Stimulation of the catalytic activity of the tyrosine kinase Btk by the adaptor protein Grb2: Part 3
Data files
Apr 25, 2023 version files 8.66 GB
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13_1nMBtk_LAT_SOSpr_Grb2_stream640_20ms_tirf561_20ms_1mW_em100_1p5x_10_MMStack.ome.tif_kept_stack.tif
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13_1nMBtk_LAT_SOSpr_Grb2_stream640_20ms_tirf561_20ms_1mW_em100_1p5x_11_MMStack.ome.tif_kept_stack.tif
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13_Btk_LATphase_10.xml
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13_Btk_LATphase_11.xml
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13_Btk_LATphase_6.xml
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13_Btk_LATphase_7.xml
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13_Btk_LATphase_8.xml
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13_Btk_LATphase_9.xml
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13_Btk_LATphase.tif
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13_LAT_SOSpr_Grb2_stream640_20ms_tirf561_20ms_1mW_em100_1p5x_6_MMStack.ome.tif_kept_stack.tif
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13_LAT_SOSpr_Grb2_stream640_20ms_tirf561_20ms_1mW_em100_1p5x_7_MMStack.ome.tif_kept_stack.tif
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13_LAT_SOSpr_Grb2_stream640_20ms_tirf561_20ms_1mW_em100_1p5x_8_MMStack.ome.tif_kept_stack.tif
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13_LAT_SOSpr_Grb2_stream640_20ms_tirf561_20ms_1mW_em100_1p5x_9_MMStack.ome.tif_kept_stack.tif
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14_1nMBtk_LAT_SOSpr_Grb2_stream640_20ms_tirf561_20ms_1mW_em100_1p5x_1_MMStack.ome.tif_kept_stack.tif
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14_1nMBtk_LAT_SOSpr_Grb2_stream640_20ms_tirf561_20ms_1mW_em100_1p5x_2_MMStack.ome.tif_kept_stack.tif
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14_1nMBtk_LAT_SOSpr_Grb2_stream640_20ms_tirf561_20ms_1mW_em100_1p5x_3_MMStack.ome.tif_kept_stack.tif
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14_1nMBtk_LAT_SOSpr_Grb2_stream640_20ms_tirf561_20ms_1mW_em100_1p5x_4_MMStack.ome.tif_kept_stack.tif
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14_1nMBtk_LAT_SOSpr_Grb2_stream640_20ms_tirf561_20ms_1mW_em100_1p5x_5_MMStack.ome.tif_kept_stack.tif
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14_1nMBtk_LAT_SOSpr_Grb2_stream640_20ms_tirf561_20ms_1mW_em100_1p5x_6_MMStack.ome.tif_kept_stack.tif
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14_1nMBtk_LAT_SOSpr_Grb2_stream640_20ms_tirf561_20ms_1mW_em100_1p5x_7_MMStack.ome.tif_kept_stack.tif
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14_Btk_LATphase_1.xml
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14_Btk_LATphase_2.xml
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14_Btk_LATphase_3.xml
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14_Btk_LATphase_4.xml
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14_Btk_LATphase_5.xml
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14_Btk_LATphase_6.xml
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14_Btk_LATphase_7.xml
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15_1nMBtk_LAT_stream640_20ms_tirf561_20ms_1mW_em100_1p5x_2_MMStack.ome.tif_kept_stack.tif
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15_1nMBtk_LAT_stream640_20ms_tirf561_20ms_1mW_em100_1p5x_3_MMStack.ome.tif_kept_stack.tif
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15_1nMBtk_LAT_stream640_20ms_tirf561_20ms_1mW_em100_1p5x_5_MMStack.ome.tif_kept_stack.tif
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15_1nMBtk_LAT_stream640_20ms_tirf561_20ms_1mW_em100_1p5x_6_MMStack.ome.tif_kept_stack.tif
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15_Btk_LAT_2.xml
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15_Btk_LAT_3.xml
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15_Btk_LAT_5.xml
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15_Btk_LAT_6.xml
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16_1nMBtk_LAT_Grb2_stream640_20ms_tirf561_20ms_1mW_em100_1p5x_1_MMStack.ome.tif_kept_stack.tif
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16_1nMBtk_LAT_Grb2_stream640_20ms_tirf561_20ms_1mW_em100_1p5x_2_MMStack.ome.tif_kept_stack.tif
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16_1nMBtk_LAT_Grb2_stream640_20ms_tirf561_20ms_1mW_em100_1p5x_3_MMStack.ome.tif_kept_stack.tif
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16_1nMBtk_LAT_Grb2_stream640_20ms_tirf561_20ms_1mW_em100_1p5x_4_MMStack.ome.tif_kept_stack.tif
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16_1nMBtk_LAT_Grb2_stream640_20ms_tirf561_20ms_1mW_em100_1p5x_5_MMStack.ome.tif_kept_stack.tif
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16_1nMBtk_LAT_Grb2_stream640_20ms_tirf561_20ms_1mW_em100_1p5x_6_MMStack.ome.tif_kept_stack.tif
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16_Btk_LAT_Grb2_1.xml
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16_Btk_LAT_Grb2_2.xml
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16_Btk_LAT_Grb2_3.xml
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16_Btk_LAT_Grb2_4.xml
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16_Btk_LAT_Grb2_5.xml
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16_Btk_LAT_Grb2_6.xml
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README.md
Abstract
The Tec-family kinase Btk contains a lipid-binding Pleckstrin homology and Tec homology (PH-TH) module connected by a proline-rich linker to a “Src module”, an SH3-SH2-kinase unit also found in Src-family kinases and Abl. We showed previously that Btk is activated by PH-TH dimerization, which is triggered on membranes by the phosphatidyl inositol phosphate PIP3, or in solution by inositol hexakisphosphate (IP6) (Wang et al. 2015, https://doi.org/10.7554/eLife.06074). We now report that the ubiquitous adaptor protein growth-factor-receptor-bound protein 2 (Grb2) binds to and substantially increases the activity of PIP3-bound Btk on membranes. Using reconstitution on supported-lipid bilayers, we find that Grb2 can be recruited to membrane-bound Btk through interaction with the proline-rich linker in Btk. This interaction requires intact Grb2, containing both SH3 domains and the SH2 domain, but does not require that the SH2 domain be able to bind phosphorylated tyrosine residues – thus Grb2 bound to Btk is free to interact with scaffold proteins via the SH2 domain. We show that the Grb2-Btk interaction recruits Btk to scaffold-mediated signaling clusters in reconstituted membranes. Our findings indicate that PIP3-mediated dimerization of Btk does not fully activate Btk, and that Btk adopts an autoinhibited state at the membrane that is released by Grb2.
Methods
The dataset was collected By TIRF microscopy, and all imaging settings were included in the metadata for each TIFF file. Description of the microscope is included in the manuscript Materials and Methods. For each tiff, an associated set of single molecule tracks is included, processed via Trackmate (FIJI, Image J).
Usage notes
We recommend FIJI (Image J) for viewing the TIFF files. Tracking files can be viewed with any text editor that can open CSV files, though Vim and Excel are both convenient.