Structural basis of Aurora-A activation by TPX2 at the mitotic spindle

Aurora-A is an oncogenic kinase essential for mitotic spindle assembly. It is activated by phosphorylation and by the microtubule-associated protein TPX2, which also localizes the kinase to spindle microtubules. We have uncovered the molecular mechanism of Aurora-A activation by determining crystal structures of its phosphorylated form both with and without a 43 residue long domain of TPX2 that we identified as fully functional for kinase activation and protection from dephosphorylation. In the absence of TPX2, the Aurora-A activation segment is in an inactive conformation, with the crucial phosphothreonine exposed and accessible for deactivation. Binding of TPX2 triggers no global conformational changes in the kinase but pulls on the activation segment, swinging the phosphothreonine into a buried position and locking the active conformation. The recognition between Aurora-A and TPX2 resembles that between the cAPK catalytic core and its flanking regions, suggesting this molecular mechanism may be a recurring theme in kinase regulation.     

The Aurora-A/TPX2 complex: A novel oncogenic holoenzyme?

The Aurora-A kinase regulates cell division by phosphorylating multiple downstream targets in the mitotic apparatus. Aurora-A is frequently overexpressed in tumor cells and it is therefore regarded as a novel candidate target in anti-cancer therapy. Its actual contribution to cell transformation, however, is not entirely clarified; furthermore, its transforming ability has been found to vary broadly depending on the systems and experimental conditions in which it was assayed. This variability suggests that Aurora-A overexpression requires the concomitant deregulation of partner factor(s) to fully elicit its oncogenic potential. Molecular and structural studies indicate that the full activation and correct mitotic localisation of Aurora-A require its interaction with the spindle regulator TPX2. In this review we propose a brief reappraisal of Aurora-A intrinsic oncogenic features. We then present literature screening data indicating that TPX2 is also overexpressed in many tumor types, and, furthermore, that Aurora-A and TPX2 are frequently co-overexpressed. We therefore propose that the association of Aurora-A and TPX2 gives rise to a novel functional unit with oncogenic properties. We also suggest that some of the roles that are conventionally attributed to Aurora-A in cell transformation and tumorigenesis could in fact be a consequence of the oncogenic activation of this unit.     

Poleward Transport of TPX2 in the Mammalian Mitotic Spindle Requires Dynein,Eg5, and Microtubule Flux

TPX2 is a Ran-regulated spindle assembly factor that is required for kinetochore fiber formation and activation of the mitotic kinase Aurora A. TPX2 is enriched near spindle poles and is required near kinetochores, suggesting that it undergoes dynamic relocalization throughout mitosis. Using photoactivation, we measured the movement of PA-GFP-TPX2 in the mitotic spindle. TPX2 moves poleward in the half-spindle and is static in the interzone and near spindle poles. Poleward transport of TPX2 is sensitive to inhibition of dynein or Eg5 and to suppression of microtubule flux with nocodazole or antibodies to Kif2a. Poleward transport requires the C terminus of TPX2, a domain that interacts with Eg5. Overexpression of TPX2 lacking this domain induced excessive microtubule formation near kinetochores, defects in spindle assembly and blocked mitotic progression. Our data support a model in which poleward transport of TPX2 down-regulates its microtubule nucleating activity near kinetochores and links microtubules generated at kinetochores to dynein for incorporation into the spindle.     

A Functional Interplay Between Aurora-A,Plk1 and TPX2 at Spindle Poles: Plk1 Controls Centrosomal Localization of Aurora-A and TPX2 Spindle Association

Aurora-A and Plk1 are centrosomal kinases involved in centrosome maturation and spindle assembly. The microtubule-binding protein TPX2 interacts with, and activates, Aurora-A. Here we have used RNA interference-mediated inactivation to investigate whether Aurora-A, Plk1 and TPX2 act independently or are part of one signalling cascade in spindle formation in mammalian cells. We have identified both specific, and overlapping, roles of each single regulator in centrosome maturation and spindle formation: (i) Aurora-A and TPX2 are required for centriole cohesion and spindle bipolarity; (ii) TPX2, besides its known role in microtubule organization, is also involved in centrosome maturation; (iii) finally, Plk1 controls the localization of Aurora-A to centrosomes, as well as TPX2 recruitment to microtubules. Based on these results therefore a hierachical functional relation between Plk1 and the Aurora-A/TPX2 pathway emerges.     

