphase transition, the dynamic balance changes: pEndos dephosphorylated by PP2A-B55 cannot be replaced, so the phosphatase can refocus its attention on CDK-phosphorylated substrates. This mechanism explains simultaneously how PP2A-B55 and Gwl together regulate pEndos, and how pEndos controls PP2A-B55 (Williams, 2014).

A model is presented for the function of the Gwl --> pEndos ---| PP2A-B55 module in cell cycle transitions. The major driver for transitions between interphase and M phase is the cyclic activation and degradation of M phase-promoting factor (MPF: Cdk1-Cyclin B). When activated (in part through a feed-forward autoregulatory loop involving the kinases Myt1 and Wee1 and the phosphatase Cdc25), MPF phosphorylates many substrates (CDKSs) that play key roles in M phase events. One such MPF substrate is the kinase Greatwall (Gwl), which in its active form phosphorylates Endosulfine (Endos). Phosphorylated Endos binds to and inhibits the phosphatase PP2A-B55. This inhibition protects MPF substrates, including components of the autoregulatory loop, from premature dephosphorylation during M phase entry. During M phase exit, MPF is inactivated by degradation of its Cyclin B component, and PP2A-B55 becomes reactivated to dephosphorylate many MPF substrates. M phase exit also requires the dephosphorylation and inactivation of both Gwl and Endos. This study show that PP2A-B55 catalyzes Endos dephosphorylation. The activities responsible for the inactivation of Gwl currently remain unknown (Williams, 2014).

Three independent lines of evidence indicate that the major activity contributing to the dephosphorylation of Gwl-phosphorylated Endos is a phosphatase that includes the three subunits of the PP2A-B55 heterotrimer. (1) Inhibitor specificities: all of the anti-Endos activity (whether in M phase or interphase) is sensitive to okadaic acid and calyculin A, and the majority is highly sensitive to fostriecin, suggesting that the catalytic subunit of the anti-Endos phosphatase is PP2A or its less abundant relatives PP4 or PP6. Furthermore, the facts that tautomycetin and the S68D Drosophila phosphomimetic Endos protein are such weak inhibitors of anti-Endos can be most easily explained if the anti-Endos phosphatase has a very low Km for pEndos-below that for either inhibitor-as is the case for PP2A-B55. (2) Ablation of phosphatase activities: depletion of PP2A-A or PP2A-C from S2 tissue culture cells removes most anti-Endos from extracts, while depletion of PP4 from HeLa cells does not affect this activity. Drosophila larvae mutant for twins, which encodes the sole B55-type regulatory subunit of PP2A in flies, exhibit very little anti-Endos, while mutations in genes for other PPP family catalytic and regulatory subunits do not impede pEndos dephosphorylation in larval extracts. (3) Biochemical purification of the activity: anti-Endos copurifies with the anti-CDKS activity previously ascribed to PP2A-B55 heterotrimer, while on the same column it resolves away from other PPP phosphatase subunits. Furthermore, highly purified PP2A-B55 heterotrimer displays robust anti-Endos activity whose properties match that of the predominant anti-Endos activity in extracts (Williams, 2014).

Because most of the experiments were performed with extracts prepared from unsynchronous mammalian or Drosophila cultured cells (the large majority of which are in interphase), it is conceivable that a phosphatase other than PP2A-B55, that is activated only for a very short period following anaphase onset, is the enzyme responsible for dephosphorylating pEndos during M phase exit. This possibility is thought to be extremely remote for several reasons. (1) the inhibition by unfair competition mechanism that is proposed is sufficiently fast to account for the observed rapidity of M phase exit. (2) The characteristics of the M phase and interphase anti-Endos activities measured in Xenopus egg extracts are very similar; PP2A-B55's ability to dephosphorylate pEndos is therefore constitutive with respect to the cell cycle. (3) Because of its extremely low Km, pEndos is so tightly bound to PP2A-B55 during M phase that no other hypothetical phosphatase would be able to inactivate it in a reasonable time frame; a theoretical simulation illustrates this point. There is no reason to postulate the existence of such a transiently activated phosphatase, and this possibility is incompatible with the observed relationship between PP2A-B55 and pEndos (Williams, 2014).

Some extracts contain a secondary activity (~10 to 30% of the total) that also targets the Gwl site in Endos. This minor activity was not characterized in detail, but some evidence suggests that it may be a form of PP1: it is relatively resistant to fostriecin, and RNAi depletion of PP1-87B, the most abundant form of the PP1 catalytic subunit, removes ~20 to 25% of anti-Endos activity from Drosophila S2 cell extracts (Williams, 2014).

