Functional dissection of the TBK1 molecular network

TANK-binding kinase 1 (TBK1) and inducible IκB-kinase (IKK-i) are central regulators of type-I interferon induction. They are associated with three adaptor proteins called TANK, Sintbad (or TBKBP1) and NAP1 (or TBKBP2, AZI2) whose functional relationship to TBK1 and IKK-i is poorly understood. We performed a systematic affinity purification-mass spectrometry approach to derive a comprehensive TBK1/IKK-i molecular network. The most salient feature of the network is the mutual exclusive interaction of the adaptors with the kinases, suggesting distinct alternative complexes. Immunofluorescence data indicated that the individual adaptors reside in different subcellular locations. TANK, Sintbad and NAP1 competed for binding of TBK1. The binding site for all three adaptors was mapped to the C-terminal coiled-coil 2 region of TBK1. Point mutants that affect binding of individual adaptors were used to reconstitute TBK1/IKK-i-deficient cells and dissect the functional relevance of the individual kinase-adaptor edges within the network. Using a microarray-derived gene expression signature of TBK1 in response virus infection or poly(I∶C) stimulation, we found that TBK1 activation was strictly dependent on the integrity of the TBK1/TANK interaction.     

Drosophila nemo promotes eye specification directed by the retinal determination gene network

Drosophila nemo (nmo) is the founding member of the Nemo-like kinase (Nlk) family of serine-threonine kinases. Previous work has characterized nmo's role in planar cell polarity during ommatidial patterning. Here we examine an earlier role for nmo in eye formation through interactions with the retinal determination gene network (RDGN). nmo is dynamically expressed in second and third instar eye imaginal discs, suggesting additional roles in patterning of the eyes, ocelli, and antennae. We utilized genetic approaches to investigate Nmo's role in determining eye fate. nmo genetically interacts with the retinal determination factors Eyeless (Ey), Eyes Absent (Eya), and Dachshund (Dac). Loss of nmo rescues ey and eya mutant phenotypes, and heterozygosity for eya modifies the nmo eye phenotype. Reducing nmo also rescues small-eye defects induced by misexpression of ey and eya in early eye development. nmo can potentiate RDGN-mediated eye formation in ectopic eye induction assays. Moreover, elevated Nmo alone can respecify presumptive head cells to an eye fate by inducing ectopic expression of dac and eya. Together, our genetic analyses reveal that nmo promotes normal and ectopic eye development directed by the RDGN.     

Functional dissection of the promoter of the pollen-specific gene NTP303 reveals a novel pollen-specific, and conserved cis-regulatory element

Regulatory elements within the promoter of the pollen-specific NTP303 gene from tobacco were analysed by transient and stable expression analyses. Analysis of precisely targeted mutations showed that the NTP303 promoter is not regulated by any of the previously described pollen-specific cis -regulatory elements. However, two adjacent regions from −103 to −86 bp and from −86 to −59 bp were shown to contain sequences which positively regulated the NTP303 promoter. Both of these regions were capable of driving pollen-specific expression from a heterologous promoter, independent of orientation and in an additive manner. The boundaries of the minimal, functional NTP303 promoter were determined to lie within the region −86 to −51 bp. The sequence AAATGA localized from −94 to −89 bp was identified as a novel cis -acting element, of which the TGA triplet was shown to comprise an active part. This element was shown to be completely conserved in the similarly regulated promoter of the Bp10 gene from Brassica napus encoding a homologue of the NTP303 gene.     

Characterization of the threonine-phosphatase of mouse eyes absent 3

Eyes absent (EYA) has tyrosine- and threonine-phosphatase activities in their C-terminal and N-terminal regions, respectively. Using various mutants of mouse EYA3, we showed that the 68-amino acid domain between positions 53 and 120 was necessary and sufficient for its threonine-phosphatase activity. Point mutations were then introduced, and residues Cys-56, Tyr-77, His-79, and Tyr-90 were essential for the EYA3s threonine-phosphatase. The 68-amino acid domain is not well conserved among the four mouse EYA members, but is evolutionally highly conserved in the orthologous EYA members of different species, suggesting that the threonine-phosphatase of EYA3 has a function distinct from that of the other EYAs.     

