Peter J. Milburn

Phosphorylation of splicing factor SF1 on Ser20 by cGMP‐dependent protein kinase regulates spliceosome assembly

Splicing factor 1 (SF1) functions at early stages of pre‐mRNA splicing and contributes to splice site recognition by interacting with the essential splicing factor U2AF65 and binding to the intron branch site. We have identified an 80 kDa substrate of cGMP‐dependent protein kinase‐I (PKG‐I) isolated from rat brain, which is identical to SF1. PKG phosphorylates SF1 at Ser20, which inhibits the SF1–U2AF65 interaction leading to a block of pre‐spliceosome assembly. Mutation of Ser20 to Ala or Thr also inhibits the interaction with U2AF65, indicating that Ser20 is essential for binding. SF1 is phosphorylated in vitro by PKG, but not by cAMP‐dependent protein kinase A (PKA). Phosphorylation of SF1 also occurs in cultured neuronal cells and is increased on Ser20 in response to a cGMP analogue. These results suggest a new role for PKG in mammalian pre‐mRNA splicing by regulating in a phosphorylation‐dependent manner the association of SF1 with U2AF65 and spliceosome assembly.

Dynamic Histone Variant Exchange Accompanies Gene Induction in T Cells

ABSTRACT Changes in chromatin composition are often a prerequisite for gene induction. Nonallelic histone variants have recently emerged as key players in transcriptional control and chromatin modulation. While the changes in chromatin accessibility and histone posttranslational modification (PTM) distribution that accompany gene induction are well documented, the dynamics of histone variant exchange that parallel these events are still poorly defined. In this study, we have examined the changes in histone variant distribution that accompany activation of the inducible CD69 and heparanase genes in T cells. We demonstrate that the chromatin accessibility increases that accompany the induction of both of these genes are not associated with nucleosome loss but instead are paralleled by changes in histone variant distribution. Specifically, induction of these genes was paralleled by depletion of the H2A.Z histone variant and concomitant deposition of H3.3. Furthermore, H3.3 deposition was accompanied by changes in PTM patterns consistent with H3.3 enriching or depleting different PTMs upon incorporation into chromatin. Nevertheless, we present evidence that these H3.3-borne PTMs can be negated by recruited enzymatic activities. From these observations, we propose that H3.3 deposition may both facilitate chromatin accessibility increases by destabilizing nucleosomes and compete with recruited histone modifiers to alter PTM patterns upon gene induction.

A pharmacological and biochemical investigation of the venom from the platypus (Ornithorhynchus anatinus)

In this study several activities of the venom of Ornithorhynchus anatinus have been investigated. Whole venom induced local oedema after subplantar injection and produced relaxation of the rat uterus in vitro. The relaxant activity was partially purified by gel permeation HPLC and subsequent analyses by SDS-PAGE revealed that this activity was associated with a 4200 mol. wt peptide. The N-terminal partial sequence of this peptide exhibited substantial identity with human and porcine C-type natriuretic peptide (CNP). Three other major proteins isolated from the venom had mol. wts of 140,000, 55,000 and 16,000. None was found to have any sequence homology with proteins listed in the SwissProt database. The 140,000 mol. wt protein exhibited hyaluronidase activity but the nature of the 55,000 and 16,000 mol. wt proteins remains to be determined. Platypus venom also exhibits protease activity, although the concentration of proteolytic enzymes was too low to be visualised by SDS-PAGE using Coomassie staining.

The natriuretic peptide (ovCNP-39) from platypus (Ornithorhynchus anatinus) venom relaxes the isolated rat uterus and promotes oedema and mast cell histamine release.

In this study we characterise the ability of a C-type natriuretic peptide from platypus (Ornithorhynchus anatinus) venom (ovCNP-39) to relax the rat uterus in vitro and we investigate the possibility that ovCNP-39 contributes to the acute effects of envenomation, which include oedema, pain and erythema. We have found that both ovCNP-39 and the endogenous C-type natriuretic peptide, CNP-22, produce oedema in the rat paw and release histamine from rat peritoneal mast cells. Two synthetic peptides, ovCNP-39(1-17) and ovCNP-39(18-39), corresponding to the N- and C-termini, respectively, are equipotent histamine releasers, suggesting that ovCNP-39 and other natriuretic peptides do not act through conventional natriuretic peptide receptors on mast cells.

Chromatin-associated protein kinase C-θ regulates an inducible gene expression program and microRNAs in human T lymphocytes.

Studies in yeast demonstrate that signaling kinases have a surprisingly active role in the nucleus, where they tether to chromatin and modulate gene expression programs. Despite these seminal studies, the nuclear mechanism of how signaling kinases control transcription of mammalian genes is in its infancy. Here, we provide evidence for a hitherto unknown function of protein kinase C-theta (PKC-θ), which physically associates with the regulatory regions of inducible immune response genes in human T cells. Chromatin-anchored PKC-θ forms an active nuclear complex by interacting with RNA polymerase II, the histone kinase MSK-1, and the adaptor molecule 14-3-3ζ. ChIP-on-chip reveals that PKC-θ binds to promoters and transcribed regions of genes, as well as to microRNA promoters that are crucial for cytokine regulation. Our results provide a molecular explanation for the role of PKC-θ not only in normal T cell function, but also in circumstances of its ectopic expression in cancer.