A straightforward model is proposed for the relationship between pEndos and PP2A-B55. In essence, pEndos competes with a large class of CDK-catalyzed mitotic phosphosites (of which pCDKS and pSer50 Fizzy are examples) for access to the phosphatase active site. pEndos can successfully compete for the site because its affinity for PP2A-B55 is much higher than those of the competing substrates; that is, the Km for pEndos is extremely low. However, PP2A-B55 dephosphorylates pEndos at a much slower rate (low kcat) than the CDK phosphosites, so the tight interaction of PP2A-B55 and pEndos would be prolonged. Thus, pEndos would soon sequester the phosphatase from competing substrates, protecting such substrates against premature dephosphorylation. pEndos in this way permits M phase to begin and to be maintained as long as Gwl kinase is active. This mechanism is called 'inhibition by unfair competition'. In its essence, the model clarifies how PP2A-B55 can be 'off' during M phase for CDK-phosphorylated substrates, but 'on' at the same time for the Gwl-phosphorylated pEndos substrate (Williams, 2014).

In the context of the cell, the inhibition by unfair competition mechanism requires that pEndos be present in molar excess over PP2A-B55 in mitosis to account for the near-total absence of anti-CDKS activity observed during M phase. Considerable evidence exists that this requirement is met in many cell types. From quantitation on Western blots, it is estimated that the intracellular concentration of the PP2A B55 subunit to be between 100 and 250 nM and that of Endos to be between 500 nM and 1 μμ, depending on the cell type. The 5:1 ratio of Endos to PP2A-B55 observed is roughly consistent with estimates from other laboratories and with estimates from mass spectrometry studies of whole cell extracts, after accounting for all Endos and PP2A-B55 family members. For example, the Pax-DB database integrating the results of many mass spectrometry experiments on a variety of tissue types estimates that the abundance of Endos in Drosophila cells is 295 ppm while that of Twins (B55) is 123 ppm; for human cells, the integrated datasets yield values of 83.2 ppm for Endos-family proteins (ENSA and ARPP-19) and 23.1 ppm for B55-family proteins. Although this study did not determined the fraction of the total Endos protein that is phosphorylated during M phase, other investigators have found that this proportion is roughly 50% in extracts of synchronized mammalian tissue culture cells (perhaps some of which may not have been in M phase), and approaches 100% in frog egg M phase extracts. Sufficient 'headroom' thus exists to conclude that the molar concentration of pEndos during M phase in fact exceeds that of the PP2A-B55 phosphatase (Williams, 2014).

A major virtue of the inhibition by unfair competition model is that it explains not only how pEndos inhibits PP2A-B55 dephosphorylation of pCDKS-class substrates, but also how pEndos can itself become inactivated: the system has an automatic reset that is intrinsic to the mechanism that inactivates PP2A-B55 phosphatase in the first place. When Gwl is inactivated at anaphase onset, the pEndos dephosphorylated by PP2A-B55 can no longer be replaced. PP2A-B55 is now free to work on CDK-phosphorylated substrates and thus to promote the M phase-to-interphase transition. Experiments in which non-radioactive pEndos and radioactive pCDKS were added to the same tube of PP2A-B55, provide a direct in vitro test of the proposed mechanism by mimicking the events that occur during M phase exit. The results show that PP2A-B55 targets pCDKS only after the enzyme has dephosphorylated almost all of the pEndos (Williams, 2014).

M phase exit is surprisingly rapid given the large number of phosphorylations which must be reversed; in Xenopus cycling extracts, for example, the M phase-to-interphase transition is completed within less than 5 min. Even though the turnover rate of pEndos dephosphorylation by PP2A-B55 is quite slow (∼0.05 s−1), the inhibition by unfair competition mechanism is nevertheless compatible with the rapidity of M phase exit. The reason is that Endos is present in cells only in at most a fivefold stoichiometric excess with respect to the phosphatase, as was just discussed. Each molecule of PP2A-B55 thus needs to dephosphorylate only a few molecules of pEndos to effect the M phase-to-interphase transition (Williams, 2014).

To explore this idea quantitatively, the dynamics of a simplified system was modeled, consisting solely of Endos and PP2A-B55 at estimated physiological conditions. Because the Km of PP2A-B55 for pEndos is four-to-five orders of magnitude smaller than the Km for other substrates, typified by pCDKS, and the pEndos concentration during M phase is in excess of the phosphatase concentration, it is suggested that the approximation of ignoring the binding of PP2A-B55 to other substrates is appropriate, and that this simplified model should capture the essence of the regulatory process. The calculation begins at the end of M-phase (t = 0) with the inactivation of Gwl. Almost all the PP2A-B55 is sequestered by pEndos until PP2A-B55-catalyzed dephosphorylation causes the pEndos concentration to decrease from 1 μM to ∼250 nM, the intracellular PP2A-B55 concentration. Beyond this point, PP2A-B55 is rapidly released from sequestration; half is available to act on other substrates within 74 s (Williams, 2014).

