STE20 phosphorylation of AMPK-related kinases revealed by biochemical purifications combined with genetics.

We have recently purified mammalian sterile 20 (STE20)-like kinase 3 (MST3) as a kinase for the multifunctional kinases, AMP-activated protein kinase-related kinases (ARKs). However, unresolved questions from this study, such as remaining phosphorylation activities following deletion of the Mst3 gene from human embryonic kidney cells and mice, led us to conclude that there were additional kinases for ARKs. Further purification recovered Ca2+/calmodulin-dependent protein kinase kinases 1 and 2 (CaMKK1 and 2), and a third round of purification revealed mitogen-activated protein kinase kinase kinase kinase 5 (MAP4K5) as potential kinases of ARKs. We then demonstrated that MST3 and MAP4K5, both belonging to the STE20-like kinase family, could phosphorylate all 14 ARKs both in vivo and in vitro. Further examination of all 28 STE20 kinases detected variable phosphorylation activity on AMP-activated protein kinase (AMPK) and the salt-inducible kinase 3 (SIK3). Taken together, our results have revealed novel relationships between STE20 kinases and ARKs, with potential physiological and pathological implications.

Biochemical purification uncovers mammalian sterile 3 (MST3) as a new protein kinase for multifunctional protein kinases AMPK and SIK3.

The AMP-activated protein kinase (AMPK) and AMPK-related kinase salt-inducible kinase 3 (SIK3) regulate many important biological processes ranging from metabolism to sleep. Liver kinase B1 is known to phosphorylate and activate both AMPK and SIK3, but the existence of other upstream kinases was unclear. In this study, we detected liver kinase B1-independent AMPK-related kinase phosphorylation activities in human embryonic kidney cells as well as in mouse brains. Biochemical purification of this phosphorylation activity uncovered mammalian sterile 20-like kinase 3 (MST3). We demonstrate that MST3 from human embryonic kidney cells could phosphorylate AMPK and SIK3 in vivo. In addition, recombinant MST3 expressed in and purified from Escherichia coli could directly phosphorylate AMPK and SIK3 in vitro. Moreover, four other members of the MST kinase family could also phosphorylate AMPK or SIK3. Our results have revealed new kinases able to phosphorylate and activate AMPK and SIK3.

TRIM14 Is a Key Regulator of the Type I IFN Response during Mycobacterium tuberculosis Infection.

Tripartite motif-containing proteins (TRIMs) play a variety of recently described roles in innate immunity. Although many TRIMs regulate type I IFN expression following cytosolic nucleic acid sensing of viruses, their contribution to innate immune signaling and gene expression during bacterial infection remains largely unknown. Because Mycobacterium tuberculosis is an activator of cGAS-dependent cytosolic DNA sensing, we set out to investigate a role for TRIM proteins in regulating macrophage responses to M. tuberculosis In this study, we demonstrate that TRIM14, a noncanonical TRIM that lacks an E3 ubiquitin ligase RING domain, is a critical negative regulator of the type I IFN response in Mus musculus macrophages. We show that TRIM14 interacts with both cGAS and TBK1 and that macrophages lacking TRIM14 dramatically hyperinduce IFN stimulated gene (ISG) expression following M. tuberculosis infection, cytosolic nucleic acid transfection, and IFN-ß treatment. Consistent with a defect in resolution of the type I IFN response, Trim14 knockout macrophages have more phospho-Ser754 STAT3 relative to phospho-Ser727 and fail to upregulate the STAT3 target Socs3, which is required to turn off IFNAR signaling. These data support a model whereby TRIM14 acts as a scaffold between TBK1 and STAT3 to promote phosphorylation of STAT3 at Ser727 and resolve ISG expression. Remarkably, Trim14 knockout macrophages hyperinduce expression of antimicrobial genes like Nos2 and are significantly better than control cells at limiting M. tuberculosis replication. Collectively, these data reveal an unappreciated role for TRIM14 in resolving type I IFN responses and controlling M. tuberculosis infection.

Expression, purification and crystallization of CLK1 kinase - A potential target for antiviral therapy.

