A Presumptive Developmental Role for a Sea Urchin Cyclin B Splice Variant

We show that a splice variant–derived cyclin B is produced in sea urchin oocytes and embryos. This splice variant protein lacks highly conserved sequences in the COOH terminus of the protein. It is found strikingly abundant in growing oocytes and cells committed to differentiation during embryogenesis. Cyclin B splice variant (CBsv) protein associates weakly in the cell with Xenopus cdc2 and with budding yeast CDC28p. In contrast to classical cyclin B, CBsv very poorly complements a triple CLN deletion in budding yeast, and its microinjection prevents an initial step in MPF activation, leading to an important delay in oocyte meiosis reinitiation. CBsv microinjection in fertilized eggs induces cell cycle delay and abnormal development. We assume that CBsv is produced in growing oocytes to keep them in prophase, and during embryogenesis to slow down cell cycle in cells that will be committed to differentiation.Cyclins are a conserved family of proteins that play a central role in eukaryotic cell division cycle progression, as regulatory subunits of cyclin dependent kinases (CDKs, whose catalytic subunits are homologues of the fission yeast cdc2 protein).¹ CDKs are downstream targets of convergent cascades of regulations at critical points of the cell cycle. M-phase–promoting factor (MPF, formerly maturation promoting factor, reference 21), the factor responsible for M-phase entry and progression in mitosis, has been purified three times by biochemical means (7, 19, 36). MPF from starfish, Xenopus, and carp oocytes has been found to be a heterodimer composed of one molecule of cdc2 and one molecule of cyclin B (CB). B type cyclins are archetypal mitotic cyclins, evolutively and functionally related to fission yeast cdc13p. Among CDKs, the regulation of MPF is by far the best understood today. Cyclin B is required for activity, as well for activation and for inhibition of MPF. The cdc2 monomer has never been found active. Its activation is conferred by the CAK-dependent T161-phosphorylation that requires cyclin B association (4, 28, 33). Inhibition of MPF during S- and G2-phases and also by the DNA replication checkpoint mechanism is achieved by wee1-catalyzed phosphorylation of the tyrosine 15 in cyclin B–bound molecules of cdc2 (9, 22). Cyclin B is also likely required for activation of the protein phosphatase cdc25p that specifically dephosphorylates tyrosine 15 and allows MPF amplification and entry into mitosis (5, 37). Finally, targeted proteolysis of cyclin B by an ubiquitin-dependent pathway is the mechanism by which MPF is inactivated and the cell returns to interphase (8). Therefore, the major part of MPF regulation is accounted for by cyclin B synthesis and proteolysis. This was emphasized in simplified early embryogenesis cycles that are composed of a succession of M- and S-phases without intervening G-phases. Cycles in acellular Xenopus egg extracts are driven by MPF as a basic oscillator, whose periodic activity is scheduled strictly by oscillating abundance of cyclin B (24). Accordingly, during the cleavage period of Xenopus embryogenesis, cdc2 tyrosine 15 is never found phosphorylated (3) and checkpoint mechanisms are downregulated.Site-directed mutagenesis as well as protein crystallization have allowed the mapping of some sequences in cyclins involved in these regulations. Crystal structure of the homologous dimer cdk2–cyclin A showed that the cyclin interacts with the cdk via sequences distributed along the so-called cyclin box, a sequence well conserved among all cyclins (14). In the NH₂ terminus of mitotic cyclins A and B, a destruction box is required to allow ubiquitination of the protein and its targeted proteolysis in anaphase (8). Mutants that are deleted for this box are stable in mitosis, and their overexpression triggers mitotic arrest. Also in the NH₂-terminal region of B type cyclins, a cytoplasmic retention signal (CRS) is presumed to account for differential early prophase localization of nuclear cyclin A and cytoplasmic cyclin B (27). A chimeric cyclin A with the first amino acids of cyclin B remains cytoplasmic until early prophase. Further on, at the beginning of the cyclin box, conserved amino acids in the P-box are thought to be involved in the specific activation of cdc2 by cdc25 (37). Finally, two reports showed that a short COOH-terminal deletion of recombinant cyclins A or B abolished the binding to cdc2 (17, 34), although this region was not found to be directly involved in the physical interaction between cyclin A and cdk2 (14).Here we show that such a COOH-terminal truncation, which removes universally conserved amino acids, is naturally realized in a splice variant of sea urchin cyclin B. Moreover, immunofluorescence experiments suggest this splice variant plays a role in embryogenesis and behaves like a marker of cell lineages in postcleavage embryos.     