Cells likely harbor a pEndos-targeting phosphatase other than PP2A-B55; because this secondary activity is fostriecin-resistant, it is speculated that it may be a form of PP1. The time courses of pEndos dephosphorylation and PP2A-B55 desequestration are modified if this second phosphatase (again called PPX) is present at the same concentration as PP2A-B55 and acts on pEndos with kinetic parameters matching the activity of PP2A-B55 on pCDKS. In this case, the lag in PP2A-B55 desequestration is shortened, because PPX can dephosphorylate the pEndos that is not bound to PP2A/B55. However, the presence of PPX makes very little difference once the pEndos concentration decreases to the PP2A-B55 concentration of 250 nM, since almost all the remaining pEndos is bound to PP2A-B55. In this scenario, half of the PPA-B55 is released from pEndos by 38 s, producing a modest acceleration in M phase exit (Williams, 2014).

Our identification of PP2A-B55 as the anti-Endos phosphatase poses a dilemma in terms of M phase exit: the inhibition by unfair competition model describes events that must occur downstream of the inactivation of Gwl kinase, but what then can inactivate Gwl upon anaphase onset? According to the model, the rate-limiting Gwl-inactivating phosphatase cannot be PP2A-B55, because the system would be futile. Gwl inactivation could not proceed until pEndos was depleted, but pEndos cannot be inactivated as long as Gwl is active. An argument can be made that PP2A-B55 should be the Gwl-inactivating phosphatase because Gwl is activated by CDKs. Indeed, one group has recently reported some evidence in favor of this idea. However, it should be emphasized that PP2A-B55 does not act on all, or even perhaps the majority, of CDK phosphosites. The obvious resolution of this dilemma is therefore that a phosphatase other than PP2A-B55 inactivates Gwl during M phase exit. Preliminary evidence in support of this idea in two ways. First, purified PP2A-B55 in vitro is very inefficient in inactivating Gwl. Second, when active Gwl is added to interphase extracts from a variety of cell types, it becomes rapidly inactivated and dephosphorylated in a process that is insensitive to okadaic acid (and cannot therefore be controlled by PP2A-B55). Some evidence in favor of the existence of an okadaic acid-resistant anti-Gwl phosphatase has also recently been obtained by another group; the identity of this Gwl-inactivating enzyme is currently unknown (Williams, 2014).


GENE STRUCTURE

Transcript sizes - 2.75 kb for DPR55-1; 2.5 kb for DPR55-4 (with minor transcripts of 3.7 and 5.0 kb)

Exons - 7


PROTEIN STRUCTURE

Amino Acids - 499 for DPR55-1 and 443 for DPR55-4

Structural Domains

The cDNAs for Twins proteins fall into two classes: the DPR55-1-type and the DPR55-4-type. Three exons (V, VI and VII) are common to all of the cDNAs. The 5' sequences of the DPR55-1-type and the the DPR55-4-type are derived from (respectively) exons I and II and III and IV. There are additional variations in this latter class, in which varying portions of the 3' end of exon III are present. The DPR55-4 protein differs only in the first 3 amino acids from the DPR55-1 protein sequence and shows 78% and 77% identity to the human PR55alpha and PR55beta sequences, respectively. The sequence alignment with the S. cerevisiae PR55 homolog CDC65 requires the introduction of several gaps but still shows 72% similarity if conserved amino acid substitutions are considered (Mayer-Jaekel, 1993).

cDNA clones encoding the catalytic subunit and the 65-kDa regulatory subunit of protein phosphatase 2A (PR65) from Drosophila have been isolated by homology screening with the corresponding human cDNAs. The Drosophila clones were used to analyze the spatial and temporal expression of the transcripts encoding these two protein subunits. The Drosophila PR65 cDNA clones contains an open reading frame of 1773 nucleotides encoding a protein of 65.5 kDa. The predicted amino acid sequence showed 75 and 71% identity to the human PR65 alpha and beta isoforms, respectively. As previously reported for the mammalian PR65 isoforms, Drosophila PR65 is composed of 15 imperfect repeating units of approximately 39 amino acids. The residues contributing to this repeat structure also show the highest sequence conservation between species, indicating a functional importance for these repeats. The gene encoding Drosophila PR65 was located at 29B1,2 on the second chromosome. A major transcript of 2.8 kilobase (kb) encoding the PR65 subunit and two transcripts of 1.6 and 2.5 kb encoding the catalytic subunit can be detected throughout Drosophila development. All of these mRNAs are most abundant during early embryogenesis and are expressed at lower levels in larvae and adult flies. In situ hybridization of different developmental stages showed a colocalization of the PR65 and catalytic subunit transcripts. The mRNA expression is high in the nurse cells and oocytes, consistent with a high equally distributed expression in early embryos. In later embryonic development, the expression remains high in the nervous system and the gonads but the overall transcript levels decrease. In third instar larvae, high levels of mRNA can be observed in the brain, imaginal discs, and salivary glands. These results indicate that protein phosphatase 2A transcript levels change during development in both a tissue and in a time-specific manner (Mayer-Jaekel, 1992).


twins: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 15 FEB 97 

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