Figure 1.
ITSN1 and ITSN2 are associated in cells.
(A) Characterization of the anti-ITSN2 antibodies produced. Nontransfected HEK293 cells or cells expressing GFP-ITSN1-S or GFP-ITSN2-S were lysed 24 h post-transfection. Total cell lysates were resolved by SDS-PAGE with subsequent immunoblot analysis using the anti-ITSN2 antibodies obtained or the commercially available anti-Intersectin/ESE-1. (B) HEK293 cells were plated on coverslips and fixed. Endogenous ITSNs were stained with anti-ITSN1/m and anti-ITSN2 antibodies, and visualized with Alexa 488-conjugated or Texas Red-conjugated secondary antibodies, respectively. Higher magnification of the area enclosed by a rectangle is shown below each image. Scale bar: 10 µm. (C) Lysates of HEK293 cells were subjected to immunoprecipitation using anti-ITSN2 antibodies (left panels). Conversely immunoprecipitation was performed using rabbit polyclonal antibodies against ITSN1 (right panels). In both cases immunoprecipitated material was probed with antibodies specific to ITSN1 and ITSN2. Representative data from three independent experiments are shown. IP, immunoprecipitation; NRS, normal rabbit serum; WB, Western blotting.
Figure 2.
Comparison of ligand-binding sites of the SH3 domains of ITSN1 and ITSN2.
(A) Alignment of protein sequences of the human ITSNs SH3 domains. The alignment was generated using the ClustalW algorithm. Identical residues are highlighted in red, and homologous amino acids are shown in red letters. Amino acid residues that form ligand-binding sites of the SH3 domains are indicated by boxes, mismatches within these regions are shown by boxes of different colours. Amino acid residues marked with an asterisk are not conserved in ITSN1 and ITSN2 orthologues. (B) Merged structures are shown for each SH3 domain pair in blue and grey for ITSN1 and ITSN2, respectively. Amino acid residues that form ligand-binding sites of the SH3 domains and are identical in ITSNs or not conserved in orthologues are depicted as lines. Divergent residues involved in ligand binding of the ITSN1 and ITSN2 SH3 domains are depicted in yellow and green, respectively. Amino acid mismatches regarded as favoured are not shown in colour. SP, specificity pocket of the SH3 domain ligand-binding site.
Table 1.
Differences within ligand-binding sites of the SH3 domains of human ITSN1 and ITSN2.
Figure 3.
ITSNs have common SH3 domain interactors.
The GST-fused SH3 domains of ITSN1 and ITSN2 were bacterially expressed and affinity purified. The GST-SH3 domains or GST alone (control) immobilized on glutathione beads were incubated with lysate of mouse brain (for dynamin 1 and SOS1) or HEK293 expressing Omni-synaptojanin 1 (SYNJ1), Omni-Reps1, Omni-N-WASP, Omni-CdGAP, Omni-Sema6A, Flag-SPRY2, Flag-Numb or Myc-POB1. Bound proteins were separated by SDS-PAGE and detected by immunoblotting with antibodies against dynamin 1 (DNM1), SOS1 or tags. GST-SH3 domains of ITSN1 were used as positive control of binding. GST-fused proteins were visualized by Coomassie or Ponceau S staining. The experiments were performed at least twice, and representative data are shown. WB, Western blotting.
Figure 4.
ITSN2 is phosphorylated on tyrosine residues.
(A) Line graphs show the total number of tyrosine residues in ITSN1 and ITSN2 in a range of vertebrate taxonomic groups. Amino acid sequences of ITSN1 and ITSN2 orthologues in fish (Danio rerio, Oreochromis niloticus and Tetraodon nigroviridis), amphibians (Xenopus tropicalis and Xenopus laevis), reptiles (Anolis carolinensis), birds (Gallus gallus, Meleagris gallopavo and Taeniopygia guttata), rodents (Mus musculus, Rattus norvegicus and Cricetulus griseus) and primates (Homo sapiens, Pan troglodita and Pongo abelii) were obtained from the NCBI protein database (http: //ncbi.nlm.nih.gov). The average number of tyrosine residues per taxon is plotted, and error bars represent standard deviations. (B) Schematic representation of ITSNs domain organization and distribution of conserved tyrosine residues in ITSN1 and ITSN2 of various vertebrates. Tyrosine residues located within domain and interdomain regions are shown as dark blue and light blue boxes, respectively. Tyrosine residues that could be phosphorylated according to phosphoproteomic data (www.phosphosite.org) are indicated by green circles. The number above each circle indicates the position of the residue in the amino acid sequence of human ITSN. Abbreviations are defined as follows: Homo, Homo sapiens; Mus, Mus musculus; Gallus, Gallus gallus; Anolis, Anolis carolinensis; Xenopus, Xenopus laevis. (C) Lysates of growing HEK293, HeLa, MCF-7 and MBA-MB-231 cells were subjected to immunoprecipitation with rabbit polyclonal anti-ITSN2 (left panel) or anti-ITSN1 (right panel) antibodies. Normal rabbit serum (NRS) was used as control. Precipitated proteins were analyzed by Western blotting using anti-phosphotyrosine antibodies and antibodies against ITSN1 or ITSN2. The experiments were repeated at least twice with reproducible results. IP, immunoprecipitation; WB, Western blotting.
Figure 5.
Tyrosine phosphorylation of ITSN2 depends on mitogenic stimulation and clathrin-mediated endocytosis.
HeLa cells were maintained with 10% FBS, starved for 16 h or starved for 16 h and treated with 20 ng/ml of EGF for 10 and 20 min (A, B). For inhibition of endocytosis, serum-starved cells were treated with 30 µM chlorpromazine (CPZ) for 20 min followed by stimulation with EGF (20 ng/ml) in the presence of chlorpromazine (C). Extracts of cells were subjected to immunoprecipitation with antibodies against ITSN2 (A, C) and ITSN1 (B). Normal rabbit serum (NRS) was used as control. The precipitated proteins were analyzed by Western blotting with anti-phosphotyrosine, anti-ITSN2 and anti-ITSN1 antibodies. The ratio of pITSN2/ITSN2 intensity values was calculated, the data are presented as mean ±SEM (n = 3);*P<0.05,**P<0.01,***P<0.001. IP, immunoprecipitation; WB, Western blotting.
Figure 6.
ITSN2 is specifically recognized by the SH2 domains of signaling proteins.
HeLa cells were maintained in DMEM supplemented with 10% FBS, starved overnight and stimulated with 20 ng/ml EGF. Bacterially expressed and affinity purified GST-SH2 domains of Grb2, Crk, Itk, Fgr, Fyn, Abl1, PI3KR1, PLCg1 or GST alone (control) were bound to glutathione beads and used as bait to pull down ITSN proteins. For in vitro binding assays GST-fused proteins were incubated with lysates of EGF-treated (left panel) and growing (right panel) HeLa cells. Bound proteins were analyzed by Western blotting using antibodies against ITSN2. GST-fused proteins were visualized by Coomassie staining. The experiments were repeated three times with reproducible results. PI3KR1-N and PI3KR1-C correspond to the N- and C-terminal SH2 domains of PI3KR1, respectively. WB, Western blotting.