Stimulation of the catalytic activity of the tyrosine kinase Btk by the adaptor protein Grb2: Part 2

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.