An essential function of the C. elegans ortholog of TPX2 is to localize activated aurora A kinase to mitotic spindles

In vertebrates, the microtubule binding protein TPX2 is required for meiotic and mitotic spindle assembly. TPX2 is also known to bind to and activate Aurora A kinase and target it to the spindle. However, the relationship between the TPX2-Aurora A interaction and the role of TPX2 in spindle assembly is unclear. Here, we identify TPXL-1, a C. elegans protein that is the first characterized invertebrate ortholog of TPX2. We demonstrate that an essential role of TPXL-1 during mitosis is to activate and target Aurora A to microtubules. Our data suggest that this targeting stabilizes microtubules connecting kinetochores to the spindle poles. Thus, activation and targeting of Aurora A appears to be an ancient and conserved function of TPX2 that plays a central role in mitotic spindle assembly.     

Spatial regulation of Aurora A activity during mitotic spindle assembly requires RHAMM to correctly localize TPX2

Construction of a mitotic spindle requires biochemical pathways to assemble spindle microtubules and structural proteins to organize these microtubules into a bipolar array. Through a complex with dynein, the receptor for hyaluronan-mediated motility (RHAMM) cross-links mitotic microtubules to provide structural support, maintain spindle integrity, and correctly orient the mitotic spindle. Here, we locate RHAMM to sites of microtubule assembly at centrosomes and non-centrosome sites near kinetochores and demonstrate that RHAMM is required for the activation of Aurora kinase A. Silencing of RHAMM delays the kinetics of spindle assembly, mislocalizes targeting protein for XKlp2 (TPX2), and attenuates the localized activation of Aurora kinase A with a consequent reduction in mitotic spindle length. The RHAMM–TPX2 complex requires a C-terminal basic leucine zipper in RHAMM and a domain that includes the nuclear localization signal in TPX2. Together, our findings identify RHAMM as a critical regulator for Aurora kinase A signaling and suggest that RHAMM ensures bipolar spindle assembly and mitotic progression through the integration of biochemical and structural pathways.     

Building a spindle of the correct length in human cells requires the interaction between TPX2 and Aurora A

To assemble mitotic spindles, cells nucleate microtubules from a variety of sources including chromosomes and centrosomes. We know little about how the regulation of microtubule nucleation contributes to spindle bipolarity and spindle size. The Aurora A kinase activator TPX2 is required for microtubule nucleation from chromosomes as well as for spindle bipolarity. We use bacterial artificial chromosome-based recombineering to introduce point mutants that block the interaction between TPX2 and Aurora A into human cells. TPX2 mutants have very short spindles but, surprisingly, are still bipolar and segregate chromosomes. Examination of microtubule nucleation during spindle assembly shows that microtubules fail to nucleate from chromosomes. Thus, chromosome nucleation is not essential for bipolarity during human cell mitosis when centrosomes are present. Rather, chromosome nucleation is involved in spindle pole separation and setting spindle length. A second Aurora A-independent function of TPX2 is required to bipolarize spindles.     

Aurora kinase-A regulates kinetochore/chromatin associated microtubule assembly in human cells

Microtubule nucleation and formation from the kinetochore/chromatin have been proposed to contribute to bipolar spindle assembly facilitating equal segregation of chromosomes in mitosis. Although two independent pathways involving the small Ran GTPase-TPX2 proteins and the chromosomal passenger complex proteins have been implicated in the formation of microtubules from the kinetochore/chromatin, detailed molecular mechanisms integrating the pathways and regulating the process have not been well elucidated. This study demonstrates that Aurora kinase-A plays a central role in the kinetochore/chromatin associated microtubule assembly in human cells by integrating the two pathways regulating the process. Silencing by siRNA and over expression of a kinase inactive mutant revealed involvement of Aurora-A at two critical steps. These include accumulation of g-tubulin in the vicinity of kinetochore/chromatin to create microtubule nucleation sites as well as INCENP and TPX2 mediated activation of Aurora-A facilitating formation and stabilization of microtubules. The findings provide the first evidence of Aurora-A, in association with INCENP and TPX2, being a key regulator of kinetochore/chromatin associated microtubule formation in human cells.     

