N-terminal Kinase Domain Research Articles

ERK5 kinase activity is dispensable for cellular immune response and proliferation

Unlike other members of the MAPK family, ERK5 contains a large C-terminal domain with transcriptional activation capability in addition to an N-terminal canonical kinase domain. Genetic deletion of ERK5 is embryonic lethal, and tissue-restricted deletions have profound effects on erythroid development, cardiac function, and neurogenesis. In addition, depletion of ERK5 is antiinflammatory and antitumorigenic. Small molecule inhibition of ERK5 has been shown to have promising activity in cell and animal models of inflammation and oncology. Here we report the synthesis and biological characterization of potent, selective ERK5 inhibitors. In contrast to both genetic depletion/deletion of ERK5 and inhibition with previously reported compounds, inhibition of the kinase with the most selective of the new inhibitors had no antiinflammatory or antiproliferative activity. The source of efficacy in previously reported ERK5 inhibitors is shown to be off-target activity on bromodomains, conserved protein modules involved in recognition of acetyl-lysine residues during transcriptional processes. It is likely that phenotypes reported from genetic deletion or depletion of ERK5 arise from removal of a noncatalytic function of ERK5. The newly reported inhibitors should be useful in determining which of the many reported phenotypes are due to kinase activity and delineate which can be pharmacologically targeted.

Genetic Screen in Drosophila Larvae Links ird1 Function to Toll Signaling in the Fat Body and Hemocyte Motility.

To understand how Toll signaling controls the activation of a cellular immune response in Drosophila blood cells (hemocytes), we carried out a genetic modifier screen, looking for deletions that suppress or enhance the mobilization of sessile hemocytes by the gain-of-function mutation Toll10b (Tl10b). Here we describe the results from chromosome arm 3R, where five regions strongly suppressed this phenotype. We identified the specific genes immune response deficient 1 (ird1), headcase (hdc) and possibly Rab23 as suppressors, and we studied the role of ird1 in more detail. An ird1 null mutant and a mutant that truncates the N-terminal kinase domain of the encoded Ird1 protein affected the Tl10b phenotype, unlike mutations that affect the C-terminal part of the protein. The ird1 null mutant suppressed mobilization of sessile hemocytes, but enhanced other Tl10b hemocyte phenotypes, like the formation of melanotic nodules and the increased number of circulating hemocytes. ird1 mutants also had blood cell phenotypes on their own. They lacked crystal cells and showed aberrant formation of lamellocytes. ird1 mutant plasmatocytes had a reduced ability to spread on an artificial substrate by forming protrusions, which may explain why they did not go into circulation in response to Toll signaling. The effect of the ird1 mutation depended mainly on ird1 expression in hemocytes, but ird1-dependent effects in other tissues may contribute. Specifically, the Toll receptor was translocated from the cell membrane to intracellular vesicles in the fat body of the ird1 mutant, and Toll signaling was activated in that tissue, partially explaining the Tl10b-like phenotype. As ird1 is otherwise known to control vesicular transport, we conclude that the vesicular transport system may be of particular importance during an immune response.

Conformational Change of Human Checkpoint Kinase 1 (Chk1) Induced by DNA Damage

Phosphorylation of Chk1 by ataxia telangiectasia-mutated and Rad3-related (ATR) is critical for checkpoint activation upon DNA damage. However, how phosphorylation activates Chk1 remains unclear. Many studies suggest a conformational change model of Chk1 activation in which phosphorylation shifts Chk1 from a closed inactive conformation to an open active conformation during the DNA damage response. However, no structural study has been reported to support this Chk1 activation model. Here we used FRET and bimolecular fluorescence complementary techniques to show that Chk1 indeed maintains a closed conformation in the absence of DNA damage through an intramolecular interaction between a region (residues 31-87) at the N-terminal kinase domain and the distal C terminus. A highly conserved Leu-449 at the C terminus is important for this intramolecular interaction. We further showed that abolishing the intramolecular interaction by a Leu-449 to Arg mutation or inducing ATR-dependent Chk1 phosphorylation by DNA damage disrupts the closed conformation, leading to an open and activated conformation of Chk1. These data provide significant insight into the mechanisms of Chk1 activation during the DNA damage response.