Cdc-like kinase 1 (CLK1) is a dual-specificity kinase capable of autophosphorylation on tyrosine residues and Ser/Thr phosphorylation of its substrates. CLK1 belongs to the CLK kinase family that regulates alternative splicing through phosphorylation of serine-arginine rich (SR) proteins. Recent studies have demonstrated that CLK1 has an important role in the replication of influenza A and chikungunya viruses. Furthermore, CLK1 was found to be relevant for the replication of HIV-1 and the West Nile virus, making CLK1 an interesting cellular candidate for the development of a host-directed antiviral therapy that might be efficient for treatment of newly emerging viruses. We describe here our attempts and detailed procedures to obtain the recombinant kinase domain of CLK1 in suitable amounts for crystallization in complex with specific inhibitors. The key solution for the reproducibility of crystals resides in devising and refining expression and purification protocols leading to homogeneous protein. Co-expression of CLK1 with λ-phosphatase and careful purification has yielded crystals of CLK1 complexed with the KH-CB19 inhibitor that diffracted to 1.65 Å. These results paved the path to the screening of more structures of CLK1 complexed compounds, leading to further optimization of their inhibitory activity. Moreover, since kinases are desired targets in numerous pathologies, the approach we report here, the co-expression of kinases with λ-phosphatase, previously used in other kinases, can be adopted as a general protocol in numerous kinase targets for obtaining reproducible and homogenic non-phosphorylated (inactive) forms suitable for biochemical and structural studies thus facilitating the development of novel inhibitors.

Expression, purification, and in vitro characterization of kinase domain of NtGCN2 from tobacco.

General control nonderepressible 2 (GCN2) can phosphorylate the α subunit of eukaryotic initiation factor eIF2 (eukaryotic translation initiation factor 2) to down-regulateprotein synthesis in response to various biotic and abiotic stresses. However, the kinase activity of plant GCN2 has not been well-characterized in vitro. In this study, the kinase domain of Nicotiana tabacum GCN2 (NtGCN2) was inserted into the pET15b vector for prokaryotic expressionin Escherichia coli BL21-CodonPlus-(DE3)-RIPL after induction by 0.5 mmol L-1 IPTG for 13 h at 16 °C. The soluble protein was collected and purified by Ni2+-NTA agarose column, anion exchange, and molecular sieve, and the purified proteinwas used for kinase assays and the preparation of a polyclonal antibody. Enzyme-linked immunosorbent assay results showed that the titer of the antiserum was 1:520K. Western blot analysis showed that the prepared antibody reacted with GCN2 in tobacco. Additionally, the kinase activity of NtGCN2 was characterized by using recombinant NteIF2α protein as a substrate in vitro. The results showed that NtGCN2 phosphorylated NteIF2α in vitro, with the level of phosphorylation positively correlated with the NtGCN2 concentration and reaction time. Our study has prepared a specific antibody, and proves NtGCN2 can phosphorylate NteIF2α in vitro, which lays a foundation for further study of the function and interaction network of NtGCN2.

Expression, purification and characterization of the plant Snf1-related protein kinase 1 from Escherichia coli.

The SnRK1 (SNF1 related protein kinase 1), a plant homologue of SNF1 (Sucrose non-fermenting 1)/AMPK (AMP-activated protein kinase), is an important metabolic sensor involving in catabolic and anabolic processes. SnRK1 is essential for plant metabolism regulation and response to environmental stresses. The plant SnRK1 consists of one catalytic (α1/α2 subunit) and two regulatory subunits (ß1/ß2/ß3 and γ/ßγ subunits), and functions as a heterotrimeric complex. Here we took advantage of a tricistronic expression vector and successfully purified the holoenzyme containing three subunits of SnRK1 from the E.coli. Using advantages of the E.coli system, we would be able to purify SnRK1 complex with high yield and high purity. Moreover, the complex is stable with high homogeneity. Using the purified complex, we confirmed that the ßγ rather than the γ subunit of the plant SnRK1 acts as the canonical regulatory subunit. Besides, some basic characters of the SnRK1 holoenzyme was studied. Together, our results provide a convenient way for purify the plant SnRK1 complexes, and this would be helpful for follow-up study on SnRK1's structure and mechanism.

Identification and sequence analysis of putative Sulla species nod factor receptor.