Regulation of Aurora-A kinase on the mitotic spindle

The error-free segregation of duplicated chromosomes during cell division is essential for the maintenance of an intact genome. This process is brought about by a highly dynamic bipolar array of microtubules, the mitotic spindle. The formation and function of the mitotic spindle during M-phase of the cell cycle is regulated by protein phosphorylation, involving multiple protein kinases and phosphatases. Prominent among the enzymes implicated in spindle assembly is the serine/threonine-specific protein kinase Aurora-A. In several common human tumors, Aurora-A is overexpressed, and deregulation of this kinase was shown to result in mitotic defects and aneuploidy. Moreover, recent genetic evidence directly links the human Aurora-A gene to cancer susceptibility. Several of the physiological substrates of Aurora-A presumably await identification, but recent studies are beginning to shed light on the regulation of this critical mitotic kinase. Here, we review these findings with particular emphasis on the role of TPX2, a prominent spindle component implicated in a Ran-GTP-mediated spindle assembly pathway.Communicated by E.A. Nigg     

TPX2 is required for postmitotic nuclear assembly in cell-free Xenopus laevis egg extracts

Cell division in many metazoa is accompanied by the disassembly of the nuclear envelope and the assembly of the mitotic spindle. These dramatic structural rearrangements are reversed after mitosis, when the mitotic spindle is dismantled and the nuclear envelope reassembles. The targeting protein for XKlp2 (TPX2) plays important roles in mitotic spindle assembly. We report that TPX2 depletion from nuclear assembly extracts prepared from Xenopus laevis eggs results in the formation of nuclei that are only about one fifth the size of control nuclei. TPX2-depleted nuclei assemble nuclear envelopes, nuclear pore complexes, and a lamina, and they perform nuclear-specific functions, including DNA replication. We show that TPX2 interacts with lamina-associated polypeptide 2 (LAP2), a protein known to be required for nuclear assembly in interphase extracts and in vitro. LAP2 localization is disrupted in TPX2-depleted nuclei, suggesting that the interaction between TPX2 and LAP2 is required for postmitotic nuclear reformation.     

Urochordate Ascidians Possess a Single Isoform of Aurora Kinase That Localizes to the Midbody via TPX2 in Eggs and Cleavage Stage Embryos

Aurora kinases are key proteins found throughout the eukaryotes that control mitotic progression. Vertebrate Aurora-A and B kinases are thought to have evolved from a single Aurora-kinase isoform closest to that found in present day urochordates. In urochordate ascidians Aurora binds both TPX2 (a vertebrate AURKA partner) and INCENP (a vertebrate AURKB partner) and localizes to centrosomes and spindle microtubules as well as chromosomes and midbody during both meiosis and mitosis. Ascidian Aurora also displays this localization pattern during mitosis in echinoderms, strengthening the idea that non-vertebrate deuterostomes such as the urochordates and echinoderms possess a single form of Aurora kinase that has properties of vertebrate Aurora-kinase A and B. In the ascidian, TPX2 localizes to the centrosome and the spindle poles also as in vertebrates. However, we were surprised to find that TPX2 also localized strongly to the midbody in ascidian eggs and embryos. We thus examined more closely Aurora localization to the midbody by creating two separate point mutations of ascidian Aurora predicted to perturb binding to TPX2. Both forms of mutated Aurora behaved as predicted: neither localized to spindle poles where TPX2 is enriched. Interestingly, neither form of mutated Aurora localized to the midbody where TPX2 is also enriched, suggesting that ascidian Aurora midbody localization required TPX2 binding in ascidians. Functional analysis revealed that inhibition of Aurora kinase with a pharmacological inhibitor or with a dominant negative kinase dead form of Aurora caused cytokinesis failure and perturbed midbody formation during polar body extrusion. Our data support the view that vertebrate Aurora-A and B kinases evolved from a single non-vertebrate deuterostome ancestor. Moreover, since TPX2 localizes to the midbody in ascidian eggs and cleavage stage embryos it may be worthwhile re-assessing whether Aurora A kinase or TPX2 localize to the midbody in eggs and cleavage stage embryos.     

