Interactions between the Fyn SH3‐domain and adaptor protein Cbp/PAG derived ligands, effects on kinase activity and affinity

Abstract

Tyrosine phosphorylation is one of the key regulatory protein modifications in multicellular organisms and tightly controls and coordinates a wide range of cellular responses such as growth, metabolism, tissue repair, migration and apoptosis [1][2][3]. Phosphorylation modulates enzymatic activity as well as creating new binding sites for the recruitment of active molecules into signalling complexes, and assists in building dynamic networks for the transduction of information from the extracellular environment to intracellular signalling pathways. Accurate and specific processing of information is vital to maintaining cellular homeostasis, and errors in signal transduction pathways are linked to a range of diseases such as cancer, autoimmunity and diabetes [4,5].Tyrosine phosphorylation is a reversible modification, regulated by protein tyrosine kinases (PTKs) and phosphatases (PTPs) [6,7]. PTKs of the Src kinase family employ a well-conserved modular arrangement of interaction domains in the regulation of kinase Csk-binding protein ⁄ phosphoprotein associated with glycosphingolipidenriched domains is a transmembrane adaptor protein primarily involved in negative regulation of T-cell activation by recruitment of C-terminal Src kinase (Csk), a protein tyrosine kinase which represses Src kinase activity through C-terminal phosphorylation. Recruitment of Csk occurs via SH2-domain binding to PAG pTyr317, thus, the interaction is highly dependent on phosphorylation performed by the Src family kinase Fyn, which docks onto PAG using a dual-domain binding mode involving both SH3-and SH2-domains of Fyn. In this study, we investigated Fyn SH3-domain binding to 14-mer peptide ligands derived from Cbp ⁄ PAG-enriched microdomains sequence using biochemical, biophysical and computational techniques. Interaction kinetics and dissociation constants for the various ligands were determined by SPR. The local structural impact of ligand association has been evaluated using CD, and molecular modelling has been employed to investigate details of the interactions. We show that data from these investigations correlate with functional effects of ligand binding, assessed experimentally by kinase assays using full-length PAG proteins as substrates. The presented data demonstrate a potential method for modulation of Src family kinase tyrosine phosphorylation through minor changes of the substrate SH3-interacting motif.Abbreviations Cbp ⁄ PAG, Csk binding protein ⁄ phosphoprotein associated with glycosphingolipids-enriched microdomains; Csk, C-terminal Src kinase; GST, glutathione S-transferase; PPII, polyproline type II; PRD, proline-rich domain; PTK, protein tyrosine kinase; SFK, Src family kinase; SH2, Src homology 2; SH3, Src homology 3; TCR, T-cell receptor.

Disruption of Fyn SH3 Domain Interaction with a Proline-Rich Motif in Liver Kinase B1 Results in Activation of AMP-Activated Protein Kinase

“…Of note, expression of these point mutants did not affect Fyn protein expression since signal detected was identical for each mutant (Fig. 2A, lower panel) although the W119A mutation did result in a reduced SDS-PAGE mobility, as already observed by others [22]. Consistent with our previous report, Fyn-WT bound to LKB1 (Fig.…”

“…S1A). These specific residues for mutagenesis were chosen as alanine substitution of the tryptophan 119 (W119) was shown to abolish SH3 function [20], [21], [22] and the substitution of the arginine 176 (R176) of Fyn SH2 domain with a lysine residue (R176K) reduces phosphotyrosine binding [23]. Similarly, we introduced a single alanine substitution in the LKB1 proline-rich (P328A) motif (Fig.…”

Phosphoprotein Associated with Glycosphingolipid-Enriched Microdomains Differentially Modulates Src Kinase Activity in Brain Maturation

“…Again, we did not find any difference in the levels of pY215 between the brains of wild type and Pag1 -/- mice. Finally, SFK activity can also be enhanced by stabilizing an open/active conformation of the Src kinases [48], [49]. We show here that the binding of Fyn to PAG involves the first proline-rich region as well as Y105 in PAG, thus confirming the findings of Solheim et al [50].…”

A Computational Model for Early Events in B Cell Antigen Receptor Signaling: Analysis of the Roles of Lyn and Fyn

“…We set p 11 = 0.6 μM −1 s −1 and p 13 = 30 s −1 , where p 11 is the forward rate constant for SFK SH3 domain binding to a PRS in PAG1 and p 13 is the reverse rate constant. These estimates yield an affinity lower than that measured by Solheim et al (18), but the affinity can still be considered typical for an SH3 domain-PRS interaction (58). …”