In legumes, LysM domains of receptors-like kinases (RLKs) mediate rhizobial NFs perception; which are required for infection and establishment of symbiosis without triggering the host immune response. In this study, we identify the LysM extracellular domain sequences of putative Sulla species Nod factor receptors (S. pallida, S. capitata and S. coronaria). The Blast search displayed high identity percentages with genes encoding LjNFR5-like of several legumes. Phylogenetic trees were built using the partial nod factor receptor and predicted amino acid sequences, which grouped Sulla in a separate clade. The multiple alignments of the LysM2 domains revealed that amino acids found to be important in other legume species are not conserved in Sulla species. Further examination of the predicted proteins sequences (LysM2 domain) showed that the three species were different in the two crucial sites for Nod factor perception.

In Silico Screen and Structural Analysis Identifies Bacterial Kinase Inhibitors which Act with ß-Lactams To Inhibit Mycobacterial Growth.

New tools and concepts are needed to combat antimicrobial resistance. Actinomycetes and firmicutes share several eukaryotic-like Ser/Thr kinases (eSTK) that offer antibiotic development opportunities, including PknB, an essential mycobacterial eSTK. Despite successful development of potent biochemical PknB inhibitors by many groups, clinically useful microbiologic activity has been elusive. Additionally, PknB kinetics are not fully described, nor are structures with specific inhibitors available to inform inhibitor design. We used computational modeling with available structural information to identify human kinase inhibitors predicted to bind PknB, and we selected hits based on drug-like characteristics intended to increase the likelihood of cell entry. The computational model suggested a family of inhibitors, the imidazopyridine aminofurazans (IPAs), bind PknB with high affinity. We performed an in-depth characterization of PknB and found that these inhibitors biochemically inhibit PknB, with potency roughly following the predicted models. A novel X-ray structure confirmed that the inhibitors bound as predicted and made favorable protein contacts with the target. These inhibitors also have antimicrobial activity toward mycobacteria and nocardia. We demonstrated that the inhibitors are uniquely potentiated by ß-lactams but not antibiotics traditionally used to treat mycobacteria, consistent with PknB's role in sensing cell wall stress. This is the first demonstration in the phylum actinobacteria that some ß-lactam antibiotics could be more effective if paired with a PknB inhibitor. Collectively, our data show that in silico modeling can be used as a tool to discover promising drug leads, and the inhibitors we discovered can act with clinically relevant antibiotics to restore their efficacy against bacteria with limited treatment options.

Synthesis and in vitro evaluation of diverse heterocyclic diphenolic compounds as inhibitors of DYRK1A.

Dual-specificity tyrosine phosphorylation-related kinase 1A (DYRK1A) is a dual-specificity protein kinase that catalyses phosphorylation and autophosphorylation. Higher DYRK1A expression correlates with cancer, in particular glioblastoma present within the brain. We report here the synthesis and biological evaluation of new heterocyclic diphenolic derivatives designed as novel DYRK1A inhibitors. The generation of these heterocycles such as benzimidazole, imidazole, naphthyridine, pyrazole-pyridines, bipyridine, and triazolopyrazines was made based on the structural modification of the lead DANDY and tested for their ability to inhibit DYRK1A. None of these derivatives showed significant DYRK1A inhibition but provide valuable knowledge around the importance of the 7-azaindole moiety. These data will be of use for developing further structure-activity relationship studies to improve the selective inhibition of DYRK1A.

Differential Phosphoproteomic Analysis of Recombinant Chinese Hamster Ovary Cells Following Temperature Shift.

Phosphorylation is one of the most important post-translational modifications, playing a crucial role in regulating many cellular processes, including transcription, cytoskeletal rearrangement, cell proliferation, differentiation, apoptosis, and signal transduction. However, to date, little work has been carried out on the phosphoproteome in CHO cells. In this study we have carried out a large scale differential phosphoproteomic analysis of recombinant CHO cells following a reduction of culture temperature (temperature shift). The reduction of culture temperature during the exponential phase of growth is commonly employed by the biopharmaceutical industry to increase product yield; however, the molecular mechanisms of temperature shift in CHO cells remain poorly understood. We have identified 700 differentially expressed phosphopeptides using quantitative label-free LC-MS/MS phosphoproteomic analysis in conjunction with IMAC and TiO2 phosphopeptide enrichment strategies, following a reduction in temperature from 37 to 31 °C. Functional assessment of the phosphoproteomic data using gene ontology analysis showed a significant enrichment of biological processes related to growth (e.g., cell cycle, cell division), ribosomal biogenesis, and cytoskeleton organization, and molecular functions related to RNA binding, transcription factor activity, and protein serine/threonine kinase activity. Differential phosphorylation of two proteins, ATF2 and NDRG1, was confirmed by Western blotting. This data suggests the importance of including the post-translational layer of regulation, such as phosphorylation, in CHO "omics" studies. This study also has the potential to identify phosphoprotein targets that could be modified using cell line engineering approaches to improve the efficiency of recombinant protein production.