Abrogation of AuroraA-TPX2 by novel natural inhibitors: molecular dynamics-based mechanistic analysis

Abstract

Introduction: Cancer is characterized by uncontrolled cell growth and genetic instabilities. The human Aurora-A kinase protein plays a crucial role in spindle assembly during mitosis and is activated by another candidate oncogene, targeting protein for Xklp2 (TPX2). It has been proposed that dissociation of Aurora A–TPX2 complex leads to disruption of mitotic spindle apparatus, thereby preventing cell division and further tumor growth. Materials and methods: A large natural compound library was docked against the active site of Aurora A–TPX2 complex. The protein–ligand complexes were subjected to molecular dynamics simulation to ascertain their binding stability. The drug properties of the compounds were analyzed to observe their drug-like properties. Results: The virtual screening of natural compound library yielded two high scoring compounds, the first compound CTOM [ZINC ID: 38143674] (Glide score: –9.49) was stable for 17 ns while the second TTOM (Glide score: ?9.07) was stable for 15 ns. While CTOM interacted with His280, Thr288 of Aurora A and Tyr34, Lys38 of TPX2, TTOM interacted with Arg285 and Arg286 in addition to the residues involved with CTOM. Conclusions: We report two natural compounds as potential drugs leads for the disruption of this complex. These ligands show a preferable docking score and have many drugs like properties within in the range of 95% of known drugs. The study provides evidence that CTOM and TTOM can efficiently inhibit the TPX2-mediated activation of Aurora A. Thus, it paves way for an elaborate investigation and establishes the importance of computational approaches as time- and cost-effective techniques.     

Phosphorylation of Targeting Protein for Xenopus Kinesin-like Protein 2 (TPX2) at Threonine 72 in Spindle Assembly

The human ortholog of the targeting protein for Xenopus kinesin-like protein 2 (TPX2) is a cytoskeletal protein that plays a major role in spindle assembly and is required for mitosis. During spindle morphogenesis, TPX2 cooperates with Aurora A kinase and Eg5 kinesin to regulate microtubule organization. TPX2 displays over 40 putative phosphorylation sites identified from various high-throughput proteomic screenings. In this study, we characterize the phosphorylation of threonine 72 (Thr⁷²) in human TPX2, a residue highly conserved across species. We find that Cdk1/2 phosphorylate TPX2 in vitro and in vivo. Using homemade antibodies specific for TPX2 phosphorylated at Thr⁷², we show that this phosphorylation is cell cycle-dependent and peaks at M phase. Endogenous TPX2 phosphorylated at Thr⁷² does not associate with the mitotic spindle. Furthermore, ectopic GFP-TPX2 T72A preferentially concentrates on the spindle, whereas GFP-TPX2 WT distributes to both spindle and cytosol. The T72A mutant also increases the proportion of cells with multipolar spindles phenotype. This effect is associated with increased Aurora A activity and abnormally elongated spindles, indicative of higher Eg5 activity. In summary, we propose that phosphorylation of Thr⁷² regulates TPX2 localization and impacts spindle assembly via Aurora A and Eg5.     

HURP is part of a Ran-dependent complex involved in spindle formation

BACKGROUND: GTP-loaded Ran induces the assembly of microtubules into aster-like and spindle-like structures in Xenopus egg extract. The microtubule-associated protein (MAP), TPX2, can mediate Ran's role in aster formation, but factors responsible for the transition from aster-like to spindle-like structures have not been described. RESULTS: Here we identify a complex that is required for the conversion of aster-like to spindle-like structures. The complex consists of two characterized MAPs (TPX2, XMAP215), a plus end-directed motor (Eg5), a mitotic kinase (Aurora A), and HURP, a protein associated with hepatocellular carcinoma. Formation and function of the complex is dependent on Aurora A activity. HURP protein was further characterized and shown to bind microtubules and affect their organization both in vitro and in vivo. In egg extract, anti-HURP antibodies disrupt the formation of both Ran-dependent and chromatin and centrosome-induced spindles. HURP is also required for the proper formation and function of mitotic spindles in HeLa cells. CONCLUSIONS: HURP is a new and essential component of the mitotic apparatus. HURP acts as part of a multicomponent complex that affects the growth or stability of spindle MTs and is required for spindle MT organization.     