Distribution of millipedes along an altitudinal gradient in the south of Lake Teletskoye,Altai Mts,Russia (Diplopoda)

The distribution of millipedes along an altitudinal gradient in the south of Lake Teletskoye, Altai, Russia based on new samples from the Kyga Profile sites, as well as on partly published and freshly revised material (Mikhaljova et al. 2007, 2008, 2014, Nefedieva and Nefediev 2008, Nefediev and Nefedieva 2013, Nefedieva et al. 2014), is established. The millipede diversity is estimated to be at least 15 species and subspecies from 10 genera, 6 families and three orders. The bulk of species diversity is confined both to low- and mid-mountain chern taiga forests and high-mountain shrub tundras, whereas the highest numbers, reaching up to 130 ind./m², is shown in subalpine Pinus sibirica sparse growths. Based on clustering studied localities on species diversity similarity two groups of sites are defined: low-mountain sites and subalpine sparse growths of Pinus sibirica ones.     

A Functional P2X7 Splice Variant with an Alternative Transmembrane Domain 1 Escapes Gene Inactivation in P2X7 Knock-out Mice

The ATP-activated P2X7 receptor channel is involved in immune function and inflammatory pain and represents an important drug target. Here we describe a new P2X7 splice variant (P2X7(k)), containing an alternative intracellular N terminus and first transmembrane domain encoded by a novel exon 1 in the rodent P2rx7 gene. Whole cell patch clamp recordings of the rat isoform expressed in HEK293 cells revealed an 8-fold higher sensitivity to the agonist Bz-ATP and much slower deactivation kinetics when compared with the P2X7(a) receptor. Permeability measurements in Xenopus oocytes show a high permeability for N-methyl-d-glucamine immediately upon activation, suggesting that the P2X7(k) channel is constitutively dilated upon opening. The rates of agonist-induced dye uptake and membrane blebbing in HEK cells were also increased. PCR analyses and biochemical analysis by SDS-PAGE and BN-PAGE indicate that the P2X7(k) variant escapes gene deletion in one of the available P2X7^−/− mice strains and is strongly expressed in the spleen. Taken together, we describe a novel P2X7 isoform with distinct functional properties that contributes to the diversity of P2X7 receptor signaling. Its presence in one of the P2X7^−/− strains has important implications for our understanding of the role of this receptor in health and disease.P2X receptors (P2XRs)³ are ATP-gated cation channels. They consist of three subunits (1, 2) each containing two transmembrane domains (TMDs) linked by an extracellular ligand-binding domain (3). The P2X7 receptor is distinguished from other P2X receptors by its long intracellular C terminus, a low ATP sensitivity (EC₅₀: 100 μm to 1 mm), and its ability to induce “cell permeabilization,” meaning that upon prolonged ATP application the opening of a permeation pathway for large molecules is induced. This process eventually leads to apoptosis, requires the C terminus (3–6), and may be mediated by interaction with pannexin hemichannels (7). In addition, “pore dilation,” which allows the passage of the large cation NMDG, is observed if extracellular sodium is replaced by NMDG (8), a property also displayed by the P2X2 (9) and P2X4 (10) receptors. This pore dilation is assumed to represent an intrinsic property of these P2X receptors (11, 12), although it can be influenced by interaction with intracellular proteins (13). However, both processes are still poorly understood.P2X7 receptors are found on cells of the hematopoietic lineage, in epithelia, and endothelia. Several studies report its expression and/or function in neurons, although its presence here is under debate (14, 15). So far, nine splice variants (P2X7(b) through P2X7(j)) have been described, only one of which was shown to be, at least partially, functional (16, 17). In addition, numerous single nucleotide polymorphisms have been identified in the human P2X7 receptor. Some of these have been found to cause either gain or loss of function and have been associated with chronic lymphocytic leukemia, bone fracture risk, and impaired immune functions (18–20). Recent genetic studies indicate an association between the Gln-460 → Arg polymorphism and familial depressive disorders (21).Two P2X7-deficient mouse lines have been described. In the mouse line generated by Glaxo, the P2rx7 gene was knocked out by insertion of a lacZ transgene into exon 1 (22). In the mouse line generated by Pfizer (23) a neomycin cassette was inserted into exon 13, replacing a region that encodes Cys-506–Pro-532 of the intracellular C terminus of the receptor. The Pfizer P2X7 KO mice demonstrated the involvement of this receptor in bone formation (24), cytokine production, and inflammation (23, 25) while the Glaxo^−/− mice established its role in inflammatory and neuropathic pain (26). All these findings and multiple subsequent studies based on these mouse models defined the P2X7R as a promising target for the development of innovative therapeutic strategies, and selective P2X7 inhibitors are already in clinical trials for the treatment of rheumatoid arthritis (27).Here, we describe a novel P2X7 isoform with an alternative N terminus and TMD 1. Compared with the originally identified P2X7(a) variant, it has increased agonist sensitivity and a higher propensity to form NMDG-permeable pores and permit dye uptake. Due to a novel alternative exon 1 and translation start, this splice variant escapes inactivation in the Glaxo P2X7^−/− mice and thus could account for possible inconsistencies between results obtained with different P2X7^−/− mouse lines (28).     