STAT6 negatively regulates TBK1-triggered IFNΦ1 activation in zebrafish

Mammalian STAT6 plays a crucial role in lymphocyte differentiation and proliferation in adaptive immunity, and recently, studies have showed that it can be regulated by TBK1 to promote the transcriptions of CCLs in innate immunity. Whether STAT6 is involved in innate immunity in lower vertebrates is unclear. In this study, we reported that zebrafish STAT6 suppressed TBK1 phosphorylation, resulting in the decrease of IFN expression. Firstly, a typical ISRE motif was found from the promoter region of zebrafish stat6, whereas it is absent in human stat6; and the transcription of zebrafish stat6 was upregulated significantly in the early stage after virus infection. These indicated that fish STAT6 might be involved in host defense to virus. Subsequently, overexpression of STAT6 impeded the IFNφ1 promoter activity induced by SVCV or poly I:C; Furthermore, TBK1-, but not MITA-mediated activation of IFNφ1 promoter was impaired by STAT6. Co-IP and Western blot experiments indicated that MITA and IRF3 were significantly phosphorylated by TBK1, and the N-terminal kinase domain of TBK1 was indispensable. Meanwhile, STAT6 could bind the kinase domain of TBK1, which dephosphorylated TBK1 and then resulted in the reduction of IFNφ1. Finally, zebrafish STAT6 distributed in both nucleus and cytosol, suggesting that it may play roles not only as a transcriptional factor, but also in cytoplasmic signaling transduction. Taken together, these data demonstrated that fish STAT6 can attenuate the kinase activity of TBK1, leading to the suppression of IFNφ1 expression, indicating that a novel function of STAT6 exists in lower vertebrate.

Discovery of Non-ATP-Competitive Inhibitors of Polo-like Kinase 1.

Polo-like kinase 1 (Plk1) is an evolutionarily conserved serine/threonine kinase, and its N-terminal kinase domain (KD) controls cell signaling through phosphorylation. Inhibitors of Plk1 are potential anticancer drugs. Most known Plk1 KD inhibitors are ATP-competitive compounds, which may suffer from low selectivity. In this study we discovered novel non-ATP-competitive Plk1 KD inhibitors by virtual screening and experimental studies. Potential binding sites in Plk1 KD were identified by using the protein binding site detection program Cavity. The identified site was subjected to molecular-docking-based virtual screening. The activities of top-ranking compounds were evaluated by in vitro enzyme assay with full-length Plk1 and direct binding assay with Plk1 KD. Several compounds showed inhibitory activity, and the most potent was found to be 3-((2-oxo-2-(thiophen-2-yl)ethyl)thio)-6-(pyridin-3-ylmethyl)-1,2,4-triazin-5(4H)-one (compound 4) with an IC50 value of 13.1 ± 1.7 μm. Our work provides new insight into the design of kinase inhibitors that target non-ATP binding sites.

Structure-based design and confirmation of peptide ligands for neuronal polo-like kinase to promote neuroregeneration

Neuronal polo-like kinase (nPLK) is an essential regular of cell cycle and differentiation in nervous system, and targeting nPLK has been established as a promising therapeutic strategy to treat neurological disorders and to promote neuroregeneration. The protein contains an N-terminal kinase domain (KD) and a C-terminal Polo-box domain (PBD) that are mutually inhibited by each other. Here, the intramolecular KD–PBD complex in nPLK was investigated at structural level via bioinformatics analysis, molecular dynamics (MD) simulation and binding affinity scoring. From the complex interface two regions representing separately two continuous peptide fragments in PBD domain were identified as the hot spots of KD–PBD interaction. Structural and energetic analysis suggested that one (PBD peptide 1) of the two peptides can bind tightly to a pocket nearby the active site of KD domain, which is thus potential as self-inhibitory peptide to target and suppress nPLK kinase activity. The knowledge harvesting from computational studies were then used to guide the structural optimization and mutation of PBD peptide 1. Consequently, two of three peptide mutants separately exhibited moderately and considerably increased affinity as compared to the native peptide. The computationally modeled complex structures of KD domain with these self-inhibitory peptides were also examined in detail to unravel the structural basis and energetic property of nPLK-peptide recognition and interaction.