Characterization and enzymatic properties of protein kinase ACR4 from Arabidopsis thaliana.

Serine/threonine-protein kinase-like protein ARABIDOPSIS CRINKLY4 (ACR4), a transmembrane protein of Arabidopsis thaliana, plays important roles in cell division and differentiation. Although accumulating studies shed light on the function of ACR4, the structure and catalytic mechanism of ACR4 remain to be elucidated. Here, we report the purification and enzymatic properties of the intracellular kinase domain (residues 464-799) of ACR4 (ACR4IKD). Through Ni-affinity chromatography and gel filter chromatography methods, we successfully obtain high-purity ACR4IKD protein from Escherichia coli. Dynamic light scattering and gel-filtration methods reveal that ACR4IKD distributes with high homogeneity and exists as a monomer in solution. In addition, the ACR4IKD protein has typical kinase activity with myelin basic protein (MBP) as the substrate. Our study may lay the foundation for structure determination of ACR4IKD and further functional research, for example, screening significant substrates of ACR4 in Arabidopsis thaliana.

PknB remains an essential and a conserved target for drug development in susceptible and MDR strains of M. Tuberculosis.

BACKGROUND: The Mycobacterium tuberculosis (M.tb) protein kinase B (PknB) which is now proved to be essential for the growth and survival of M.tb, is a transmembrane protein with a potential to be a good drug target. However it is not known if this target remains conserved in otherwise resistant isolates from clinical origin. The present study describes the conservation analysis of sequences covering the inhibitor binding domain of PknB to assess if it remains conserved in susceptible and resistant clinical strains of mycobacteria picked from three different geographical areas of India. METHODS: A total of 116 isolates from North, South and West India were used in the study with a variable profile of their susceptibilities towards streptomycin, isoniazid, rifampicin, ethambutol and ofloxacin. Isolates were also spoligotyped in order to find if the conservation pattern of pknB gene remain consistent or differ with different spoligotypes. The impact of variation as found in the study was analyzed using Molecular dynamics simulations. RESULTS: The sequencing results with 115/116 isolates revealed the conserved nature of pknB sequences irrespective of their susceptibility status and spoligotypes. The only variation found was in one strains wherein pnkB sequence had G to A mutation at 664 position translating into a change of amino acid, Valine to Isoleucine. After analyzing the impact of this sequence variation using Molecular dynamics simulations, it was observed that the variation is causing no significant change in protein structure or the inhibitor binding. CONCLUSIONS: Hence, the study endorses that PknB is an ideal target for drug development and there is no pre-existing or induced resistance with respect to the sequences involved in inhibitor binding. Also if the mutation that we are reporting for the first time is found again in subsequent work, it should be checked with phenotypic profile before drawing the conclusion that it would affect the activity in any way. Bioinformatics analysis in our study says that it has no significant effect on the binding and hence the activity of the protein.

Development of Bifunctional Inhibitors of Polo-Like Kinase†1 with Low-Nanomolar Activities Against the Polo-Box Domain.

Polo-like kinase 1 (Plk1), a validated cancer target, harbors a protein-protein interaction domain referred to as the polo-box domain (PBD), in addition to its enzymatic domain. Although functional inhibition either of the enzymatic domain or of the PBD has been shown to inhibit Plk1, so far there have been no reports of bifunctional agents with the potential to target both protein domains. Here we report the development of Plk1 inhibitors that incorporate both an ATP-competitive ligand of the enzymatic domain, derived from BI 2536, and a functional inhibitor of the PBD, based either on the small molecule poloxin-2 or on a PBD-binding peptide. Although these bifunctional agents do not seem to bind both protein domains simultaneously, the most potent compound displays low-nanomolar activity against the Plk1 PBD, with excellent selectivity over the PBDs of Plk2 and Plk3. Our data provide insights into challenges and opportunities relating to the optimization of Plk1 PBD ligands as potent Plk1 inhibitors.

Complement activation by ceramide transporter proteins.