Dbf4-Cdc7 Phosphorylation of Mcm2 Is Required for Cell Growth

The Dbf4-Cdc7 kinase (DDK) is required for the activation of the origins of replication, and DDK phosphorylates Mcm2 in vitro. We find that budding yeast Cdc7 alone exists in solution as a weakly active multimer. Dbf4 forms a likely heterodimer with Cdc7, and this species phosphorylates Mcm2 with substantially higher specific activity. Dbf4 alone binds tightly to Mcm2, whereas Cdc7 alone binds weakly to Mcm2, suggesting that Dbf4 recruits Cdc7 to phosphorylate Mcm2. DDK phosphorylates two serine residues of Mcm2 near the N terminus of the protein, Ser-164 and Ser-170. Expression of mcm2-S170A is lethal to yeast cells that lack endogenous MCM2 (mcm2Δ); however, this lethality is rescued in cells harboring the DDK bypass mutant mcm5-bob1. We conclude that DDK phosphorylation of Mcm2 is required for cell growth.The Cdc7 protein kinase is required throughout the yeast S phase to activate origins (1, 2). The S phase cyclin-dependent kinase also activates yeast origins of replication (3–5). It has been proposed that Dbf4 activates Cdc7 kinase in S phase, and that Dbf4 interaction with Cdc7 is essential for Cdc7 kinase activity (6). However, it is not known how Dbf4-Cdc7 (DDK)² acts during S phase to trigger the initiation of DNA replication. DDK has homologs in other eukaryotic species, and the role of Cdc7 in activation of replication origins during S phase may be conserved (7–10).The Mcm2-7 complex functions with Cdc45 and GINS to unwind DNA at a replication fork (11–15). A mutation of MCM5 (mcm5-bob1) bypasses the cellular requirements for DBF4 and CDC7 (16), suggesting a critical physiologic interaction between Dbf4-Cdc7 and Mcm proteins. DDK phosphorylates Mcm2 in vitro with proteins purified from budding yeast (17, 18) or human cells (19). Furthermore, there are mutants of MCM2 that show synthetic lethality with DBF4 mutants (6, 17), suggesting a biologically relevant interaction between DBF4 and MCM2. Nevertheless, the physiologic role of DDK phosphorylation of Mcm2 is a matter of dispute. In human cells, replacement of MCM2 DDK-phosphoacceptor residues with alanines inhibits DNA replication, suggesting that Dbf4-Cdc7 phosphorylation of Mcm2 in humans is important for DNA replication (20). In contrast, mutation of putative DDK phosphorylation sites at the N terminus of Schizosaccharomyces pombe Mcm2 results in viable cells, suggesting that phosphorylation of S. pombe Mcm2 by DDK is not critical for cell growth (10).In budding yeast, Cdc7 is present at high levels in G₁ and S phase, whereas Dbf4 levels peak in S phase (18, 21, 22). Furthermore, budding yeast DDK binds to chromatin during S phase (6), and it has been shown that Dbf4 is required for Cdc7 binding to chromatin in budding yeast (23, 24), fission yeast (25), and Xenopus (9). Human and fission yeast Cdc7 are inert on their own (7, 8), but Dbf4-Cdc7 is active in phosphorylating Mcm proteins in budding yeast (6, 26), fission yeast (7), and human (8, 10). Based on these data, it has been proposed that Dbf4 activates Cdc7 kinase in S phase and that Dbf4 interaction with Cdc7 is essential for Cdc7 kinase activity (6, 9, 18, 21–24). However, a mechanistic analysis of how Dbf4 activates Cdc7 has not yet been accomplished. For example, the multimeric state of the active Dbf4-Cdc7 complex is currently disputed. A heterodimer of fission yeast Cdc7 (Hsk1) in complex with fission yeast Dbf4 (Dfp1) can phosphorylate Mcm2 (7). However, in budding yeast, oligomers of Cdc7 exist in the cell (27), and Dbf4-Cdc7 exists as oligomers of 180 and 300 kDa (27).DDK phosphorylates the N termini of human Mcm2 (19, 20, 28), human Mcm4 (10), budding yeast Mcm4 (26), and fission yeast Mcm6 (10). Although the sequences of the Mcm N termini are poorly conserved, the DDK sites identified in each study have neighboring acidic residues. The residues of budding yeast Mcm2 that are phosphorylated by DDK have not yet been identified.In this study, we find that budding yeast Cdc7 is weakly active as a multimer in phosphorylating Mcm2. However, a low molecular weight form of Dbf4-Cdc7, likely a heterodimer, has a higher specific activity for phosphorylation of Mcm2. Dbf4 or DDK, but not Cdc7, binds tightly to Mcm2, suggesting that Dbf4 recruits Cdc7 to Mcm2. DDK phosphorylates two serine residues of Mcm2, Ser-164 and Ser-170, in an acidic region of the protein. Mutation of Ser-170 is lethal to yeast cells, but this phenotype is rescued by the DDK bypass mutant mcm5-bob1. We conclude that DDK phosphorylation of Ser-170 of Mcm2 is required for budding yeast growth.     

