Figure 1.
Structures of the modeled Tip-STAT complexes.
(A) Tip and STAT structural domains (not to scale). (Top) From amino-terminus to carboxy-terminus of Tip the four tyrosine residues are displayed, the major phosphotyrosine residues Y114 and Y127 are also highlighted, along with the Lck binding domains; transmembrane region (TM). (Bottom) Schematic arrangement of STAT domains; N terminal domain (NTD), Coiled coil domain (CCD), DNA binding domain (DBD), Src homology 2 (SH2) domain, Linker domain (LD), Y tyrosine that stabilizes antiparallel conformation upon phosphorylation, and Transactivation domain (TD). (B) Multiple sequence alignment of human STAT SH2 domain sequences. Human STAT SH2 domains were aligned using CLUSTAL Omega [47] and displayed with Vektor NTi (version 9). The consensus is based on residues present in more than 75% of sequences; fully conserved residues are colored in yellow, those present in more than 75% in blue, homologous amino acids are colored in green (C) Structures of the modeled Tip-STAT complexes highlighting the overall complex geometry and the main interacting regions. (top left) Tip114-STAT3, (top right) Tip127-STAT3, (bottom left) Tip114-STAT6, and (bottom right) Tip127-STAT6. The STAT SH2 domain is shown in grey space-filled presentation. The three main interacting regions are defined and colored as follows: region I (binds ligand residues pY, pY+1; orange), region II (binds pY+2, pY+3; red), region III (binds pY+4 to pY+8; cyan). The Tip ligand is shown in stick presentation and key residues belonging to different interaction regions are labeled.
Figure 2.
Details of the Tip-STAT interaction in the four investigated complexes.
(A) Tip114-STAT3, (B) Tip127-STAT3, (C) Tip114-STAT6, and (D) Tip127-STAT6. For each complex, three panels are shown that highlight the interactions formed in region I, II, and III. The backbone topology of STAT is shown as cyan ribbon and the interacting residues are shown in ball-and-stick presentation. Tip those residues that bind to STAT are shown in stick presentation and labeled in italics. Polar interactions (hydrogen bonds, salt-bridges) are indicated as dotted red lines.
Table 1.
Tip-Stat interactions detected in the molecular dynamics simulations.
Figure 3.
Electrostatic surface of the Stat SH2 domains and interaction sites with the Tip ligand.
(A) Tip114-STAT3, (B) Tip127-STAT3, (C) Tip114-STAT6, and (D) Tip127-STAT6. Positively and negatively charged regions of the STAT surface are colored in blue and red, respectively, and the residues that interact with Tip are labeled in black. The position of the bound residues from Tip (labeled in green) is indicated by a black circle.
Figure 4.
STAT3 and STAT6 factors are activated in Tip and Lck transfected cells, and the transcriptional activity STAT6 mediated by Tip.
Active STAT, phosphorylated at the respective tyrosine residue required for dimerization and activation, was detected by antibodies recognizing only the phosphorylated domain. (A) STAT3 phosphorylated at Y705; pSTAT6, Tip (HA) and GAPDH were detected by multicolor fluorescent immunoblot without stripping of the membrane; STAT3 was detected in a parallel experiment using the same cell protein lysate (B) STAT6 phosphorylated at Y641. STAT6, pSTAT6, Tip(HA) and GAPDH were detected by multicolor fluorescent immunoblot without stripping of the membrane. A representative example of several independent immunoblotting experiments is shown. (C) For each experiment dataset (triplicate values) firefly luciferase activity was normalized to the activity of renilla luciferase transfection control, the mean then mean and SD values were calculated and are shown for a representative experiment.
Figure 5.
Specific inhibition of Src-Tyrosine kinase reduces STAT6 activation.
(A) Tip, Lck and STAT6 transfected 293T cells were treated with PP2 or the non-active control substance PP3 (10 µM in DMSO each). STAT6, pSTAT6, Tip and GAPDH were detected by fluorescent immunoblotting on the same membrane. (B) The ratio of pSTAT6 to total STAT6 was normalized to the GAPDH signal. The ratio of STAT6-pY641 (clone 18, detected with anti mouse Dylight-647), total STAT6 (polyclonal rb-anti-STAT6 (M-20) with anti-rabbit-Dylight-488) was calculated and normalized to the GAPDH loading control (biotinylated goat anti-GAPDH/Strepavidin-Alexa-555).
Figure 6.
Viruses expressing Tip deficient in STAT6 activation fail to growth transform human T cells.
(A) Pairs of cell lines from identical donors are shown. Values of three different HVS transformed cell lines (2 donors #1587, 1611) are shown relative to the minimal transcript level detected for each cell line. (B). Human cord blood lymphocytes were infected with recombinant HVS strain C488 viruses, and maintained in medium supplemented with Interleukin-2. Cell growth was monitored and growth curves were calculated as described previously [28]. YYYY - recombinant wild type HVS in which Tip has four tyrosine residues Y94, Y114, Y127 and Y155; YFYY - mutant Y114F (tyrosine Y114 mutated to phenylalanine F); YYFY- mutant Y127F (Y127 mutated to F); YFFY- mutant Y114F and Y127F; FYYF - mutant Y94F and Y155F; FFFF - all four tyrosines mutated.