Characterization of a Unique Myosin IIIa Deafness Mutation which Enhances Actin-Sliding Velocity but Abolishes Filopodia Tip Localization

MYO3A, an unconventional myosin that localizes to the distal tips of stereocilia in the sensory epithelia of the vertebrate auditory and vestibular systems, consists of an N-terminal kinase domain, an actin-activated motor domain, and a class-specific tail domain. Previous work indicates that MYO3A tip-localization is regulated through autophosphorylation. MYO3A is thought to transport the actin regulatory protein, ESPIN1, to the stereocilia tips in inner ear hair cells and mutations in MYO3A lead to non-syndromic deafness in humans. We investigated the G488E mutation in MYO3A, a deafness associated mutation located near the conserved switch I region. The G488E mutation reduces the maximum actin-activated ATPase rate 2-fold and increases 10-fold the actin concentration required to reach one-half maximal ATPase. Interestingly, G488E MYO3A increases 2-fold the actin filament sliding velocity measured with the in vitro motility assay. We also investigated the impact of the G488E mutation on cellular localization by examining GFP tagged MYO3A dK constructs transfected into COS7 cells. The G488E mutation completely abolished MYO3A dK filopodia tip localization both in the presence and absence of ESPIN1. In addition, co-transfection of G488E MYO3A dK, ESPIN1 and wild-type (WT) MYO3A dK demonstrated that the presence of WT MYO3A does not rescue the tip localization of G488E. We hypothesize that the G488E mutation disrupts the duty ratio which prevents processive movement to the tips of parallel actin bundles. In support, chimeras of the MYO3A tail with other myosin motors (MYO1A, MYO19, MYO5A, and MYO10) demonstrate that only high duty-ratio motors are capable of filopodia tip localization. Our results demonstrate that intra-filopodia motility requires high duty ratio motor activity, which may be crucial for the formation, maintenance, and ultrastructure of parallel actin bundles.

Inhibitors of the Polo-Box Domain of Polo-Like Kinase 1.

Polo-like kinase 1 (Plk1), a key player in mitosis, is overexpressed in a wide range of tumor types and has been validated as a target for tumor therapy. In addition to its N-terminal kinase domain, Plk1 harbors a C-terminal protein-protein interaction domain, referred to as the polo-box domain (PBD). Because the PBD is unique to the five-member family of polo-like kinases, and its inhibition is sufficient to inhibit the enzyme, the Plk1 PBD is an attractive target for the inhibition of Plk1 function. Although peptide-based inhibitors are invaluable tools for elucidating the nature of the binding interface, small molecules are better suited for the induction of mitotic arrest and apoptosis in tumor cells by Plk1 inhibition. This review describes the considerable progress that has been made in developing small-molecule and peptide-based inhibitors of the Plk1 PBD.

Regulation of RIPK1's cell death function by phosphorylation

RIPK1 is a key molecule determining cellular fate downstream of several innate immune receptors. It is a serine/threonine kinase consisting of an N-terminal kinase domain linked by a largely unstru...

Structural insight into the mechanism of synergistic autoinhibition of SAD kinases.

The SAD/BRSK kinases participate in various important life processes, including neural development, cell cycle and energy metabolism. Like other members of the AMPK family, SAD contains an N-terminal kinase domain followed by the characteristic UBA and KA1 domains. Here we identify a unique autoinhibitory sequence (AIS) in SAD kinases, which exerts autoregulation in cooperation with UBA. Structural studies of mouse SAD-A revealed that UBA binds to the kinase domain in a distinct mode and, more importantly, AIS nestles specifically into the KD-UBA junction. The cooperative action of AIS and UBA results in an ‘αC-out' inactive kinase, which is conserved across species and essential for presynaptic vesicle clustering in C. elegans. In addition, the AIS, along with the KA1 domain, is indispensable for phospholipid binding. Taken together, these data suggest a model for synergistic autoinhibition and membrane activation of SAD kinases.