Differential 14-3-3 Affinity Capture Reveals New Downstream Targets of Phosphatidylinositol 3-Kinase Signaling

We devised a strategy of 14-3-3 affinity capture and release, isotope differential (d₀/d₄) dimethyl labeling of tryptic digests, and phosphopeptide characterization to identify novel targets of insulin/IGF1/phosphatidylinositol 3-kinase signaling. Notably four known insulin-regulated proteins (PFK-2, PRAS40, AS160, and MYO1C) had high d₀/d₄ values meaning that they were more highly represented among 14-3-3-binding proteins from insulin-stimulated than unstimulated cells. Among novel candidates, insulin receptor substrate 2, the proapoptotic CCDC6, E3 ubiquitin ligase ZNRF2, and signaling adapter SASH1 were confirmed to bind to 14-3-3s in response to IGF1/phosphatidylinositol 3-kinase signaling. Insulin receptor substrate 2, ZNRF2, and SASH1 were also regulated by phorbol ester via p90RSK, whereas CCDC6 and PRAS40 were not. In contrast, the actin-associated protein vasodilator-stimulated phosphoprotein and lipolysis-stimulated lipoprotein receptor, which had low d₀/d₄ scores, bound 14-3-3s irrespective of IGF1 and phorbol ester. Phosphorylated Ser¹⁹ of ZNRF2 (RTRAYpS¹⁹GS), phospho-Ser⁹⁰ of SASH1 (RKRRVpS⁹⁰QD), and phospho- Ser⁴⁹³ of lipolysis-stimulated lipoprotein receptor (RPRARpS⁴⁹³LD) provide one of the 14-3-3-binding sites on each of these proteins. Differential 14-3-3 capture provides a powerful approach to defining downstream regulatory mechanisms for specific signaling pathways.Activated tyrosine kinase receptors generally drive cells to assimilate nutrients; regulate partitioning of the assimilate to make storage polymers and biosynthetic precursors and for energy production; and promote cellular survival, growth, division, movement, and differentiation. From this spectrum, each cell displays a specific subset of responses depending on the hormone, specific receptors, cross-talk from other signaling pathways, metabolic conditions, and cellular complement of effector proteins. For example, insulin stimulates glucose uptake and glycogen synthesis in skeletal muscle, whereas IGF1¹ promotes survival, growth, and proliferation of many cell types (1, 2).Many of these cellular responses are mediated via PI 3-kinase, which generates phosphatidylinositol 3,4,5-trisphosphate, promoting the activation of AGC protein kinases such as PKB/Akt and other signaling components (1, 3). PI 3-kinase is activated by binding to tyrosine-phosphorylated receptors such as the platelet-derived growth factor receptor or via adaptor molecules such as insulin receptor substrates, which are phosphorylated by the activated insulin receptor. Deregulated PI 3-kinase and downstream signaling has been linked to problems with wound healing, immune responses, neurodegeneration, and cardiovascular disease; decreased PI 3-kinase signaling may underlie insulin resistance and type II diabetes; and this pathway is often activated in human tumors (4, 5). To help pinpoint drug targets for these diseases we must define the mechanisms linking PI 3-kinase and other signaling pathways to downstream effectors and understand specificity with respect to different hormone/cell type combinations.Many missing substrates of PI 3-kinase/AGC kinases must be found to explain all the