Functional dissection of a conserved motif within the pilus retraction protein PilT

PilT is a hexameric ATPase required for type IV pilus retraction in gram-negative bacteria. Retraction of type IV pili mediates intimate attachment to and signaling in host cells, surface motility, biofilm formation, natural transformation, and phage sensitivity. We investigated the in vivo and in vitro roles of each amino acid of the distinct, highly conserved C-terminal AIRNLIRE motif in PilT. Substitution of amino acids A288, I289, L292, and I293 as well as a double substitution of R290 and R294 abolished Pseudomonas aeruginosa PilT function in vivo, as measured by a loss of surface motility and phage sensitivity. When introduced into purified Aquifex aeolicus PilT, substitutions in the AIRNLIRE motif did not disrupt ATPase activity or oligomerization. In contrast, a K136Q substitution in the broadly conserved nucleotide binding motif prevented PilT function in vivo as well as in vitro. We propose that the AIRNLIRE motif forms an amphipathic alpha helix which transmits signals between a surface-exposed protein interaction site and the ATPase core of PilT, and we recognize a potential functional homology in other type II secretion ATPases.     

Structure of the retinal determination protein Dachshund reveals a DNA binding motif

The Dachshund proteins are essential components of a regulatory network controlling cell fate determination. They have been implicated in eye, limb, brain, and muscle development. These proteins cannot be assigned to any recognizable structural or functional class based on amino acid sequence analysis. The 1.65 A crystal structure of the most conserved domain of human DACHSHUND is reported here. The protein forms an alpha/beta structure containing a DNA binding motif similar to that found in the winged helix/forkhead subgroup of the helix-turn-helix family. This unexpected finding alters the previously proposed molecular models for the role of Dachshund in the eye determination pathway. Furthermore, it provides a rational framework for future mechanistic analyses of the Dachshund proteins in several developmental contexts.     

Functional protein network activation mapping reveals new potential molecular drug targets for poor prognosis pediatric BCP-ALL

Background

In spite of leukemia therapy improvements obtained over the last decades, therapy is not yet effective in all cases. Current approaches in Acute Lymphoblastic Leukemia (ALL) research focus on identifying new molecular targets to improve outcome for patients with a dismal prognosis. In this light phosphoproteomics seems to hold great promise for the identification of proteins suitable for targeted therapy.

Methodology/Principal Findings

We employed Reverse Phase Protein Microarrays to identify aberrantly activated proteins in 118 pediatric B-cell precursor (BCP)-ALL patients. Signal transduction pathways were assayed for activation/expression status of 92 key signalling proteins. We observed an increased activation/expression of several pathways involved in cell proliferation in poor clinical prognosis patients. MLL-rearranged tumours revealed BCL-2 hyperphosphorylation through AMPK activation, which indicates that AMPK could provide a functional role in inhibiting apoptosis in MLL-rearranged patients, and could be considered as a new potential therapeutic target. Second, in patients with poor clinical response to prednisone we observed the up-modulation of LCK activity with respect to patients with good response. This tyrosine-kinase can be down-modulated with clinically used inhibitors, thus modulating LCK activity could be considered for further studies as a new additional therapy for prednisone-resistant patients. Further we also found an association between high levels of CYCLIN E and relapse incidence. Moreover, CYCLIN E is more expressed in early relapsed patients, who usually show an unfavourable prognosis.

Conclusions/Significance

We conclude that functional protein pathway activation mapping revealed specific deranged signalling networks in BCP-ALL that could be potentially modulated to produce a better clinical outcome for patients resistant to standard-of-care therapies.