Gonadotropic activity of a second relaxin-type peptide in starfish.

In starfish, a relaxin-like gonad-stimulating peptide (RGP) acts as a gonadotropin that triggers gamete maturation and spawning. In common with other relaxin/insulin superfamily peptides, RGP consists of an A- and a B-chain, with cross-linkages mediated by one intra- and two inter-chain disulfide bonds. In this study, a second relaxin-like peptide (RLP2) was identified in starfish species belonging to the orders Valvatida, Paxillosida, and Forcipulatida. Like RGP, RLP2 precursors comprise a signal peptide and a C-peptide in addition to the A- and B-chains. However, a unique cysteine motif [CC-(3X)-C-(10X)-C] is present in the A-chain of RLP2, which contrasts with the cysteine motif in other members of the relaxin/insulin superfamily [CC-(3X)-C-(8X)-C]. Importantly, in vitro pharmacological tests revealed that Patiria pectinifera RLP2 (Ppe-RLP2) and Asterias rubens RLP2 (Aru-RLP2) trigger shedding of mature eggs from ovaries of P. pectinifera and A. rubens, respectively. Furthermore, the potencies of Ppe-RLP2 and Aru-RLP2 as gonadotropic peptides were similar to those of Ppe-RGP and Aru-RGP, respectively, and the effect of RLP2 exhibited partial species-specificity. These findings indicate that two relaxin-type peptides regulate spawning in starfish and therefore we propose that RGP and RLP2 are renamed RGP1 and RGP2, respectively.

A Relaxin-Like Gonad-Stimulating Peptide Appears in the Early Development of the Starfish Patiria pectinifera.

Relaxin-like gonad-stimulating peptide (RGP) is a hormone with gonadotropin-like activity in starfish. This study revealed that spawning inducing activity was detected in an extract of brachiolaria larvae of Patiria pectinifera. Spawning inducing activity in the extract was due to P. pectinifera RGP (PpeRGP), not 1-methyladenine. The expression of PpeRGP mRNA was also found in brachiolaria. Immunohistochemical observation with specific antibodies for PpeRGP showed that PpeRGP was distributed in the peripheral adhesive papilla of the brachiolaria arms. In contrast, PpeRGP was not detected in the adult rudiment or ciliary band regions, which are present in the neural system. These findings strongly suggest that RGP exists in the larvae before metamorphosis. Because gonads are not developed in starfish larvae, it seems likely that RGP plays another role other than gonadotropic action in the early development of starfish.

The Starfish Asterina pectinifera: Collection and Maintenance of Adults and Rearing and Metamorphosis of Larvae.

Here we describe methods for (a) collecting starfish during their breeding period; (b) maintaining adults with fully grown gonads in laboratory aquaria; (c) rearing fertilized eggs to brachiolaria larvae, and (d) inducing larvae to metamorphose into juveniles under laboratory conditions. Such protocols should facilitate various analyses of starfish development throughout the entire life cycle of these model organisms.

SGK phosphorylates Cdc25 and Myt1 to trigger cyclin B-Cdk1 activation at the meiotic G2/M transition.

The kinase cyclin B-Cdk1 complex is a master regulator of M-phase in both mitosis and meiosis. At the G2/M transition, cyclin B-Cdk1 activation is initiated by a trigger that reverses the balance of activities between Cdc25 and Wee1/Myt1 and is further accelerated by autoregulatory loops. In somatic cell mitosis, this trigger was recently proposed to be the cyclin A-Cdk1/Plk1 axis. However, in the oocyte meiotic G2/M transition, in which hormonal stimuli induce cyclin B-Cdk1 activation, cyclin A-Cdk1 is nonessential and hence the trigger remains elusive. Here, we show that SGK directly phosphorylates Cdc25 and Myt1 to trigger cyclin B-Cdk1 activation in starfish oocytes. Upon hormonal stimulation of the meiotic G2/M transition, SGK is activated by cooperation between the Gßγ-PI3K pathway and an unidentified pathway downstream of Gßγ, called the atypical Gßγ pathway. These findings identify the trigger in oocyte meiosis and provide insights into the role and activation of SGK.

Effects of Polar Steroids from the Starfish Patiria (=Asterina) pectinifera in Combination with X-Ray Radiation on Colony Formation and Apoptosis Induction of Human Colorectal Carcinoma Cells.