Expression profiles and interaction suggest TBK1 can be regulated by Nrdp1 in response to immune stimulation in large yellow croaker Larimichthys crocea.

TBK1 has been extensively studied in mammals because of its important roles as a molecular bridge, linking the TLRs (TLR3 and TLR4) and RLRs signals to activate transcriptional factors IRF3 and IRF7 for IFN-I production. However, the information on molecular and functional characteristics of TBK1 in teleosts is limited. In this study, the molecular characterization and immune response of TBK1 in Larimichthys crocea (named as LcTBK1) as well as its interaction with Nrdp1 were investigated. Sequence analysis demonstrated that LcTBK1 included four functional motifs, the N-terminal protein kinase domain and ATP-binding site, middle ULD and C-terminal coiled-coil domain. The tissue expression profiles indicated that LcTBK1 gene was constitutively expressed in the twelve tissues examined, with high expression in brain. Temporal expression analysis showed that LcTBK1 mRNA was obviously increased in the liver after injection of LPS, Poly I:C and inactive Vibrio parahaemolyticus, however, declined at some time points in spleen and head-kidney. Furthermore, we found that LcTBK1 can interact with LcNrdp1, an E3 ubiquitin ligase that involved in immune response to Cryptocaryon irritans infection in L. crocea. The qPCR showed that LcNrdp1 was also significantly up-regulated in liver, down-regualted at some time points in spleen and head-kidney after LPS, Poly I:C and inactive V. parahaemolyticus injection, although the expression patterns of the two genes after the three treatments were different in change magnitude and up-regulation timespan. These results suggested that LcTBK1 was involved in L. crocea defense against the pathogen infection and can be regulated by Nrdp1 in PPRs signaling pathway of fishes.

Preferential Binding of the Marine β‐carboline Manzamine A to the NTDK Domain of RSK1

We reported that manzamine A (MZA) inhibited LPS‐treated rat microglia TXB2 (IC50<0.016 µM) and O2‐ (IC50=0.1 µM) generation (BMC Pharmacology 5(1) 6, 2005), LPS‐treated human BMΦ TXB2 (apparent IC50<0.7 µM) (Inflam Res. 53(S3): S217, 2004) and rat p90 ribosomal S6 kinase 1 (RSK1) with a 10‐fold selectivity in potency versus RSK2 (IC50 of 15.01 μM and 108.4 μM, respectively) in biochemical studies (Program 545.5, Society of Neuroscience, 2009). We also hypothesized that MZA's differential binding and selectivity toward RSK1 and RSK2 could be explored by computational docking experiments employing both the N‐terminal (NTDK) and C‐terminal kinase domains (CTKD). Results: The RSK1‐MZA (NTDK and CTKD‐termini) complexes appeared to have stronger interactions and preferable energetics compared to respective RSK2‐MZA complexes. In addition, our computational strategy suggested that MZA bound preferentially to the NTDK domain of RSK1 rather than the CTKD domain. RSK is a vertebrate family of cytosolic serine‐threonine kinases that act downstream of the ras‐ERK1/2 (extracellular‐signal‐regulated kinase 1/2) pathway, which phosphorylates substrates shown to regulate several cellular processes including growth, survival and proliferation. Consequently, our findings lead us to hypothesize that there may be additional novel pharmacological properties of MZA and the 80 currently known manzamine‐type alkaloids isolated from several sponge genera. Support by the Office of Research, Midwestern University (AMSM) is gratefully acknowledged.

The clathrin-binding and J-domains of GAK support the uncoating and chaperoning of clathrin by Hsc70 in the brain.

