Phospho‐site mapping,genetic and in planta activation studies reveal key aspects of the different phosphorylation mechanisms involved in activation of SnRK2s

Snf1‐related protein kinases 2 (SnRK2s) are major positive regulators of drought stress tolerance. The kinases of this family are activated by hyperosmotic stress, but only some of them are also responsive to abscisic acid (ABA). Moreover, genetic evidence has indicated the ABA‐independence of SnRK2 activation in the fast response to osmotic stress. Although phosphorylation was demonstrated to be crucial for the activation or activity of the kinases of both subgroups, different phosphorylation mechanisms were suggested. Here, using one kinase from each subgroup (SnRK2.6 and SnRK2.10), two phosphorylation sites within the activation loop were identified by mass spectrometry after immunoprecipitation from Arabidopsis cells treated by ABA or osmolarity. By site‐directed mutagenesis, the phosphorylation of only one of the two sites was shown to be necessary for the catalytic activity of the kinase, whereas both sites are necessary for the full activation of the two SnRK2s by hyperosmolarity or ABA. Phosphoprotein staining together with two‐dimensional PAGE followed by immunoblotting indicated distinct phosphorylation mechanisms of the two kinases. While SnRK2.6 seems to be activated through the independent phosphorylation of these two sites, a sequential process occurs in SnRK2.10, where phosphorylation of one serine is required for the phosphorylation of the other. In addition, a subgroup of protein phosphatases 2C which interact and participate in the regulation of SnRK2.6 do not interact with SnRK2.10. Taken together, our data bring evidence for the involvement of distinct phosphorylation mechanisms in the activation of SnRK2.6 and SnRK2.10, which may be conserved between the two subgroups of SnRK2s depending on their ABA‐responsiveness.     

An enriched structural kinase database to enable kinome‐wide structure‐based analyses and drug discovery

The development of a kinase structural database, the kinase knowledge base (KKB), is described. It covers all human kinase domain structures that have been deposited in the Protein Data Bank. All structures are renumbered using a common scheme, which enables efficient cross‐comparisons and multiple queries of interest to the kinase field. The common numbering scheme is also used to automatically annotate conserved residues and motifs, and conformationally classify the structures based on the DFG‐loop and Helix C. Analyses of residue conservation in the ATP binding site using the full human‐kinome–sequence alignment lead to the identification of a conserved hydrogen bond between the hinge region backbone and a glycine in the specificity surface. Furthermore, 90% of kinases are found to have at least one stabilizing interaction for the hinge region, which has not been described before.     

Selection for individual recognition and the evolution of polymorphic identity signals in Polistes paper wasps

Individual recognition (IR) requires individuals to uniquely identify their social partners based on phenotypic variation. Because IR is so specific, distinctive phenotypes that stand out from the crowd facilitate efficient recognition. Over time, the benefits of unique appearances are predicted to produce a correlation between IR and phenotypic variation. Here, we test whether there is an association between elevated phenotypic polymorphism and IR in paper wasps. Previous work has shown that Polistes fuscatus use variable colour patterns for IR. We test whether two less variable wasp species, Polistes dominulus and Polistes metricus, are capable of IR. As predicted, neither species is capable of IR, suggesting that highly variable colour patterns are confined to Polistes species with IR. This association suggests that elevated phenotypic variation in taxa with IR may be the result of selection for identity signals rather than neutral processes. Given that IR is widespread among social taxa, selection for identity signalling may be an underappreciated mechanism for the origin and maintenance of polymorphism.     

Ontogenetic and spatial variation in size‐selective mortality of a marine fish

Although body size can affect individual fitness, ontogenetic and spatial variation in the ecology of an organism may determine the relative advantages of size and growth. During an 8‐year field study in the Bahamas, we examined selective mortality on size and growth throughout the entire reef‐associated life phase of a common coral‐reef fish, Stegastes partitus (the bicolour damselfish). On average, faster‐growing juveniles experienced greater mortality, though as adults, larger individuals had higher survival. Comparing patterns of selection observed at four separate populations revealed that greater population density was associated with stronger selection for larger adult size. Large adults may be favoured because they are superior competitors and less susceptible to gape‐limited predators. Laboratory experiments suggested that selective mortality of fast‐growing juveniles was likely because of risk‐prone foraging behaviour. These patterns suggest that variation in ecological interactions may lead to complex patterns of lifetime selection on body size.     

Pollinator shifts and the loss of style polymorphism in Narcissus papyraceus (Amaryllidaceae)

Darwin proposed that the driving force for the evolution of style polymorphisms is the promotion of cross‐pollination between style morphs, through accurate placement of pollen on the pollinator’s body. This hypothesis has received much attention, but the effect of different pollinators in the fitness of morphs remains poorly understood. Narcissus papyraceus is a style dimorphic species (long ‐L‐ and short ‐S‐ styled) with isoplethic (1 : 1) and L‐monomorphic populations, mainly visited by long‐tongued (LT) nocturnal and short‐tongued (ST) diurnal pollinators, respectively. We studied natural female fertility of morphs, and assessed the role of diurnal and nocturnal pollinators. We also quantified female fertility of the morphs in experimental populations with different morph ratio, exposed to predominately long‐ or short‐tongued pollinators. We found that with LT pollinators, both morphs were successfully pollinated in all morph ratio conditions, suggesting that these insects could be involved in maintenance of the polymorphism, although other factors may also play a role. However, with ST pollinators, S‐plants displayed less fertility than L‐plants, and mating among L‐plants was favoured, implying that the polymorphism is lost. These results underscore the role of pollinators on variations in style polymorphism.     