Despite significant advances in the understanding, prevention, and treatment of cancer, the disease continues to affect millions of people worldwide. Chemoradiation therapy is a rational approach that has already proven beneficial for several malignancies. However, the existence of toxicity to normal tissue is a serious limitation of this treatment modality. The aim of the present study is to investigate the ability of polar steroids from starfish Patiria (=Asterina) pectinifera to enhance the efficacy of radiation therapy in colorectal carcinoma cells. The cytotoxic activity of polar steroids and X-ray radiation against DLD-1, HCT 116, and HT-29 cells was determined by an MTS assay. The effect of compounds, X-ray, and their combination on colony formation was studied using the soft agar method. The molecular mechanism of the radiosensitizing activity of asterosaponin P1 was elucidated by western blotting and the DNA comet assay. Polar steroids inhibited colony formation in the tested cells, and to a greater extent in HT-29 cells. Asterosaponin P1 enhanced the efficacy of radiation and, as a result, reduced the number and size of the colonies of colorectal cancer cells. The radiosensitizing activity of asterosaponin P1 was realized by apoptosis induction through the regulation of anti- and pro-apoptotic protein expression followed by caspase activation and DNA degradation.

Interaction of starfish gonadotropin with its receptor: Effect of chimeric relaxin-like gonad-stimulating peptides.

A relaxin-like gonad-stimulating peptide (RGP) of starfish Patiria (Asterina) pectinifera is the first identified invertebrate gonadotropin for final gamete maturation. Recently, we found three orthologs of RGP in the class Asteroida; PpeRGP in P. pectinifera, AamRGP in Asterias amurensis, and AjaRGP in Aphelasterias japonica. In this study, nine kinds of RGP derivatives with exchanged each A- and B-chain were synthesized chemically to analyze the interaction of RGP with its receptor. Among these RGP derivatives, PpeRGP and its chimeric RGPs with B-chains from AamRGP or AjaRGP could induce oocyte maturation and ovulation in P. pectinifera ovaries. In contrast, other RGP derivatives were failed to induce spawning in P. pectinifera ovaries. Circular dichroism spectra of PpeRGP were similar to those of chimeric RGPs with the B-chains from AamRGP or AjaRGP. Furthermore, the predicted three-dimensional structure models of the B-chains from RGP derivatives have almost the same conformation. These findings suggest that the B-chain of PpeRGP is involved in binding to its receptor. Thus, it is likely that the A-chain of AamRGP or AjaRGP disturbs the binding of the PpeRGP B-chain to its receptor.

Starfish Apaf-1 activates effector caspase-3/9 upon apoptosis of aged eggs.

Caspase-3-related DEVDase activity is initiated upon apoptosis in unfertilized starfish eggs. In this study, we cloned a starfish procaspase-3 corresponding to mammalian effector caspase containing a CARD that is similar to the amino terminal CARD of mammalian capsase-9, and we named it procaspase-3/9. Recombinant procaspase-3/9 expressed at 15 °C was cleaved to form active caspase-3/9 which has DEVDase activity. Microinjection of the active caspase-3/9 into starfish oocytes/eggs induced apoptosis. An antibody against the recombinant protein recognized endogenous procaspase-3/9 in starfish oocytes, which was cleaved upon apoptosis in aged unfertilized eggs. These results indicate that caspase-3/9 is an effector caspase in starfish. To verify the mechanism of caspase-3/9 activation, we cloned starfish Apaf-1 containing a CARD, a NOD, and 11 WD40 repeat regions, and we named it sfApaf-1. Recombinant sfApaf-1 CARD interacts with recombinant caspase-3/9 CARD and with endogenous procaspase-3/9 in cell-free preparations made from starfish oocytes, causing the formation of active caspase-3/9. When the cell-free preparation without mitochondria was incubated with inactive recombinant procaspase-3/9 expressed at 37 °C, DEVDase activity increased and apoptosome-like complexes were formed in the high molecular weight fractions containing both sfApaf-1 and cleaved caspase-3/9. These results suggest that sfApaf-1 activation is not dependent on cytochrome c.

Enzyme-linked immunosorbent assay of relaxin-like gonad-stimulating peptide in the starfish Patiria (Asterina) pectinifera.