Molecular dynamics of Aurora-A kinase in living mitotic cells simultaneously visualized with histone H3 and nuclear membrane protein importinalpha

Aurora-A is known to be a mitotic kinase required for spindle assembly. We constructed a human stable cell-line in which Aurora-A, histone H3 and importinalpha were differentially expressed as fusions to green, cyan, and red fluorescent proteins (GFP, CFP and DsRed). In interphase cells, GFP-Aurora-A was localized in the centrosome. Its molecular behavior in living mitotic cells was extensively analyzed by an advanced timelapse image analyzing system. In G2 phase, duplicated centrosomal dots of Aurora-A separated and moved to the opposite poles, a process requiring 18 min. In prophase, the Aurora-A dots approached closer and the nuclear membrane of DsRed-importinalpha beneath them became thick and invaginated, resulting in a "dumb-bell" shaped nucleus with condensed chromatin. As the importinalpha membrane further shrank and disappeared, the condensed chromatin was excluded from the nucleus and the Aurora-A dots grew rapidly into a spindle-like structure. Congression of mitotic chromosomes continued for 20-50 min until they were properly aligned at the spindle equator and then the sister chromatids started to segregate, taking 4-6 min for them to reach the poles. An importinalpha membrane reappeared around the surface of chromatin 10 min after anaphase onset. Aurora-A gradually decreased in size in telophase and returned to the surface of the newly formed small sister nuclei. These observations showed that the morphological change of Aurora-A was cooperated with the breakdown and reformation of nuclear membrane. Immunostaining with anti-alpha or gamma-tubulin further indicated that Aurora-A was involved in the formation of mitotic spindle in metaphase as well as the subsequent chromosome movement in anaphase.     

A novel mechanism for activation of the protein kinase Aurora A

Segregation of chromosomes during mitosis requires interplay between several classes of protein on the spindle, including protein kinases, protein phosphatases, and microtubule binding motor proteins [1-4]. Aurora A is an oncogenic cell cycle-regulated protein kinase that is subject to phosphorylation-dependent activation [5-11]. Aurora A localization to the mitotic spindle depends on the motor binding protein TPX2 (Targeting Protein for Xenopus kinesin-like protein 2), but the protein(s) involved in Aurora A activation are unknown [11-13]. Here, we purify an activator of Aurora A from Xenopus eggs and identify it as TPX2. Remarkably, Aurora A that has been fully deactivated by Protein Phosphatase 2A (PP2A) becomes phosphorylated and reactivated by recombinant TPX2 in an ATP-dependent manner. Increased phosphorylation and activation of Aurora A requires its own kinase activity, suggesting that TPX2 stimulates autophosphorylation and autoactivation of the enzyme. Consistently, wild-type Aurora A, but not a kinase inactive mutant, becomes autophosphorylated on the regulatory T loop residue (Thr 295) after TPX2 treatment. Active Aurora A from bacteria is further activated at least 7-fold by recombinant TPX2, and TPX2 also impairs the ability of protein phosphatases to inactivate Aurora A in vitro. This concerted mechanism of stimulation of activation and inhibition of deactivation implies that TPX2 is the likely regulator of Aurora A activity at the mitotic spindle and may explain why loss of TPX2 in model systems perturbs spindle assembly [14-16]. Our finding that a known binding protein, and not a conventional protein kinase, is the relevant activator for Aurora A suggests a biochemical model in which the dynamic localization of TPX2 on mitotic structures directly modulates the activity of Aurora A for spindle assembly.     

Aurora-A phosphorylates Augmin complex component Hice1 protein at an N-terminal serine/threonine cluster to modulate its microtubule binding activity during spindle assembly

Proper assembly of mitotic spindles requires Hice1, a spindle-associated protein. Hice1 possesses direct microtubule binding activity at its N-terminal region and contributes to intraspindle microtubule nucleation as a subunit of the Augmin complex. However, whether microtubule binding activity of Hice1 is modulated by mitotic regulators remains unexplored. Here, we found that Aurora-A kinase, a major mitotic kinase, specifically binds to and phosphorylates Hice1. We identified four serine/threonine clusters on Hice1 that can be phosphorylated by Aurora-A in vitro. Of the four clusters, the Ser/Thr-17-21 cluster was the most critical for bipolar spindle assembly, whereas other phospho-deficient point mutants had a minimal effect on spindle assembly. Immunostaining with a phospho-Ser-19/20 phospho-specific antibody revealed that phosphorylated Hice1 primarily localizes to spindle poles during prophase to metaphase but gradually diminishes after anaphase. Consistently, the phospho-mimic 17-21E mutant reduced microtubule binding activity in vitro and diminished localization to spindles in vivo. Furthermore, expression of the 17-21E mutant led to decreased association of Fam29a, an Augmin component, with spindles. On the other hand, expression of the phospho-deficient 17-21A mutant permitted intraspindle nucleation but delayed the separation of early mitotic spindle poles and the timely mitotic progression. Taken together, these results suggest that Aurora-A modulates the microtubule binding activity of Hice1 in a spatiotemporal manner for proper bipolar spindle assembly.     