Cyclin-G-associated kinase (GAK), the ubiquitously expressed J-domain protein, is essential for the chaperoning and uncoating of clathrin that is mediated by Hsc70 (also known as HSPA8). Adjacent to the C-terminal J-domain that binds to Hsc70, GAK has a clathrin-binding domain that is linked to an N-terminal kinase domain through a PTEN-like domain. Knocking out GAK in fibroblasts caused inhibition of clathrin-dependent trafficking, which was rescued by expressing a 62-kDa fragment of GAK, comprising just the clathrin-binding and J-domains. Expressing this fragment as a transgene in mice rescued the lethality and the histological defects caused by knocking out GAK in the liver or in the brain. Furthermore, when both GAK and auxilin (also known as DNAJC6), the neuronal-specific homolog of GAK, were knocked out in the brain, mice expressing the 62-kDa GAK fragment were viable, lived a normal life-span and had no major behavior abnormalities. However, these mice were about half the size of wild-type mice. Therefore, the PTEN-like domains of GAK and auxilin are not essential for Hsc70-dependent chaperoning and uncoating of clathrin, but depending on the tissue, these domains appear to increase the efficiency of these co-chaperones.

Crystal structure of the polo-box domain of polo-like kinase 2

Polo-like kinase 2 (PLK2) is a crucial regulator in cell cycle progression, DNA damage response, and neuronal activity. PLK2 is characterized by the conserved N-terminal kinase domain and the unique C-terminal polo-box domain (PBD). The PBD mediates diverse functions of PLK2 by binding phosphorylated Ser-pSer/pThr motifs of its substrates. Here, we report the first crystal structure of the PBD of PLK2. The overall structure of the PLK2 PBD is similar to that of the PLK1 PBD, which is composed by two polo boxes each contain β6α structures that form a 12-stranded β sandwich domain. The edge of the interface between the two polo boxes forms the phosphorylated Ser-pSer/pThr motifs binding cleft. On the hand, the peripheral regions around the core binding cleft of the PLK2 PBD is distinct from that of the PLK1 PBD, which might confer the substrate specificity of the PBDs of the polo-like kinase family.

3-Phosphoinositide-Dependent Protein Kinase 1 (PDPK1) Is a Pivotal Cell Signaling Mediator in Multiple Myeloma