A decrease in cyclin B1 levels leads to polyploidization in DNA damage‐induced senescence

Adriamycin, an anthracycline antibiotic, has been used for the treatment of various types of tumours. Adriamycin induces at least two distinct types of growth repression, such as senescence and apoptosis, in a concentration‐dependent manner. Cellular senescence is a condition in which cells are unable to proliferate further, and senescent cells frequently show polyploidy. Although abrogation of cell division is thought to correlate with polyploidization, the mechanisms underlying induction of polyploidization in senescent cells are largely unclear. We wished, therefore, to explore the role of cyclin B1 level in polyploidization of Adriamycin‐induced senescent cells. A subcytotoxic concentration of Adriamycin induced polyploid cells having the features of senescence, such as flattened and enlarged cell shape and activated β‐galactosidase activity. In DNA damage‐induced senescent cells, the levels of cyclin B1 were transiently increased and subsequently decreased. The decrease in cyclin B1 levels occurred in G2 cells during polyploidization upon treatment with a subcytotoxic concentration of Adriamycin. In contrast, neither polyploidy nor a decrease in cyclin B1 levels was induced by treatment with a cytotoxic concentration of Adriamycin. These results suggest that a decrease in cyclin B1 levels is induced by DNA damage, resulting in polyploidization in DNA damage‐induced senescence.     

Mapping interactions of gastric inhibitory polypeptide with GIPR N‐terminus using NMR and molecular dynamics simulations

Glucose‐dependent insulinotropic polypeptide (gastric inhibitory polypeptide, or GIP), a 42‐amino acid incretin hormone, modulates insulin secretion in a glucose‐concentration‐dependent manner. Its insulinotropic action is highly dependent on glucose concentration that surmounts the hypoglycemia side effects associated with current therapy. In order to develop a GIP‐based anti‐diabetic therapy, it is essential to establish the 3D structure of the peptide and study its interaction with the GIP receptor (GIPR) in detail. This will give an insight into the GIP‐mediated insulin release process. In this article, we report the solution structure of GIP(1–42, human)NH₂ deduced by NMR and the interaction of the peptide with the N‐terminus of GIPR using molecular modelling methods. The structure of GIP(1–42, human)NH₂ in H₂O has been investigated using 2D‐NMR (DQF‐COSY, TOCSY, NOESY, ¹H‐¹³C HSQC) experiments, and its conformation was built by constrained MD simulations with the NMR data as constraints. The peptide in H₂O exhibits an α‐helical structure between residues Ser8 and Asn39 with some discontinuity at residues Gln29 to Asp35; the helix is bent at Gln29. This bent gives the peptide an ‘L’ shape that becomes more pronounced upon binding to the receptor. The interaction of GIP with the N‐terminus of GIPR was modelled by allowing GIP to interact with the N‐terminus of GIPR under a series of decreasing constraints in a molecular dynamics simulation, culminating with energy minimization without application of any constraints on the system. The canonical ensemble obtained from the simulation was subjected to a detailed energy analysis to identify the peptide–protein interaction patterns at the individual residue level. These interaction energies shed some light on the binding of GIP with the GIPR N‐terminus in a quantitative manner. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Perspectives for the use of structural information and chemical genetics to develop inhibitors of Janus kinases

Gain-of-function mutations in the genes encoding Janus kinases have been discovered in various haematologic diseases. Jaks are composed of a FERM domain, an SH2 domain, a pseudokinase domain and a kinase domain, and a complex interplay of the Jak domains is involved in regulation of catalytic activity and association to cytokine receptors. Most activating mutations are found in the pseudokinase domain. Here we present recently discovered mutations in the context of our structural models of the respective domains. We describe two structural hotspots in the pseudokinase domain of Jak2 that seem to be associated either to myeloproliferation or to lymphoblastic leukaemia, pointing at the involvement of distinct signalling complexes in these disease settings. The different domains of Jaks are discussed as potential drug targets. We present currently available inhibitors targeting Jaks and indicate structural differences in the kinase domains of the different Jaks that may be exploited in the development of specific inhibitors. Moreover, we discuss recent chemical genetic approaches which can be applied to Jaks to better understand the role of these kinases in their biological settings and as drug targets.     

Les isoformes neuronales des tyrosines kinases Src,Fyn et Lck : rôle particulier de la p56lckN dans la survie neuronale

The two main tyrosine kinases (TK) in the brain are p60Src and p59Fyn, expressed as specific isoforms (p60SrcNI, p60SrcNI + NII and p59fynB). They play a pivotal role in some major processes such as neuronal growth and myelinisation. Another member of this TK family was then reported in brain, the p56lck. Its name Lck (lymphocyte cell kinase) indicates its cellular specificity observed initially, so its presence in the brain was intriguing. But no further studies were performed to understand its role in brain until recent clinical studies on Alzheimer patients’ brains. One study reveals a decreased p56lck level in the brains of these patients while another study shows an association between one peculiar SNP (single nucleotide polymorphism) of the lck gene and some cases of the disease. These new data prompt us to reinvestigate the original biochemical data and to confront them with the present knowledge. This analysis suggests some hypothesis concerning both the Lck protein expressed in the brain (rather an isoform than the lymphocyte protein itself) and its role (to maintain the neuronal survival presumably by protecting them from inflammation, the main pathway that leads to neuron degeneracy).