A Ran signalling pathway mediated by the mitotic kinase Aurora A in spindle assembly

The activated form of Ran (Ran-GTP) stimulates spindle assembly in Xenopus laevis egg extracts, presumably by releasing spindle assembly factors, such as TPX2 (target protein for Xenopus kinesin-like protein 2) and NuMA (nuclear-mitotic apparatus protein) from the inhibitory binding of importin-alpha and -beta. We report here that Ran-GTP stimulates the interaction between TPX2 and the Xenopus Aurora A kinase, Eg2. This interaction causes TPX2 to stimulate both the phosphorylation and the kinase activity of Eg2 in a microtubule-dependent manner. We show that TPX2 and microtubules promote phosphorylation of Eg2 by preventing phosphatase I (PPI)-induced dephosphorylation. Activation of Eg2 by TPX2 and microtubules is inhibited by importin-alpha and -beta, although this inhibition is overcome by Ran-GTP both in the egg extracts and in vitro with purified proteins. As the phosphorylation of Eg2 stimulated by the Ran-GTP-TPX2 pathway is essential for spindle assembly, we hypothesize that the Ran-GTP gradient established by the condensed chromosomes is translated into the Aurora A kinase gradient on the microtubules to regulate spindle assembly and dynamics.     

Interaction of Aurora-A and centrosomin at the microtubule-nucleating site in Drosophila and mammalian cells

A mitosis-specific Aurora-A kinase has been implicated in microtubule organization and spindle assembly in diverse organisms. However, exactly how Aurora-A controls the microtubule nucleation onto centrosomes is unknown. Here, we show that Aurora-A specifically binds to the COOH-terminal domain of a Drosophila centrosomal protein, centrosomin (CNN), which has been shown to be important for assembly of mitotic spindles and spindle poles. Aurora-A and CNN are mutually dependent for localization at spindle poles, which is required for proper targeting of gamma-tubulin and other centrosomal components to the centrosome. The NH2-terminal half of CNN interacts with gamma-tubulin, and induces cytoplasmic foci that can initiate microtubule nucleation in vivo and in vitro in both Drosophila and mammalian cells. These results suggest that Aurora-A regulates centrosome assembly by controlling the CNN's ability to targeting and/or anchoring gamma-tubulin to the centrosome and organizing microtubule-nucleating sites via its interaction with the COOH-terminal sequence of CNN.     

Activation of Aurora-A kinase by protein partner binding and phosphorylation are independent and synergistic

Protein kinases are activated by phosphorylation and by the binding of activator proteins. The interplay of these two factors is incompletely understood. We applied energetic analysis to this question and characterized the activation process of the serine/threonine kinase Aurora-A by phosphorylation and by its protein partner, targeting protein for Xenopus kinesin-like protein 2 (TPX2). We discovered that these two activators act synergistically and without a predefined order: each can individually increase the activity of Aurora-A, and the effect of both bound together is the exact sum of their individual contributions to catalysis. Unexpectedly, the unphosphorylated enzyme has catalytic activity that is increased 15-fold by the binding of TPX2 alone. The energetic contribution of phosphorylation to catalysis is 2-fold greater than that of TPX2 binding, which is independent of the phosphorylation state of the enzyme. Based on this analysis, we propose a revised, fluid model of Aurora-A activation in which the first step is a reduction in the mobility of the activation loop by either TPX2 binding or phosphorylation. Furthermore, our results suggest that unphosphorylated Aurora-A bound to the mitotic spindle by TPX2 is catalytically active and that the phosphorylation state of Aurora-A is an inaccurate surrogate for its activity. Extending this form of analysis will allow us to compare quantitatively the effects of the whole network of kinase-activating partners. Comparison with other kinases showed that kinetic characterization detects those kinases whose activation loops undergo a rearrangement upon phosphorylation and thus whose unphosphorylated state offers a distinct target for the development of Type II inhibitors.