C1q is the initiator of the classical complement pathway and, as such, is essential for efficient opsonization and clearance of pathogens, altered self-structures, and apoptotic cells. The ceramide transporter protein (CERT) and its longer splicing isoform CERTL are known to interact with extracellular matrix components, such as type IV collagen, and with the innate immune protein serum amyloid P. In this article, we report a novel function of CERT in the innate immune response. Both CERT isoforms, when immobilized, were found to bind the globular head region of C1q and to initiate the classical complement pathway, leading to activation of C4 and C3, as well as generation of the membrane attack complex C5b-9. In addition, C1q was shown to bind to endogenous CERTL on the surface of apoptotic cells. These results demonstrate the role of CERTs in innate immunity, especially in the clearance of apoptotic cells.

FANCM and FAAP24 function in ATR-mediated checkpoint signaling independently of the Fanconi anemia core complex.

The Fanconi anemia (FA) pathway is implicated in DNA repair and cancer predisposition. Central to this pathway is the FA core complex, which is targeted to chromatin by FANCM and FAAP24 following replication stress. Here we show that FANCM and FAAP24 interact with the checkpoint protein HCLK2 independently of the FA core complex. In addition to defects in FA pathway activation, downregulation of FANCM or FAAP24 also compromises ATR/Chk1-mediated checkpoint signaling, leading to defective Chk1, p53, and FANCE phosphorylation; 53BP1 focus formation; and Cdc25A degradation. As a result, FANCM and FAAP24 deficiency results in increased endogenous DNA damage and a failure to efficiently invoke cell-cycle checkpoint responses. Moreover, we find that the DNA translocase activity of FANCM, which is dispensable for FA pathway activation, is required for its role in ATR/Chk1 signaling. Our data suggest that DNA damage recognition and remodeling activities of FANCM and FAAP24 cooperate with ATR/Chk1 to promote efficient activation of DNA damage checkpoints.

Quantitative phosphoproteome analysis of Bacillus subtilis reveals novel substrates of the kinase PrkC and phosphatase PrpC.

Reversible protein phosphorylation on serine, threonine, and tyrosine (Ser/Thr/Tyr) residues plays a critical role in regulation of vital processes in the cell. Despite of considerable progress in our understanding of the role of this modification in bacterial physiology, the dynamics of protein phosphorylation during bacterial growth has rarely been systematically addressed. In addition, little is known about in vivo substrates of bacterial Ser/Thr/Tyr kinases and phosphatases. An excellent candidate to study these questions is the Gram-positive bacterium Bacillus subtilis, one of the most intensively investigated bacterial model organism with both research and industrial applications. Here we employed gel-free phosphoproteomics combined with SILAC labeling and high resolution mass spectrometry to study the proteome and phosphoproteome dynamics during the batch growth of B. subtilis. We measured the dynamics of 1666 proteins and 64 phosphorylation sites in five distinct phases of growth. Enzymes of the central carbon metabolism and components of the translation machinery appear to be highly phosphorylated in the stationary phase, coinciding with stronger expression of Ser/Thr kinases. We further used the SILAC workflow to identify novel putative substrates of the Ser/Thr kinase PrkC and the phosphatase PrpC during stationary phase. The overall number of putative substrates was low, pointing to a high kinase and phosphatase specificity. One of the phosphorylation sites affected by both, PrkC and PrpC, was the Ser281 on the oxidoreductase YkwC. We showed that PrkC phosphorylates and PrpC dephosphorylates YkwC in vitro and that phosphorylation at Ser281 abolishes the oxidoreductase activity of YkwC in vitro and in vivo. Our results present the most detailed phosphoproteomic analysis of B. subtilis growth to date and provide the first global in vivo screen of PrkC and PrpC substrates.

Isolation and Purification of Recombinant Serine/Threonine Protein Kinases of the Strain Bifidobacterium longum B379M and Investigation of Their Activity.

Previously, we identified six serine/threonine protein kinases (STPK) of Bifidobacterium and named them Pkb1-Pkb6. In the present study, we optimized methods for isolation of the six STPK catalytic domains proteins of B. longum B379M: a method for isolation of Pkb3 and Pkb4 in native conditions, a method for isolation of Pkb5 in denaturing conditions, and a method for isolation of Pkb1, Pkb2, and Pkb6 from inclusion bodies. The dialysis conditions for the renaturation of the proteins were optimized. All of the enzymes were isolated in quantities sufficient for study of the protein activity. The proteins were homogeneous according to SDS-PAGE. The autophosphorylation ability of Pkb1, Pkb3, Pkb4, and Pkb6 was investigated for the first time. Autophosphorylation was detected only for the Pkb3 catalytic domain.