A relaxin-like gonad-stimulating peptide (RGP) from starfish Patiria (Asterina) pectinifera is the first identified invertebrate gonadotropin for final gamete maturation. Recently, we succeeded in obtaining specific antibodies against P. pectinifera RGP (PpeRGP). In this study, the antibodies were used for the development of a specific and sensitive enzyme-linked immunosorbent assay (ELISA) for the measurement of PpeRGP. A biotin-conjugated peptide that binds to peroxidase-conjugated streptavidin is specifically detectable using 3,3',5,5'-tetramethylbenzidine (TMB)/hydrogen peroxide as a substrate; therefore, biotin-conjugated RGP (biotin-PpeRGP) was synthesized chemically. Similarly to PpeRGP, synthetic biotin-PpeRGP bound to the antibody against PpeRGP. In binding experiments with biotin-PpeRGP using wells coated with the antibody, a displacement curve was obtained using serial concentrations of PpeRGP. The ELISA system showed that PpeRGP could be measured in the range 0.01-10pmol per 50µl assay buffer. On the contrary, the B-chains of PpeRGP, Asterias amurensis RGP, Aphelasterias japonica RGP, and human relaxin showed minimal cross-reactivity in the ELISA, except that the A-chain of PpeRGP affected it slightly. These results strongly suggest that this ELISA system is highly specific and sensitive with respect to PpeRGP.

Fermented Asterina pectinifera with Cordyceps militaris Mycelia Induced Apoptosis in B16F10 Melanoma Cells.

This prime objective of this study was to explore the anti-cancer activity of fermented Asterina pectinifera with Cordyceps militaris mycelia (FACM) in B16F10 murine melanoma cells. The effect of FACM on cell viability was assessed using MTT assay. Furthermore, the effect of FACM was compared with unfermented A. pectinifera on cell viability. The results demonstrated that the fermented FACM extract has a higher inhibitory activity on the proliferation of B16F10 murine melanoma cells than unfermented A. pectinifera. In addition, FACM also promoted the expression of pro-apoptotic protein Bax leading to stimulate apoptosis in B16F10 cells. Therefore the present study demonstrates that the FACM might be a potential effective anti-cancer agent, as a result of its stronger anti-proliferative effect and apoptosis inducing effect than A. pectinifera or C. militaris on melanoma cells.

Chemical and structural characterization of α-N-acetylgalactosaminidase I and II from starfish, asterina amurensis.

BACKGROUND: The marine invertebrate starfish was found to contain a novel α-N-acetylgalactosaminidase, α-GalNAcase II, which catalyzes removal of terminal α-N-acetylgalactosamine (α-GalNAc), in addition to a typical α-N-acetylgalactosaminidase, α-GalNAcase I, which catalyzes removal of terminal α-N-acetylgalactosamine (α-GalNAc) and, to a lesser extent, galactose. The interrelationship between α-GalNAcase I and α-GalNAcase II and the molecular basis of their differences in substrate specificity remain unknown. RESULTS: Chemical and structural comparisons between α-GalNAcase I and II using immunostaining, N-terminal amino acid sequencing and peptide analysis showed high homology to each other and also to other glycoside hydrolase family (GHF) 27 members. The amino acid sequence of peptides showed conserved residues at the active site as seen in typical α-GalNAcase. Some substitutions of conserved amino acid residues were found in α-GalNAcase II that were located near catalytic site. Among them G171 and A173, in place of C171 and W173, respectively in α-GalNAcase were identified to be responsible for lacking intrinsic α-galactosidase activity of α-GalNAcase II. Chemical modifications supported the presence of serine, aspartate and tryptophan as active site residues. Two tryptophan residues (W16 and W173) were involved in α-galactosidase activity, and one (W16) of them was involved in α-GalNAcase activity. CONCLUSIONS: The results suggested that α-GalNAcase I and II are closely related with respect to primary and higher order structure and that their structural differences are responsible for difference in substrate specificities.

Inhibitory mechanism of l-glutamic acid on spawning of the starfish Patiria (Asterina) pectinifera.

l-Glutamic acid was previously identified as an inhibitor of spawning in the starfish Patiria (Asterina) pectinifera; this study examined how l-glutamic acid works. Oocyte release from ovaries of P. pectinifera occurred after germinal vesicle breakdown (GVBD) and follicular envelope breakdown (FEBD) when gonads were incubated ex vivo with either relaxin-like gonad-stimulating peptide (RGP) or 1-methyladenine (1-MeAde). l-Glutamic acid blocked this spawning phenotype, causing the mature oocytes to remain within the ovaries. Neither RGP-induced 1-MeAde production in ovarian follicle cells nor 1-MeAde-induced GVBD and FEBD was affected by l-glutamic acid. l-Glutamic acid may act through metabotropic receptors in the ovaries to inhibit spawning, as l-(+)-2-amino-4-phosphonobutyric acid, an agonist for metabotropic glutamate receptors, also inhibited spawning induced by 1-MeAde. Application of acetylcholine (ACH) to ovaries under inhibitory conditions with l-glutamic acid, however, brought about spawning, possibly by inducing contraction of the ovarian wall to discharge mature oocytes from the ovaries concurrently with GVBD and FEBD. Thus, l-glutamic acid may inhibit ACH secretion from gonadal nerve cells in the ovary. Mol. Reprod. Dev. 84: 246-256, 2017. © 2017 Wiley Periodicals, Inc.