Multiple myeloma (MM) is a highly molecularly heterogeneous hematologic malignancy and remains mostly incurable despite recent improvement of treatment outcome by novel agents. Therefore, the identification of novel and universal targetable therapeutic molecules is a core component for the therapeutic development. Herein, we identified that 3-phosphoinositide-dependent protein kinase 1 (PDPK1), a member of serine threonine kinase, is a rationalistic candidate of a novel therapeutic target against MM. PDPK1 is universally phosphorylated in all eleven MM-derived cell lines examined regardless of type of cytogenetic/genetic abnormalities or the activation/mutation state of FGFR3, RAS, ERK and AKT. PDPK1 promotes the cell proliferation of myeloma cells by activating RSK2Ser227 at N-terminal kinase domain which is a pivotal regulator of molecules that are essential for myelomagenesis, such as c-MYC, IRF4, or Cyclin Ds (Shimura Y, Mol Cancer Ther 2011), and AKTThr308. In contrast, PDPK1 inhibition by a selective inhibitor, BX-912, caused G2/M arrest, which was accompanied by the inactivation of PLK1, and resulted in cell death via induction of apoptosis which was accompanied by the activation of pro-apoptotic BH3-only proteins, BIM and BAD, in myeloma-derived cell lines. These molecular and cytological effects of PDPK1 inhibition in myeloma cells were also validated by gene knockdown by means of RNA interference using two different siRNAs specific for PDPK1. In addition, the cytotoxic effect of BX-912 was not hampered in two cell lines acquiring resistance to bortezomib (BTZ) (Ri M, Leukemia 2010), and PDPK1 inhibition by BX-912 showed additive to synergistic in vitro cytotoxic effects on myeloma cells with melphalan, etoposide, bortezomib or BAY11-7085, an inhibitor for NF-κB. BX-912 also induced cell death in eleven patient-derived primary myeloma cells those were positively isolated by CD138-labelling from bone marrow aspirates, while normal peripheral lymphocytes were significantly less sensitive to PDPK1 inhibitor compared with MM cells. In the clinical setting, PDPK1 was active in myeloma cells of 57 of 65 (87.7%) symptomatic MM patients at diagnosis. While patients backgrounds, such as age, gender, and the type of M-protein, were not significantly different between PDPK1-negative (PDPK1(-)) and PDPK1-positive (PDPK1(+)) patients, patients with the disease stage III according to International Staging System were significantly more frequent in the PDPK1(+) cohort compared with PDPK1(-) cohort. The PDPK1(-) patients tended to exhibit longer overall survival (OS) than the patients with PDPK1(+) (median OS: 2925 days vs. 2155 days, p=0.069) with a median follow-up period of 1310 days (range: 228–3317 days). Of particular, the 8-year OS of PDPK1(-) patients was statistically significantly more favorable than those of PDPK1(+) patients (83% vs. 17%, p=0.041). In addition, when we analyzed the impact of PDPK1 activity on the treatment outcome of patients treated by BTZ and dexamethasone therapy (BD), our results revealed that progression free survival (PFS) of patients with PDPK1(-) was a significantly longer than those of patients with PDPK1(+) (median PFS: PDPK1(-) vs. PDPK1(+), not reached vs. 167 days, p=0.049). The PFS of PDPK1(-) patients treated by high-dose therapy/autologous stem cell transplantation (HDT/ASCT) tended to be longer than those of PDPK1(+) (median PFS: PDPK1(-) vs. PDPK1(+), 972 days vs. 567 days, p=0.125). In conclusion, our study provides the rationale for PDPK1 as a possible universal molecular target for MM with various molecular/cytogenetic features. Especially, PDPK1 is a potential therapeutic target for not only newly diagnosed patients but also patients who are resistant or refractory to currently available anti-myeloma therapies. This study was conducted in accordance with the Declaration of Helsinki and with the approval of the Institutional Review Board. Patient-derived samples were obtained with informed consent. DisclosuresNo relevant conflicts of interest to declare.

Regulation of Greatwall kinase by protein stabilization and nuclear localization

Greatwall (Gwl) functions as an essential mitotic kinase by antagonizing protein phosphatase 2A. In this study we identified Hsp90, Cdc37 and members of the importin α and β families as the major binding partners of Gwl. Both Hsp90/Cdc37 chaperone and importin complexes associated with the N-terminal kinase domain of Gwl, whereas an intact glycine-rich loop at the N-terminus of Gwl was essential for binding of Hsp90/Cdc37 but not importins. We found that Hsp90 inhibition led to destabilization of Gwl, a mechanism that may partially contribute to the emerging role of Hsp90 in cell cycle progression and the anti-proliferative potential of Hsp90 inhibition. Moreover, in agreement with its importin association, Gwl exhibited nuclear localization in interphase Xenopus S3 cells, and dynamic nucleocytoplasmic distribution during mitosis. We identified KR456/457 as the locus of importin binding and the functional NLS of Gwl. Mutation of this site resulted in exclusion of Gwl from the nucleus. Finally, we showed that the Gwl nuclear localization is indispensable for the biochemical function of Gwl in promoting mitotic entry.

Interdomain allosteric regulation of Polo kinase by Aurora B and Map205 is required for cytokinesis.

Drosophila melanogaster Polo and its human orthologue Polo-like kinase 1 fulfill essential roles during cell division. Members of the Polo-like kinase (Plk) family contain an N-terminal kinase domain (KD) and a C-terminal Polo-Box domain (PBD), which mediates protein interactions. How Plks are regulated in cytokinesis is poorly understood. Here we show that phosphorylation of Polo by Aurora B is required for cytokinesis. This phosphorylation in the activation loop of the KD promotes the dissociation of Polo from the PBD-bound microtubule-associated protein Map205, which acts as an allosteric inhibitor of Polo kinase activity. This mechanism allows the release of active Polo from microtubules of the central spindle and its recruitment to the site of cytokinesis. Failure in Polo phosphorylation results in both early and late cytokinesis defects. Importantly, the antagonistic regulation of Polo by Aurora B and Map205 in cytokinesis reveals that interdomain allosteric mechanisms can play important roles in controlling the cellular functions of Plks.