A protein kinase screen of Neurospora crassa mutant strains reveals that the SNF1 protein kinase promotes glycogen synthase phosphorylation.

Glycogen functions as a carbohydrate reserve in a variety of organisms and its metabolism is highly regulated. The activities of glycogen synthase and glycogen phosphorylase, the rate-limiting enzymes of the synthesis and degradation processes, respectively, are regulated by allosteric modulation and reversible phosphorylation. To identify the protein kinases affecting glycogen metabolism in Neurospora crassa, we performed a screen of 84 serine/threonine kinase knockout strains. We identified multiple kinases that have already been described as controlling glycogen metabolism in different organisms, such as NcSNF1, NcPHO85, NcGSK3, NcPKA, PSK2 homologue and NcATG1. In addition, many hypothetical kinases have been implicated in the control of glycogen metabolism. Two kinases, NcIME-2 and NcNIMA, already functionally characterized but with no functions related to glycogen metabolism regulation, were also identified. Among the kinases identified, it is important to mention the role of NcSNF1. We showed in the present study that this kinase was implicated in glycogen synthase phosphorylation, as demonstrated by the higher levels of glycogen accumulated during growth, along with a higher glycogen synthase (GSN) ±glucose 6-phosphate activity ratio and a lesser set of phosphorylated GSN isoforms in strain Ncsnf1KO, when compared with the wild-type strain. The results led us to conclude that, in N. crassa, this kinase promotes phosphorylation of glycogen synthase either directly or indirectly, which is the opposite of what is described for Saccharomyces cerevisiae. The kinases also play a role in gene expression regulation, in that gdn, the gene encoding the debranching enzyme, was down-regulated by the proteins identified in the screen. Some kinases affected growth and development, suggesting a connection linking glycogen metabolism with cell growth and development.

Drug Discovery of Host CLK1 Inhibitors for Influenza Treatment.

The rapid evolution of influenza virus makes antiviral drugs less effective, which is considered to be a major bottleneck in antiviral therapy. The key proteins in the host cells, which are related with the replication cycle of influenza virus, are regarded as potential drug targets due to their distinct advantage of lack of evolution and drug resistance. Cdc2-like kinase 1 (CLK1) in the host cells is responsible for alternative splicing of the M2 gene of influenza virus during influenza infection and replication. In this study, we carried out baculovirus-mediated expression and purification of CLK1 and established a reliable screening assay for CLK1 inhibitors. After a virtual screening of CLK1 inhibitors was performed, the activities of the selected compounds were evaluated. Finally, several compounds with strong inhibitory activity against CLK1 were discovered and their in vitro anti-influenza virus activities were validated using a cytopathic effect (CPE) reduction assay. The assay results showed that clypearin, corilagin, and pinosylvine were the most potential anti-influenza virus compounds as CLK1 inhibitors among the compounds tested. These findings will provide important information for new drug design and development in influenza treatment, and CLK1 may be a potent drug target for anti-influenza drug screening and discovery.

Greatwall kinase is required for meiotic maturation in porcine oocytes.

Meiotic maturation in many species is initiated by the activation of maturation-promoting factor (MPF) with concomitant inactivation of counteracting phosphatases, most notably protein phosphatase 2A (PP2A). Recently, Greatwall (GWL) has been identified as a cell cycle regulator that inhibits PP2A activity. In this study, we demonstrate that GWL is required for meiotic maturation in porcine oocytes. GWL expression increases from germinal vesicle (GV) to metaphase II (MII) stages of porcine oocytes and dramatically decreases with progression of the meiotic cell cycle. GWL is initially localized in the nucleus of GV oocytes and is associated with spindle fibers following GV breakdown. Depletion of GWL inhibited or delayed meiotic maturation secondary to defects in chromosome congression and spindle formation. Conversely, overexpression of GWL overcame meiotic arrest and initiated progression to MII stage. However, these oocytes had severe spindle defects. Furthermore, MII oocytes depleted of GWL progressed to pronuclear formation. Taken together, our data demonstrate that GWL is required not only for meiotic maturation but also for maintenance of MII arrest in porcine oocytes.