Radioimmunoassay of relaxin-like gonad-stimulating peptide in the starfish Patiria (=Asterina) pectinifera.

A relaxin-like gonad-stimulating peptide (RGP) from starfish Patiria (=Asterina) pectinifera is the first identified invertebrate gonadotropin for final gamete maturation. An antiserum against P. pectinifera RGP (PpeRGP) was produced by immunizing rabbits with a PpeRGP sulfanyl-polyethylene glycol derivative conjugated with keyhole limpet hemocyanin (KLH) as the antigen. The antiserum was used for the development of a specific and sensitive radioimmunoassay (RIA) for the measurement of RGP. In binding experiments using radioiodinated PpeRGP and antiserum against PpeRGP, a displacement curve was obtained using radioinert PpeRGP. The sensitivity of the RIA, defined as the amount of PpeRGP that significantly decreased the counts by 2 SD from the 100% bound point, averaged 0.040±0.002pmol PpeRGP per 100µl assay buffer (0.40±0.02nM) in 10 assays. Intra-assay and inter-assay coefficients of variation were 6.1% and 2.7%, respectively. Serial dilution of whole homogenates from the radial nerve cords and circumoral nerve-rings of P. pectinifera produced displacement curves parallel to the PpeRGP standard. Thus, the amounts of PpeRGP were determined as 1.54±0.09pmol/mg wet weight of radial nerves and 0.87±0.04pmol/mg wet weight of nerve-rings, respectively. On contrary, pyloric stomach, pyloric caeca, tube-feet, ovaries, testes, and ovarian follicle cells did not react in the RIA system. Furthermore, the A- and B-chains of PpeRGP, Asterias amurensis RGP, bovine insulin, and human relaxin did not show cross-reactivity in the RIA. These results strongly suggest that the RIA system is a highly specific and sensitive with respect to PpeRGP.

Impact of natural and calcined starfish (Asterina pectinifera) on the stabilization of Pb, Zn and As in contaminated agricultural soil.

Metal stabilization using soil amendments is an extensively applied, economically viable and environmentally friendly remediation technique. The stabilization of Pb, Zn and As in contaminated soils was evaluated using natural starfish (NSF) and calcined starfish (CSF) wastes at different application rates (0, 2.5, 5.0 and 10.0 wt%). An incubation study was conducted over 14 months, and the efficiency of stabilization for Pb, Zn and As in soil was evaluated by the toxicity characteristic leaching procedure (TCLP) test. The TCLP-extractable Pb was reduced by 76.3-100 and 91.2-100 % in soil treated with NSF and CSF, respectively. The TCLP-extractable Zn was also reduced by 89.8-100 and 93.2-100 % in soil treated with NSF and CSF, respectively. These reductions could be associated with the increased metal adsorption and the formation of insoluble metal precipitates due to increased soil pH following application of the amendments. However, the TCLP-extractable As was increased in the soil treated with NSF, possibly due to the competitive adsorption of phosphorous. In contrast, the TCLP-extractable As in the 10 % CSF treatment was not detectable because insoluble Ca-As compounds might be formed at high pH values. Thermodynamic modeling by visual MINTEQ predicted the formation of ettringite (Ca6Al2(SO4)3(OH)12·26H2O) and portlandite (Ca(OH)2) in the 10 % CSF-treated soil, while SEM-EDS analysis confirmed the needle-like structure of ettringite in which Pb was incorporated and stabilized in the 10 % CSF treatment.

Development of Ca2+-release mechanisms during oocyte maturation of the starfish Asterina pectinifera.