cellular responses to insulin and growth factors (3). Several targets of PI 3-kinase/PKB signaling, including TSC2 (6), PRAS40 (7), AS160 (8), and FYVE domain-containing phosphatidylinositol 3-phosphate 5-kinase (9) were identified using the anti-PAS antibody, which loosely recognizes the minimal phosphorylated consensus for PKB, which is RXRXX(pS/pT) where pS is phosphoserine and pT is phosphothreonine. Another helpful feature for identifying new downstream targets is that phosphorylation by PKB sometimes creates binding sites for 14-3-3s, which are dimeric proteins that bind to specific phosphorylated sites on target proteins. Thus PKB promotes the binding of 14-3-3s to proteins including PFKFB2 cardiac PFK-2 (10, 11), BimEL (12), β-catenin (13), p27(Kip1) (14), PRAS40 (7), FOXO1 (15), Miz1 (16), TBC1D4 (AS160 (17, 18), and TBC1D1 (19). Functionally 14-3-3s can trigger changes in the conformations of their targets and alter how targets interact with other proteins. Consistent with 14-3-3/target interactions being important in cellular responses to growth factors and insulin, reagents that compete with targets for binding to 14-3-3s inhibit the IGF1-stimulated increase in the glycolytic stimulator fructose-2,6-bisphosphate (10) and PKB-dependent cell survival (20).Some 14-3-3-binding sites on the above named proteins can also be phosphorylated by other basophilic protein kinases (21). For example, AS160 and TBC1D1 are two related RabGAPs (GTPase-activating protein for Rabs) regulated by multisite phosphorylation that regulate trafficking of GluT4 transporter to the plasma membrane for uptake of glucose. The two 14-3-3-binding sites on AS160 can be phosphorylated by PKB, p90RSK, serum- and glucocorticoid-inducible kinase, and other kinases, whereas one of the 14-3-3-binding sites on TBC1D1 is also a substrate of the energy-sensing kinase AMP-activated protein kinase (17–19). Thus, the relative sensitivity of glucose trafficking to insulin and AMP-activated protein kinase activators in different tissues may depend in part on the distribution of AS160 and TBC1D1. Other insulin-regulated 14-3-3 targets, such as myosin 1C (22), are also convergence points for phosphorylation by more than one AGC and/or Ca²⁺/calmodulin-dependent protein kinase.Here many more proteins than those already identified were found to display 14-3-3 and/or PAS binding signals when the PI 3-kinase pathway was activated in cells against a “background” of other proteins whose 14-3-3 and PAS binding status was unaffected by PI 3-kinase signaling. We aimed to pick out the PI 3-kinase-regulated proteins, which was challenging given the hundreds of 14-3-3 binding partners in mammalian cells (10, 23–27). We used 14-3-3 affinity capture and release, identified phosphopeptides, and devised a quantitative proteomics approach in which 14-3-3-binding proteins from insulin-stimulated versus unstimulated cells were labeled with formaldehyde containing light or heavy isotopes, respectively. Biochemical checking of candidates from these screens, which included proteins with links to diabetes, cancers, and neurodegenerative disorders, confirmed the identification of novel downstream targets of PI 3-kinase, some of which are also convergence points for regulation by MAPK/p90RSK signaling.     

