Mutations in the effector region of Ras disrupt PI3KC2β binding.

<p>(A) Point mutations in the effector region of Ras12V disrupt interactions with specific Ras targets. (B) VC-tagged PI3KC2β was co-transfected with either VN-tagged Ras17N, 17N/69N, or one the effector mutants in the background of Ras17N/69N. BiFC signal is pseudo-colored green. Effector mutations that disrupt Class I PI3K binding to Ras12V disrupt Class II PI3K binding to Ras17N/69N. CFP (red) was used as a transfection control. (C) Graph represents the average fluorescence intensity per cell ± S.E.M. from at least three independent experiments. (*p = 0.02). (D) Western blot analysis demonstrates equal expression of all constructs. (E) Mutation of Thr392 to Asp or Lys379 to Ala in full-length PI3KC2β disrupts interaction with Ras17N. The ΔRBD mutant was also included as a negative control. Graph represents the average of three independent experiments (*p<0.05).</p

Biological activity of PI3KC2β versus Raf RBDs.

<p>(A) Expression of the Raf-RBD but not the PI3KC2β-RBD or GST alone inhibited EGF-stimulation of a Gal-Elk reporter assay <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045360#pone.0045360-Adams1" target="_blank">[23]</a>. GST-RBDs were expressed equally. GST alone migrated at a faster rate and was not visible in this image. Results represent the average relative activation ± S.E.M. from at least three independent experiments. (*p<0.05 compared to unstimulated GST, **p<0.01 compared to EGF stimulated GST). (B). PI3KC2β-RBD dose-dependently inhibits the effect of Ras17N on Src-mediated transformation. NIH/3T3 cells were transfected with 100 ng of SrcY527F expression construct in the presence or absence of Ras17N. Co-expression of the PI3KC2β-RBD reverses the inhibitory effect of Ras17N on Src transformation whereas the Raf-RBD does not. In contrast, expression of the Raf-RBD alone, but not the PI3KC2β-RBD, significantly inhibited Src-mediated transformation. The results represent the average relative focus forming activity ± S.E.M. from three independent experiments performed in triplicate. Asterisks denote samples that were significantly different from Src alone (*p<0.05).</p

Ras is necessary for ITSN1 activation of AKT.

<p>(A) YFP-ITSN1 overexpression stimulates AKT activation as measured by levels of phospho-AKT as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045360#pone.0045360-Das1" target="_blank">[22]</a>. Co-expression of Ras17N or Ras17N/69N inhibits this response. Results represent the average fold activation of AKT ± S.E.M. from at least three independent experiments. (B) Western blot analysis of AKT activation from a representative experiment. Top two panels represent Western blots of HA immunoprecipitates of cell lysates to assess AKT activation as described in the Materials and Methods section. The lower three panels indicate the level of expression of ITSN1, Ras, and actin (a loading control).</p

Ras forms a complex with PI3KC2β.

<p>(A) PI3KC2β preferentially interacts with Ras17N. VC-tagged PI3KC2β was co-transfected with one of the following VN-tagged Ras constructs: WT, 61L, 17N, or 17N/69N. BiFC signal (green) demonstrates that PI3KC2β interacted with Ras17N >17N/69N >WT >61L. CFP (red) was used as a transfection control. The graph represents the average fluorescence intensity per cell ± S.E.M. from at least three independent (*p<0.05). Western blot analysis demonstrates equivalent expression of all constructs. (B) PI3KC2β does not interact with Ras12V. VC-tagged PI3KC2β was co-transfected with one of the following VN-tagged Ras constructs: WT,17N, or 12V. BiFC signal (green) demonstrates that PI3KC2β interacted with Ras17N >WT >12V. CFP (red) was used as a transfection control. The graph represents the average fluorescence intensity per cell ± S.E.M. from at least three independent experiments (*p<0.05). Western blot analysis demonstrates expression of all constructs (size bar  = 20 μm).</p

Binding of PI3KC2β-RBD to Ras.

