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

ITSNs form homo and heterodimers.

<p>Bimolecular fluorescence complementation was used to assess the dimerization of ITSN1 and ITSN2 isoforms in COS cells. A–E. The BiFC emission from the reconstituted Venus fluorophore was pseudocolored green (left panel). CFP (pseudocolored red, right panel) was co-transfected to mark transfected cells and only CFP-positive cells were imaged BiFC. A. ITSN1-S forms homodimers. VN-ITSN1-S was co-expressed with VC-ITSN1-S. B. The short and long isoforms of ITSN1 form heterodimers. VN-ITSN1-S was co-expressed with VC-ITSN1-L. C. ITSN2-S forms homodimers. VN-ITSN2-S was co-expressed with VC-ITSN2-S. D. ITSN1 and ITSN2 heterodimerize. VN-ITSN1-S was co-expressed with VN-ITSN2-S. E. As a negative control for BiFC, VN-ITSN1-S was co-expressed with a non-specific peptide fused to VC (VC-pep). F. For comparison with the BiFC results, YFP-ITSN1-S was expressed in COS cells. Shown are two different panels of positive cells. The fluorescence patterns shown in all panels (A–F) are representative of localization observed throughout the plate. These results are representative of at least three independent experiments. Note: white scale bar represent 10? m.</p

ITSN1 interacts with components of the Rab5 GTPase pathway.

<p>A. ITSN1 interacts with Rabaptin5. Endogenous ITSN1 (red) in HeLa cells co-localizes with myc-tagged Rabaptin5 (green). Antibodies to Rabaptin5 did not detect the endogenous protein. Overlap of proteins is shown in yellow. Regions of overlap were extracted from the image using ImageJ. B. Endogenous ITSN1 (green) co-localizes with endogenous Rab5 (red) in COS cells. Overlap of proteins is shown in yellow. Regions of overlap were extracted from the image using ImageJ. C. ITSN1-S forms a complex with Rab5 on early endosomes. HeLa cells were transfected with VN-Rab5 and VC-ITSN1-S and then stained with antibody to EEA-1 (red). The BiFC complex of Rab5 and ITSN1-S is pseudocolored green. Overlap of the BiFC complex with EEA1 is shown in yellow. Regions of overlap were extracted from the image using ImageJ. Note: white scale bars represent 20? m. The fluorescence patterns shown in all panels are representative of localization observed throughout the plate.</p

ITSN1 interacts with components of the Arf6 GTPase pathways.

<p>A. ITSN1 interacts with Arf6 as well as its effector Arfaptin2. BiFC was used to analyze the interaction of ITSN1S with components of the Arf6 pathway. VN-ITSN1-S was co-expressed in COS cells with VC-Arf6 (top panels), VC-Arfaptin2 (middle panels), or VC-pep (bottom panels) as a negative control. The emission from the reconstituted Venus fluorophore was pseudocolored green. CFP (pseudocolored red) was co-transfected to mark transfected cells and only CFP-positive cells were imaged. Scale bar, 20 um. The fluorescence pattern shown in all panels are representative of localization observed throughout the plate. B. ITSN1 forms a trimolecular complex with Arf6 and Arfaptin2 in COS cells. Top panels: VN-ITSN1-S and VC-Arf6 were co-expressed in the absence of CFP-Arfaptin2 to demonstrate lack of bleed through of the BiFC signal into the CFP channel. Middle panels: CFP-Arfaptin2 was expressed alone to demonstrate lack of bleed through of the CFP signal into the BiFC channel. Bottom panels: VN-ITSN1-S, VC-Arf6, and CFP-Arfaptin2 were co-expressed in COS cells. The BiFC complex of VN-ITSN1-S and VC-Arf6 co-localizes with CFP-Arfaptin2. A region of overlap of the BiFC complex with CFP-Arf6 is enlarged in the inset in the bottom right panel. Note: white scale bar represents 20? m. The fluorescence pattern shown in all panels are representative of localization observed throughout the plate. C. VC-Arfaptin2 co-precipitates with ITSN1. HA-epitope tagged ITSN1-S or various domains of ITSN1 were co-expressed with VN-Arfaptin2 which contains a FLAG epitope tag. HA-immunoprecipitates were probed for either HA (top panel) or FLAG (bottom) to detect association of Arfaptin2. The CC region interacts most strongly with VN-Arfaptin2 consistent with the Y2H results (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036023#pone.0036023.s001" target="_blank">Tables S1</a>).</p