An extracellular receptor tyrosine kinase motif orchestrating intracellular STAT activation

The ErbB4 receptor isoforms JM-a and JM-b differ within their extracellular juxtamembrane (eJM) domains. Here, ErbB4 isoforms are used as a model to address the effect of structural variation in the eJM domain of receptor tyrosine kinases (RTK) on downstream signaling. A specific JM-a-like sequence motif is discovered, and its presence or absence (in JM-b-like RTKs) in the eJM domains of several RTKs is demonstrated to dictate selective STAT activation. STAT5a activation by RTKs including the JM-a like motif is shown to involve interaction with oligosaccharides of N-glycosylated cell surface proteins such as β1 integrin, whereas STAT5b activation by JM-b is dependent on TYK2. ErbB4 JM-a- and JM-b-like RTKs are shown to associate with specific signaling complexes at different cell surface compartments using analyses of RTK interactomes and super-resolution imaging. These findings provide evidence for a conserved mechanism linking a ubiquitous extracellular motif in RTKs with selective intracellular STAT signaling.


Data analysis
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Data
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Life sciences study design
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Sample size
For image analysis FiJi (Schindelin, J. et al. Fiji: An open-source platform for biological-image analysis. Nature Methods 9, 676-682 (2012), versions 1.50 and 1.51) was used. For quantification of Western blot analyses, Image Studio Lite, version 5.2 (LI-COR Biosciences, NE, USA) was used. For live-imaging analysis, Incucyte ZOOM 2018b (Sartorius, Germany) software was used. The study was conducted with cell lines, which is why the traditional sense of sample size does not apply. For quantitative studies, the amount of analyzed samples was determined by how many replicates were needed to gain a significant P-value (under 0.05) for observed differences.

Antibodies
Antibodies used Data were excluded from analyses due to technical concerns. In live-imaging experiments, cell plate wells with a significantly different confluence in the beginning of the experiments were excluded. Western blot bands with smeared wells or unfortunate air bubbles or very low signal against the background were excluded. In immunofluorescence imaging experiments, cells that had highly irregular morphology against other cells in the slide were excluded. Experiments with failed positive and/or negative controls were excluded.
The findings were replicated in different cell lines to ensure reproducibility. All experiments were replicated at least twice in the exact same setting to ensure reproducibility. Different approaches to show the same phenomenon were applied to ensure reproducibility. All attempts at replication that were not successful were due to technical concerns.
Randomization of samples to group allocation is not relevant to the study. The experimental model used was cell lines. By using cell lines the background of each treatment is identical.
Blinding to group allocation during collection or analysis was not feasible since most experiments were conducted independently by one investigator and the sample order needed to be recorded for accurate interpretation of results (western analyses, immunofluorescence analyses). The experiments conducted with more than one investigator were blinded by not divulging the details of the samples to the second investigator running the analyses (mass spectrometry experiments). Goat anti-rabbit IgG Abberior STAR 635, Abberior, Catalog no: 2-0012-002-7, Lot no: 14012016Cw, 1:100 dilution for immunofluorescence analyses; Abberior STAR 580, goat anti-mouse IgG, Abberior, Catalog no: 2-0002-005-1, Lot no: 09072018CW/JR, 1:100 dilution for immunofluorescence analyses;

October 2018
Mouse monoclonal anti-"1-integrin (K-20), Validated for mmunofluorescence at manufacturer's website for human, immunofluorescence staining pattern similar to the one in manufacturer's datasheet for green monkey cells Rabbit monoclonal anti-"4-integrin (D8P6C), Validated for Western analysis at manufacturer's website for human, In Fig. 4D Fig. 4C produced a band of correct size in green monkey cells that was recognized also with a pSTAT3 antibody Rabbit monoclonal anti-Phospho-Stat3 (Tyr705) (D3A7) XP®,Cell Signaling, #9145; Validated for western analysis at manufacture's sheet for human. In Fig. 4C produced a band of correct size in green monkey cells that was recognized also with a STAT3 antibody Mouse monoclonal Phospho-Stat5 (Tyr694) (14H2) , Validated for Western analysis at manufacturer's website for human and mouse, In Fig. 3D the antibody recognized a clear band of correct size that was enriched by immunoprecipitation with a STAT5b antibody Rabbit polyclonal anti-Phospho-STAT5 (Tyr694) , Validated for Western analysis at manufacturer's website for human and mouse, In Fig. 3D, 4A, 4C, S8C and S10A-B the antibody recognized a clear band of correct size that was enriched by immunoprecipitation with STAT5a and STAT5b antibodies and recognized by other STAT5 antibodies in Western analysis in green monkey cell samples Rabbit polyclonal anti-STAT5a (L-20) , Validated for Western analysis, immunoprecipitation and immunofluorescence at manufacturer's datasheet for human and mouse Mouse monoclonal anti-STAT5a (C-6) , Validated for Western analysis at manufacturer's website for human and mouse, was validated in house for immunoprecipitation in green monkey samples ( Fig. S10A-C) by detecting enrichment of a band that was detected with other STAT5a and pSTAT5 antibodies in Western analysis Rabbit polyclonal anti-STAT5a (Ab 780) , Validated for Western analysis at manufacturer's website for human, was validated in house for immunoprecipitation in green monkey samples (Fig. S8C) by detecting enrichment of a band that was detected with other STAT5a and pSTAT5 antibodies in Western analysis Rabbit polyclonal anti-STAT5a Prestige Antibodies , Validated for Western analysis at manufacturer's website for human, was validated in house for immunoprecipitation in and green monkey samples (Fig. 4A) by detecting enrichment of a band that was detected with other STAT5a and pSTAT5 antibodies in Western analysis (more data available upon request) Rabbit polyclonal anti-STAT5 (C-17) (recognizes STAT5b) , Validated for Western analysis, immunoprecipitation and immunofluorescence at manufacturer's datasheet for human and mouse, was validated in house with Western analysis of cells treated with STAT5a-and STAT5b-specific siRNAs to selectively recognize STAT5b (data available upon request), in Fig. 2B,4C,