Proteomics Identification of Drosophila Small Interfering RNA-associated Factors

The Drosophila melanogaster RNA-induced silencing complex (RISC) forms a large ribonucleoprotein particle on small interfering RNAs (siRNAs) and catalyzes target mRNA cleavage during RNA interference (RNAi). Dicer-2, R2D2, Loquacious, and Argonaute-2 are examples of RISC-associated factors that are involved in RNAi. Holo-RISC is an ∼80 S small interfering ribonucleoprotein, which suggests that there are many additional proteins that participate in the RNAi pathway. In this study, we used siRNA affinity capture combined with mass spectrometry to identify novel components of the Drosophila RNAi machinery. Our study identified both established RISC components and novel siRNA-associated factors, many of which contain domains that are consistent with potential roles in RNAi. Functional analysis of these novel siRNA-associated proteins suggests that these factors may play an important role in RNAi.Small RNAs can regulate gene expression through a collection of mechanisms broadly termed RNA silencing. Small RNA-mediated silencing mechanisms occur in most species (1–5). The ability to silence the expression of specific genes using small RNAs via RNA interference (RNAi)¹ has greatly facilitated our understanding of gene function in eukaryotes. In addition, small RNA-mediated gene silencing has therapeutic potential and holds promise for the treatment of specific diseases (6). Understanding the mechanism of RNAi and identifying the components of the RNAi machinery are essential for harnessing its full potential in both genome-wide screens and therapeutic applications.Recently, high throughput sequencing technology has revealed the presence of endogenous siRNAs in plant, fly, worm, and mammalian cells (7–16). These endogenous siRNAs target transposable element RNAs, pseudogene RNAs, and protein-coding mRNAs (17). Therefore, the endogenous siRNA pathway seems to have evolved as a mechanism of cellular defense against selfish genetic elements. The roles of these siRNAs in development and cell physiology are poorly understood.Drosophila melanogaster is a well characterized model system for studying RNAi. In Drosophila, long double-stranded RNAs (dsRNAs) are processed by the endonuclease Dicer-2 into 21-nucleotide siRNAs (18). After processing, these siRNAs form an initiator complex with Dicer-2 and the dsRNA-binding domain (dsRBD)-containing protein R2D2 (19–23). This R2D2-Dicer-2 Initiator (RDI) complex transitions to a larger siRNP called the RISC loading complex (21, 22, 24, 25) and then to pre-RISC (26). Subsequently, pre-RISC matures into holo-RISC, which includes the catalytic activity necessary for target mRNA cleavage (21, 25, 27). The endonuclease subunit responsible for target cleavage in holo-RISC is Argonaute-2 (Ago2) (28, 29), which uses the guide strand of the siRNA duplex to target complementary mRNA sequences for cleavage and degradation.Studies of the RDI complex strongly suggest that it includes no other proteins besides Dicer-2 and R2D2 (22). Additional proteins such as Ago2 are present in pre-RISC and holo-RISC, but nonetheless the complete compositions of the RISC loading complex, pre-RISC, and holo-RISC are unknown. Furthermore, holo-RISC sediments at ∼80 S during sucrose gradient centrifugation (30). These observations indicate that additional protein factors associate with siRNAs. In this study, we identified siRNA-binding proteins from Drosophila embryo extracts. Target cleavage assays and immunoblotting of our siRNA affinity-selected proteins suggest that we purified active holo-RISC components. Proteomics analysis of the affinity matrix revealed both established and novel siRNA-associated proteins. Functional analyses of a subset of these factors suggest that they play important roles in RNAi.