An important step for successful fertilization and further development is the increase in intracellular Ca2+ in the activated oocyte. It has been known that starfish oocytes become increasingly sensitive to inositol 1,4,5-trisphosphate (IP3) during meiotic maturation to exhibit highly efficient IP3-induced Ca2+ release (IICR) by the time of germinal vesicle breakdown (GVBD). However, we noted that the peak level of intracellular Ca2+ increase after insemination is already high in the maturing oocytes before GVBD. Using maturing oocytes before GVBD, we investigated Ca2+ release mechanisms other than IICR. We report here that Ca2+-release mechanisms dependent on nicotinic acid adenine dinucleotide phosphate (NAADP) and nicotinamide adenine dinucleotide (NADP), the precursor of NAADP, became functional prior to the development of IICR mechanisms. As with IP3, but unlike NAADP, the Ca2+ stores responsive to NADP are sensitized during the meiotic maturation induced by 1-methyladenine (1-MA). This suggests that the process may represent a physiological response to the maturation hormone. NADP-dependent Ca2+ release in immature oocytes, however, did not induce oocyte maturation by itself, but was enhanced by the conditions mimicking the increases of intracellular Ca2+ and pH that take place in the maturing oocytes of starfish.

Nucleotide sequence and expression of relaxin-like gonad-stimulating peptide gene in starfish Asterina pectinifera.

Starfish gonad-stimulating substance (GSS) is the only known invertebrate peptide hormone responsible for final gamete maturation, rendering it functionally analogous to gonadotropins in vertebrates. Because GSS belongs to the relaxin-like peptide family, we propose renaming for starfish gonadotropic hormone as relaxin-like gonad-stimulating peptide (RGP). This study examined the primary structure and expression regulation of the RGP gene in starfish Asterina pectinifera. RGP consisted of 3896 base pairs (bp) divided over two exons, exon 1 of 208 bp and exon 2 of 2277 bp, and one intron of 1411 bp. Promoter sequences, CAAT and TATA boxes, were present in the 5'-upstream region of the coding DNA sequence of RGP. The transcript was 2485 bases (b) in length. The AAUAAA polyadenylation signal was found in 3'-untranslated region over 2kb away from the stop codon. This showed that only 14% of the RGP mRNA was translated into the peptide, because a size of the open-reading frame was 351 b. Furthermore, an analysis by using real-time quantitative PCR with specific primers for RGP showed that mRNA of RGP was expressed at high levels in the radial nerves. Expression was also observed in the cardiac stomachs, although the level was low, and trace levels were detected in the gonads, pyloric caeca and tube feet. This result suggests that the RGP gene is transcribed mainly in the radial nerves of A. pectinifera.

Reinvestigation of epithelial lining of the genital coelomic sinus in asteroids. An ultrastructural study.

Ultrastructural study of gonadal muscles in sea star, Asterina pectinifera, showed that myoepithelial cells were located only in the epithelial lining of the genital coelomic sinus. No myoepithelial cells were found in the visceral peritoneal epithelium or within connective tissue layer of the outer sac. Morphology of the myoepithelial cells in gonads of A. pectinifera varies during the reproductive cycle. During the gametogenic phase of the reproductive cycle, the myoepithelial cells get an elongated, spindle-like shape having a length of 20­30 m. In prespawning gonads, many of the myoepithelial cells form cytoplasmic extensions of 3­5 m in length, filled with myofilaments and penetrating into the underlying connective tissue of the outer sac or haemal sinus. Besides, myoepithelial cells, simultaneously anchored in the inner and outer sacs, were also observed. These changes result in development of more elaborated musculature and increase in contractility of the gonadal wall in prespawning gonads as compared to that during other stages of the reproductive cycle.

A method for microinjection of Patiria miniata zygotes.

Echinoderms have long been a favorite model system for studies of reproduction and development, and more recently for the study of gene regulation and evolution of developmental processes. The sea star, Patiria miniata, is gaining prevalence as a model system for these types of studies which were previously performed almost exclusively in the sea urchins, Strongylocentrotus purpuratus and Lytechinus variegatus. An advantage of these model systems is the ease of producing modified embryos in which a particular gene is up or downregulated, labeling a group of cells, or introducing a reporter gene. A single microinjection method is capable of creating a wide variety of such modified embryos. Here, we present a method for obtaining gametes from P. miniata, producing zygotes, and introducing perturbing reagents via microinjection. Healthy morphant embryos are subsequently isolated for quantitative and qualitative studies of gene function. The availability of genome and transcriptome data for this organism has increased the types of studies that are performed and the ease of executing them.

Starase: A bi-functional fibrinolytic protease from hepatic caeca of Asterina pectinifera displays antithrombotic potential.