<p>(A) Nucleotide loaded Ras does not directly interact with the RBD of PI3KC2β. Ras-GDP or Ras-GTPγS were incubated with GST-Raf-RBD, GST-PI3KC2β-RBD or GST alone as a negative control. Bound proteins were then analyzed by Western blot with a Ras antibody. The RBD of Raf specifically bound Ras-GTPγS. Neither GST-PI3KC2β-RBD nor GST alone interacted with Ras-GDP or Ras-GTPγS. Top panel, Ras bound to GST proteins. Bottom panels, input amounts of proteins. (B) Nucleotide-free Ras was generated in vitro as described and then tested for binding to the various GST proteins as in (A). GST-PI3KC2β-RBD directly binds nucleotide-free Ras while little association was seen with the GST-Raf-RBD or GST alone. Panels are same as in A. (C) Repeat of (B) except nucleotide (1 mM) was present during the binding reaction. Panels are same as in (A). (D) Addition of nucleotide (1 mM) does not disrupt pre-bound PI3KC2β-RBD- nucleotide-free Ras. GST-PI3KC2β-RBD was first bound to nucleotide-free Ras. Following binding, the complex was incubated with 1 mM GDP or GTPγS at RT for 30 min and then washed with buffer. Bound proteins were then analyzed as in (A).</p

Ras, PI3KC2β, and ITSN1 co-localize on intracellular vesicles.

<p>(A) VN-Ras and VC-PI3KC2β (green) were co-transfected with CFP-ITSN1 (red) into COS cells. a. The PI3KC2β-Ras BiFC complex co-localizes with ITSN, represented by yellow in the overlay panel; b. The VN-Ras and VC-PI3KC2β YFP signal (green) does not bleed into the CFP channel; c. The CFP-ITSN1 signal does not bleed into the YFP channel (size bars  = 20 μm). Note: the differences in signal strength of the BiFC signal in (a) vs (b) are due to a lower power setting for the laser in (b) so that pixel intensities can be accurately quantified and are not saturated. In (a) a higher laser power was used to illustrate the punctate localization of the Ras-PI3KC2β complex throughout the cell. (B) Ras interaction with PI3KC2β is disrupted by deletion of the RBD (ΔRBD) but not by mutation of the Pro-rich, ITSN1 binding sites (PRD-PA). The graph represents the average fluorescence intensity per cell ± S.E.M. from at least three independent experiments (*p<0.05, WT vs ΔRBD, PRD-PA vs ΔRBD). Western blot analysis demonstrates equal expression of all constructs (size bars  = 20 μm).</p

ITSN1 and Ras form a BiFC complex.

<p>(A) VC-tagged ITSN1 was co-transfected with one of the following VN-tagged Ras constructs: WT, 61L, 17N, or 17N/69N. ITSN1 formed a complex (green) with Ras17N >WT >17N69N >61L. CFP (red) was used as a transfection control. (B) The graph represents the average fluorescence intensity per cell ± S.E.M. from at least three independent experiments (*p<0.05, **p<0.01). (C) A Western blot was performed to demonstrate equal expression of all constructs. (size bar  = 20 μm).</p

Model for ITSN1-Ras-PI3KC2β pathway.

<p>Growth factor stimulation of receptor tyrosine kinases leads to the recruitment of the Grb2-Sos complex resulting in dissociation of GDP from Ras. We propose that PI3KC2β competes for binding this transient nucleotide-free (nf) Ras trapping it in the nucleotide-free state and preventing GTP loading. In addition, binding of nucleotide-free Ras to PI3KC2β inhibits its lipid kinase activity. The PI3KC2β-Ras complex may then translocate to distal sites such as early endosomes (EE) where ITSN1 then binds to PI3KC2β leading to the release of nucleotide-free Ras and activation of the lipid kinase activity of PI3KC2β. In addition, once released from the ITSN1-PI3KC2β complex, Ras binds GTP resulting in Ras activation and recruitment of effectors, e.g., Raf, Class I PI3Ks, etc. Although the above model describes a potential role for nf-Ras in ITSN1 and PI3KC2β function, we propose that nf-Ras may be regulated by additional proteins besides these molecules. Our findings also raise the possibility that the nucleotide-free forms of other GTPases may play a similar role in cell signaling.</p

