Biochemical and structural comparison of spleen tyrosine kinase interaction with integrin and the immunoreceptor tyrosine-based activation motif

Abstract

Spleen tyrosine kinase (Syk) is a non-receptor tyrosine kinase involved in many different signalling pathways activated by immunoreceptors and integrins. The Syk activation mediated by phosphorylated Immunoreceptor Tyrosine-based Activation Motif (pITAM) receptors involves Src homology 2 (SH2) regulatory domains leading to Syk structural rearrangements. Differently, integrin cytoplasmic domains bind to the regulatory domain of Syk and the interaction does not require the phosphorylation, but the molecular mechanism is still unknown. This work focussed on describing the mechanism of integrin-Syk interaction and on how integrins activate Syk. First, using a fluorescent-based kinetic assay we showed that the soluble integrin peptide had no detectable effect on Syk activity, whereas Syk was activated by clustered integrin peptides. This suggests that autophosphorylation is involved in the integrin-induced activation process. Clustered integrins had also a synergic effect combined with pITAM. Next, we wanted to elucidate the molecular mechanism of Syk-integrin interaction. Surface plasmon resonance techniques was used to measure the binding affinities of different Syk constructs towards the integrin peptide. The N-terminal SH2 domain (N-SH2) plus the interdomain A (IA) segment had a comparable binding affinity to the two SH2 domains, whereas N-SH2 was a weak binder. Using nuclear magnetic resonance spectroscopy, we solved the structure of the N-SH2 domain of Syk and analysed the changes on the chemical environments of the N-SH2 domain induced by the integrin peptide and pITAM. The results indicate that integrin and pITAM binding induce changes on the same surface of the protein. In line with this, surface plasmon resonance experiments showed that the integrin and pITAM peptides compete for binding to the regulatory domain of Syk. We compared the binding affinity of pITAM to N-SH2 with C-terminal SH2 (C-SH2) domains observed that N- SH2 had a very low binding affinity compared to C-SH2. These studies suggest that integrin and pITAM-mediated Syk activation are independent of each other but may cause synergistic activation responses at the cellular level.