A bi-functional fibrinolytic serine protease, Starase exhibiting thrombolytic potency was purified from hepatic caeca of Asterina pectinifera. Starase showed a single band of approximately 48 kDa by SDS-PAGE and fibrin zymography. The N-terminal sequence of Starase was AIPTEFDARTKKHNN, which does not match with any known fibrinolytic enzyme. Starase had optimum amidolytic activity at 50 °C and pH 8.0 and the activity was inhibited by PMSF and APMSF. Starase showed the highest specificity toward the substrate H-D-Val-Leu-Lys-pNA for plasmin followed by pyroGlu-Gly-Arg-pNA for urokinase. The apparent Km and Vmax values of Starase toward a chromogenic substrate for plasmin H-D-Val-Leu-Lys-pNA were determined as 1.37 mM and 6.8 mM/min/mg respectively. The fibrinolytic activity of Starase by fibrin plate assay displayed that it could not only directly degrade fibrin clot but also activate plasminogen. Starase showed a strong fibrinogenolytic activity, cleaving all three major chains of fibrinogen rapidly. In addition, Starase with more than 1 µg could cleave extracellular matrix component type VII collagen, and plasma proteins such as bovine albumin and bovine gamma globulin. It could also inhibit factor Xa and thrombin activity. Starase at a dose of 0.8 mg/kg was devoid of hemorrhagic activity and it demonstrated antithrombotic effect in three animal models; FeCl2-induced carotid arterial thrombus model, carrageenan-induced tail thrombosis model and collagen and epinephrine induced pulmonary thromboembolism mice model. These results suggest that Starase has the potential to be a potent thrombolytic agent due to its bi-functional properties (containing both direct-acting and plasminogen-activating activities) and lack of hemorrhagic activity. Although Starase might interfere with the normal composition of the plasma proteins, it may be used not only for the treatment and prevention of thrombosis, but also in a number of biomedical applications.

Involvement of Gαs-proteins in the action of relaxin-like gonad-stimulating substance on starfish ovarian follicle cells.

Gonad-stimulating substance (GSS) in starfish is the only known invertebrate peptide hormone responsible for final gamete maturation, rendering it functionally analogous to gonadotropins in vertebrates. In breeding season (stage V), GSS stimulates oocyte maturation to induce 1-methyladenine (1-MeAde) by ovarian follicle cells. The hormonal action of GSS is mediated through the activation of its receptor, G-proteins and adenylyl cyclase. It has been reported that GSS fails to induce 1-MeAde and cyclic AMP (cAMP) production in follicle cells of ovaries during oogenesis (stage IV). This study examined the regulatory mechanism how ovarian follicle cells acquire the potential to respond to GSS by producing 1-MeAde and cAMP. Because the failure of GSS action was due to G-proteins of follicle cells, the molecular structures of Gαs, Gαi, Gαq and Gß were identified in follicle cells of starfish Asterina pectinifera. The cDNA sequences of Gαs, Gαi, Gαq and Gß consisted of ORFs encoding 379, 354, 353 and 353 amino acids. The expression levels of Gαs were extremely low in follicle cells at stage IV, whereas the mRNA levels increased markedly in stage V. On contrary, the mRNA levels of Gαi were almost constant regardless of stage IV and V. These findings strongly suggest that de novo synthesis of Gαs-proteins is contributed to the action of GSS on follicle cells to produce 1-MeAde and cAMP.

Cyclin B-Cdk1 inhibits protein phosphatase PP2A-B55 via a Greatwall kinase-independent mechanism.

Entry into M phase is governed by cyclin B-Cdk1, which undergoes both an initial activation and subsequent autoregulatory activation. A key part of the autoregulatory activation is the cyclin B-Cdk1-dependent inhibition of the protein phosphatase 2A (PP2A)-B55, which antagonizes cyclin B-Cdk1. Greatwall kinase (Gwl) is believed to be essential for the autoregulatory activation because Gwl is activated downstream of cyclin B-Cdk1 to phosphorylate and activate α-endosulfine (Ensa)/Arpp19, an inhibitor of PP2A-B55. However, cyclin B-Cdk1 becomes fully activated in some conditions lacking Gwl, yet how this is accomplished remains unclear. We show here that cyclin B-Cdk1 can directly phosphorylate Arpp19 on a different conserved site, resulting in inhibition of PP2A-B55. Importantly, this novel bypass is sufficient for cyclin B-Cdk1 autoregulatory activation. Gwl-dependent phosphorylation of Arpp19 is nonetheless necessary for downstream mitotic progression because chromosomes fail to segregate properly in the absence of Gwl. Such a biphasic regulation of Arpp19 results in different levels of PP2A-B55 inhibition and hence might govern its different cellular roles.