Human DNA polymerase-η, an A-T mutator in somatic hypermutation of rearranged immunoglobulin genes, is a reverse transcriptase

We have proposed previously that error‐prone reverse transcription using pre‐mRNA of rearranged immunoglobulin variable (IgV) regions as templates is involved in the antibody diversifying mechanism of somatic hypermutation (SHM). As patients deficient in DNA polymerase‐η exhibit an abnormal spectrum of SHM, we postulated that this recently discovered Y‐family polymerase is a reverse transcriptase (RT). This possibility was tested using a product‐enhanced RT (PERT) assay that uses a real time PCR step with a fluorescent probe to detect cDNA products of at least 27−37 nucleotides. Human pol‐η and two other Y‐family enzymes that are dispensable for SHM, human pols‐ι and ‐κ, copied a heteropolymeric DNA‐primed RNA template in vitro under conditions with substantial excesses of template. Repeated experiments gave highly reproducible results. The RT activity detected using one aliquot of human pol‐η was confirmed using a second sample from an independent source. Human DNA pols‐β and ‐µ, and T4 DNA polymerase repeatedly demonstrated no RT activity. Pol‐η was the most efficient RT of the Y‐family enzymes assayed but was much less efficient than an HIV‐RT standard in vitro. It is thus feasible that pol‐η acts as both a RNA‐ and a DNA‐dependent DNA polymerase in SHM in vivo, and that Y‐family RT activity participates in other mechanisms of physiological importance.

No Associations Between Telomere Length and Age-Sensitive Indicators of Physical Function in Mid and Later Life

Telomere length, which declines with age, has been hypothesized to act as an indicator of biological aging. If it fulfills this purpose, shorter telomere length should correlate with age-related loss of physical function, independent of age. In this cross-sectional Australian population study, the associations between peripheral blood leukocyte telomere length and age-sensitive indicators of physical function (lung function, blood pressure, and grip strength) were examined in two narrow age range cohorts aged 44-49 years (n = 351) and 64-70 years (n = 295). Telomere length was correlated with systolic blood pressure but only for women of the younger cohort and in the opposite direction to that expected (partial r = .181, p = .017). This evidence does not provide support for the hypothesis that telomere length is related to age-associated changes in physical function.

Pertussis toxin inhibits EGF-, phorbol ester- and insulin-stimulated DNA synthesis in BALBc3T3 cells: Evidence for post-receptor activation of Giα

The contribution of the GTP-binding protein, Gi, to EGF, phorbol dibutyrate (PdBu)-, and insulin-stimulated DNA synthesis was examined in BALB/c3T3 cells. Pertussis toxin inhibited DNA synthesis by each agonist, particularly at suboptimal agonist concentrations, but the inhibition could be partially overcome with higher agonist concentrations and combinations of these agonists. This suggested that (1) some, but not all, of the mitogenic signals for all three agonists were transduced by Gi (2) Gi may be activated by post-receptor mechanisms involving protein kinase C. Gi alpha-specific antibodies and ADP-ribosylation by pertussis toxin using 32P-NAD each labelled a single protein band, representing one or more species of Gi alpha. Pertussis toxin treatment increased the synthesis of Gi alpha. These results are discussed in relation to possible direct effects of Gi alpha on nuclear control during division.

Phosphorylation of septin 3 on Ser-91 by cGMP-dependent protein kinase-I in nerve terminals.

The septins are a family of GTPase enzymes required for cytokinesis and play a role in exocytosis. Among the ten vertebrate septins, Sept5 (CDCrel-1) and Sept3 (G-septin) are primarily concentrated in the brain, wherein Sept3 is a substrate for PKG-I (cGMP-dependent protein kinase-I) in nerve terminals. There are two motifs for potential PKG-I phosphorylation in Sept3, Thr-55 and Ser-91, but phosphoamino acid analysis revealed that the primary site is a serine. Derivatization of phosphoserine to S-propylcysteine followed by N-terminal sequence analysis revealed Ser-91 as a major phosphorylation site. Tandem MS revealed a single phosphorylation site at Ser-91. Substitution of Ser-91 with Ala in a synthetic peptide abolished phosphorylation. Mutation of Ser-91 to Ala in recombinant Sept3 also abolished PKG phosphorylation, confirming that Ser-91 is the major site in vitro. Antibodies raised against a peptide containing phospho-Ser-91 detected phospho-Sept3 only in the cytosol of nerve terminals, whereas Sept3 was located in a peripheral membrane extract. Therefore Sept3 is phosphorylated on Ser-91 in nerve terminals and its phosphorylation may contribute to the regulation of its subcellular localization in neurons.

Social organisation of thornbill-dominated mixed-species flocks using social network analysis

Mixed-species associations are a widespread phenomenon, comprising interacting heterospecific individuals which gain predator, foraging or social benefits. Avian flocks have traditionally been classified as monolithic species units, with species-wide functional roles, such as nuclear, active, passive, or follower. It has also been suggested that flocks are mutualistic interactions, where niches of participating species converge. However the species-level perspective has limited previous studies, because both interactions and benefits occur at the level of the individual. Social network analysis provides a set of tools for quantitative assessment of individual participation. We used mark-resighting methods to develop networks of nodes (colour-marked individuals) and edges (their interactions within flocks). We found that variation in flock participation across individuals within species, especially in the buff-rumped thornbill, encompassed virtually the entire range of variation across all individuals in the entire set of species. For example, female, but not male, buff-rumped thornbills had high network betweenness, indicating that they interact with multiple flocks, likely as part of a female-specific dispersal strategy. Finally, we provide new evidence that mixed-species flocking is mutualistic, by quantifying an active shift in individual foraging niches towards those of their individual associates, with implications for trade-off between costs and benefits to individuals derived from participating in mixed-species flocks. This study is, to our knowledge, the first instance of a heterospecific social network built on pairwise interactions.