Intersectin (ITSN) Family of Scaffolds Function as Molecular Hubs in Protein Interaction Networks

<div><p>Members of the intersectin (ITSN) family of scaffold proteins consist of multiple modular domains, each with distinct ligand preferences. Although ITSNs were initially implicated in the regulation of endocytosis, subsequent studies have revealed a more complex role for these scaffold proteins in regulation of additional biochemical pathways. In this study, we performed a high throughput yeast two-hybrid screen to identify additional pathways regulated by these scaffolds. Although several known ITSN binding partners were identified, we isolated more than 100 new targets for the two mammalian ITSN proteins, ITSN1 and ITSN2. We present the characterization of several of these new targets which implicate ITSNs in the regulation of the Rab and Arf GTPase pathways as well as regulation of the disrupted in schizophrenia 1 (DISC1) interactome. In addition, we demonstrate that ITSN proteins form homomeric and heteromeric complexes with each other revealing an added level of complexity in the function of these evolutionarily conserved scaffolds.</p> </div

ITSNs and DISC1 interact with common proteins.

<p>A. ITSN1 was identified as a binding partner for DISC1 by Y2H <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036023#pone.0036023-Morris1" target="_blank">[31]</a>. However, a number of additional connections between ITSN1/2 and DISC1 were identified in our Y2H screen. Proteins shaded yellow were identified as ITSN1 or 2 binding partners by Y2H. PPFIA2 is a member of the liprinα family of scaffolds (PPFIA1-4) which bind the LAR family of transmembrane protein phosphatases and are known to form heteromeric complexes with each other <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036023#pone.0036023-SerraPages1" target="_blank">[57]</a>. Thus, ITSNs and DISC1 may interact through a heteromeric complex of PPFIA2 and PPFIA4. PDE4 (red), a known DISC1 interacting protein, was examined for interaction with ITSN1. B. Interaction of ITSN1 with phosphodiesterase 4D3 isoform. HEK293T cells grown on coverslips were transfected with VN-ITSN1-S and either VC-PDE4D3 or VC-pep as a negative control. CFP was co-transfected to mark transfected cells and only CFP-positive cells were imaged. The fluorescence pattern shown is representative of localization observed throughout the plate. Note: white scale bar represent 10? m. C. ITSN1-S and PDE4D3 interaction was confirmed by co-immunoprecipitation. HEK293T cells were transfected with VSV-tagged PDE4D3 and either VN-ITSN1-S or CFP. ITSN1 and CFP were immunoprecipitated with FLAG antibody and the specific co-immunoprecipitation of PDE4D3 was determined by Western blot analysis with αVSV antibody. Input (bottom panel labeled “Cell lysate") shows the level of PDE4D3 was expressed equally in both cell lysates. Note that an empty lane was between the CFP and VN-ITSN1-S samples on the gel. The weak signal for PDE4D3 in that lane resulted from overflow of the sample. The top 3 panels in C were from the same membrane which had been separated into the indicated size ranges and probed with the indicated antibodies. CFP and VN-ITSN1-S each possess a FLAG epitope tag. Similar results were obtained from three independent experiments. D. ITSN1 SH3 domains bind DISC1. HEK293T cells were transfected with V5-tagged full length DISC1 (DISC1 FL; aa 1–854) or a truncated DISC1 (DISC1-TR; aa 1–597) corresponding to the deletion resulting from a translocation breakpoint that disrupts the DISC1 locus <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036023#pone.0036023-Millar2" target="_blank">[58]</a>. GST-SH3 (encoding all 5 SH3 domains) but not GST or GST-EH (encoding both EH domains) pulls down DISC1-FL but not DISC 1-TR (top panels). Expression of DISC1 proteins in cell lysates is shown in the Western blot of cell lysates with αV5 antibody (bottom panels). Input GST fusion proteins are indicated in the Coomassie-stained gel to the far right.</p