Cloning and characterization of tesk1, a novel spermatogenesis-related gene, in the tongue sole (Cynoglossus semilaevis).

Testis-specific protein kinase 1 (Tesk1) is a serine/threonine kinase with unique structural features. In the present study, we cloned and characterized the tesk1 gene of tongue sole, Cynoglossus semilaevis. The full-length tesk1 cDNA consists of 1,672 nucleotides, encoding a 331 amino acid polypeptide with a characteristic structure composed of an N-terminal kinase domain and a C-terminal proline-rich domain. The tesk1 genomic sequence contains eight exons and seven introns. Real-time quantitative PCR revealed that tesk1 mRNA is expressed predominantly in the testis, though the level of expression varied throughout development. We used in situ hybridization to show that tesk1 mRNA is expressed in the spermatids of males and pseudo-males, but not in triploid males. Our results suggest that tongue sole Tesk1 may play a role in spermatogenesis.

The Vip1 inositol polyphosphate kinase family regulates polarized growth and modulates the microtubule cytoskeleton in fungi.

Microtubules (MTs) are pivotal for numerous eukaryotic processes ranging from cellular morphogenesis, chromosome segregation to intracellular transport. Execution of these tasks requires intricate regulation of MT dynamics. Here, we identify a new regulator of the Schizosaccharomyces pombe MT cytoskeleton: Asp1, a member of the highly conserved Vip1 inositol polyphosphate kinase family. Inositol pyrophosphates generated by Asp1 modulate MT dynamic parameters independent of the central +TIP EB1 and in a dose-dependent and cellular-context-dependent manner. Importantly, our analysis of the in vitro kinase activities of various S. pombe Asp1 variants demonstrated that the C-terminal phosphatase-like domain of the dual domain Vip1 protein negatively affects the inositol pyrophosphate output of the N-terminal kinase domain. These data suggest that the former domain has phosphatase activity. Remarkably, Vip1 regulation of the MT cytoskeleton is a conserved feature, as Vip1-like proteins of the filamentous ascomycete Aspergillus nidulans and the distantly related pathogenic basidiomycete Ustilago maydis also affect the MT cytoskeleton in these organisms. Consistent with the role of interphase MTs in growth zone selection/maintenance, all 3 fungal systems show aspects of aberrant cell morphogenesis. Thus, for the first time we have identified a conserved biological process for inositol pyrophosphates.

Identification of green tea catechins as potent inhibitors of the polo-box domain of polo-like kinase 1.

Polo-like kinase 1 (PLK1) plays crucial functions in multiple stages of mitosis and is considered to be a potential drug target for cancer therapy. The functions of PLK1 are mediated by its N-terminal kinase domain and C-terminal polo-box domain (PBD). Most inhibitors targeting the kinase domain of PLK1 have a selectivity issue because of a high degree of structural conservation within kinase domains of all protein kinases. Here, we combined virtual and experimental screenings to identify green tea catechins as potent inhibitors of the PLK1 PBD. Initially, (-)-epigallocatechin, one of the main components of green tea polyphenols, was found to significantly block the binding of fluorescein-labeled phosphopeptide to the PBD at a concentration of 10 μm. Next, additional catechins were evaluated for their dose-dependent inhibition of the PBD and preliminary structure-activity relationships were derived. Cellular analysis further showed that catechins interfere with the proper subcellular localization of PLK1, lead to cell-cycle arrest in the S and G2M phases, and induce growth inhibition of several human cancer cell types, such as breast adenocarcinoma (MCF7), lung adenocarcinoma (A549), and cervical adenocarcinoma (HeLa). Our data provides new insight into understanding the anticancer activities of green tea catechins.