The Lnk/SH2B adaptor provides a fail-safe mechanism to establish the Insulin receptor-Chico interaction

Background Insulin/insulin-like growth factor signalling (IIS) has been described as one of the major pathways involved in growth control and homeostasis in multicellular organisms. Whereas its core components are well established, less is known about the molecular functions of IIS regulators. The adaptor molecule Lnk/SH2B has been implicated in IIS but the mechanism by which it promotes IIS activity has remained enigmatic. Results In this study, we analyse genetic and physical interactions among InR, Chico and Lnk in Drosophila tissues. FRET analysis reveals in vivo binding between all three molecules. Genetically, Lnk acts upstream of Chico. We demonstrate that Chico’s plasma membrane localisation is ensured by both its PH domain and by the interaction with Lnk. Furthermore, Lnk is able to recruit an intracellular InR fragment to the membrane. Conclusions Thus, by acting as a scaffolding molecule that ensures InR and Chico enrichment at the membrane, Lnk provides a fail-safe mechanism for IIS activation.ISSN:1478-811

The WW domain protein Kibra acts upstream of Hippo in Drosophila

SummaryThe conserved Hippo kinase pathway plays a pivotal role in organ size control and tumor suppression by restricting proliferation and promoting apoptosis. Whereas the function of the core kinase cascade, consisting of the serine/threonine kinases Hippo and Warts, in phosphorylating and thereby inactivating the transcriptional coactivator Yorkie is well established, much less is known about the upstream events that regulate Hippo signaling activity. The FERM domain proteins Expanded and Merlin appear to represent two different signaling branches that feed into the Hippo pathway. Signaling by the atypical cadherin Fat may act via Expanded, but how Merlin is regulated has remained elusive. Here, we show that the WW domain protein Kibra is a Hippo signaling component upstream of Hippo and Merlin. Kibra acts synergistically with Expanded, and it physically interacts with Merlin. Thus, Kibra predominantly acts in the Merlin branch upstream of the core kinase cascade to regulate Hippo signaling

Effects of starvation on hemolymph proteome.

<p>The magnitude versus amplitude (MA) plot shows the log2 fold change of the expression of the identified <i>D. melanogaster</i> proteins in the starved versus fed condition against the mean normalized spectral count. The top 10% differentially expressed proteins are highlighted, including 50 up-regulated proteins (red dots) and 22 down-regulated proteins (green dots). Protein identifiers are shown for selected proteins discussed in the text. Unambiguous protein identifications by class 1a, 1b, and 3a peptides are shown as full circles. Protein groups identified by class 2a or 2b peptides (which unambiguously imply a gene model) are shown as open circles, ambiguous identifications by 3b peptides are shown as open diamonds (the respective identifiers are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067208#pone.0067208.s002" target="_blank">Table S2</a>).</p

Starvation protocol and developmental effects.

<p>(A) At 65 hours after egg deposition (AED), half of the larvae were transferred to starvation medium (20% sucrose). Twenty-four hours later, hemolymph from fed and starved larvae was collected for deep shotgun proteomics. Developmental timing of ecdysone titer, larval stages L2 and L3, acquisition of critical weight, wandering behavior and pupation under optimal conditions is indicated as well. Numbers indicate time in hours AED. (B) Size of fed and starved larvae at time of hemolymph collection. (C) At 65 hours AED, larvae were either shifted to starvation medium or further maintained on rich medium followed by analysis of the fraction of pupae over time (n = 278 fed and 141 starved) (D) Size of pupae formed by either fed or starved larvae. Bars = 0.5 mm.</p

Starvation-associated protein abundance changes in larval hemolymph.

a)<p>Change in transcript levels during development in rich medium was estimated based on expression profiling data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067208#pone.0067208-Burmester2" target="_blank">[77]</a>. For transcript levels around the time when starvation was started (early) the values observed at L2 and L3/12hours were averaged. For transcript levels around the time of hemolymph collection (late) the values at L3/puff stage 1–2 were used. The given values correspond to log2(early/late).</p

Characterization of the larval hemolymph proteome.

<p>(A) Workflow of the analyses. Hemolymph samples from fed and starved larvae were digested in solution. Tryptic peptides were separated by isoelectric focusing for complexity reduction. Peptides were analyzed using microcapillary liquid chromatography–electrospray ionization–tandem MS (µLC-ESI-MS/MS). SEQUEST spectral search was performed for peptide spectrum matching. (B) Venn diagram illustrating the number of gene models detected in hemolymph from fed and starved larvae, respectively.</p

Abundance of larval serum proteins.

<p>Hemolymph was isolated from fed (f) and starved (s) larvae (see Fig. 1). Proteins in samples of 10, 3.3, 1.7 or 1 µl hemolymph were resolved by SDS-PAGE and stained with Coomassie Blue. The position of the major larval serum proteins (LSPs) is indicated by an arrowhead. Position and size (kDa) of molecular weight markers (m) are indicated on the right side.</p

Summary of identified spectra, peptides, proteins and estimated FDR levels.

a)<p>According to our peptide classification scheme <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067208#pone.0067208-Qeli1" target="_blank">[38]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067208#pone.0067208-Grobei1" target="_blank">[46]</a>, class 1a peptides unambiguously identify a single unique protein sequence encoded by a unique transcript. Class 1b peptides also unambiguously identify a unique protein sequence encoded by several transcripts of the same gene model with identical coding region and differences in the 5′ and/or 3′ untranslated regions. Class 2a peptides identify a subset and class 2b peptides all protein sequences encoded by a gene model. Class 3a peptides unambiguously identify one protein sequence, but this sequence could be encoded by several gene models from distinct loci (e.g. histones). Finally, class 3b peptides can be derived from different protein sequences encoded by several gene models from distinct loci and have the least information content.</p>b)<p>For protein groups identified by class 2a or 2b peptides (a gene model identification) all possible protein accessions are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067208#pone.0067208.s001" target="_blank">Table S1</a>.</p>c)<p>The minimal number of additional protein identifications by 3b peptides is shown.</p>d)<p>Based on the total hits in target and decoy databases (DB), the FDR was estimated at the spectra, peptide and protein level.</p