Kinetics of Src homology 3 domain association with the proline-rich domain of dynamins: specificity, occlusion, and the effects of phosphorylation.

Dynamin function is mediated in part through association of its proline-rich domain (PRD) with the Src homology 3 (SH3) domains of several putative binding proteins. To assess the specificity and kinetics of this process, we undertook surface plasmon resonance studies of the interaction between isolated PRDs of dynamin-1 and -2 ...
and several purified SH3 domains. Glutathione S-transferase-linked SH3 domains bound with high affinity (K(D) approximately 10 nm to 1 microm) to both dynamin-1 and -2. The simplest interaction appeared to take place with the amphiphysin-SH3 domain; this bound to a single high affinity site (K(D) approximately 10 nm) in the C terminus of dynamin-1 PRD, as predicted by previous studies. Binding to the dynamin-2 PRD was also monophasic but with a slightly lower affinity (K(D) approximately 25 nm). Endophilin-SH3 binding to both dynamin-1 and -2 PRDs was biphasic, with one high affinity site (K(D) approximately 14 nm) in the N terminus of the PRD and another lower affinity site (K(D) approximately 60 nm) in the C terminus of dynamin-1. The N-terminal site in dynamin-2 PRD had a 10-fold lower affinity for endophilin-SH3. Preloading of dynamin-1 PRD with the amphiphysin-SH3 domain partially occluded binding of the endophilin-SH3 domain, indicating overlap between the binding sites in the C terminus, but endophilin was still able to interact with the high affinity N-terminal site. This shows that more than one SH3 domain can simultaneously bind to the PRD and suggests that competition probably occurs in vivo between different SH3-containing proteins for the limited number of PXXP motifs. Endophilin-SH3 binding to the high affinity site was disrupted when dynamin-1 PRD was phosphorylated with Cdk5, indicating that this site overlaps the phosphorylation sites, but amphiphysin-SH3 binding was unaffected. Other SH3 domains showed similarly complex binding characteristics, and substantial differences were noted between the PRDs from dynamin-1 and -2. For example, SH3 domains from c-Src, Grb2, and intersectin bound only to the C-terminal half of dynamin-2 PRD but to both the N- and C-terminal portions of dynamin-1 PRD. Thus, differential binding of SH3 domain-containing proteins to dynamin-1 and -2 may contribute to the distinct functions performed by these isoforms.
Mesh Terms:
Adaptor Proteins, Signal Transducing, Adaptor Proteins, Vesicular Transport, Amino Acid Motifs, Amino Acid Sequence, Animals, Binding Sites, Cyclin-Dependent Kinase 5, Cyclin-Dependent Kinases, Dynamin I, Dynamin II, Dynamins, Endocytosis, GRB2 Adaptor Protein, Glutathione Transferase, Kinetics, Models, Chemical, Molecular Sequence Data, Nerve Tissue Proteins, Phosphorylation, Polymerase Chain Reaction, Proline, Protein Binding, Protein Isoforms, Protein Structure, Tertiary, Protein-Tyrosine Kinases, Rats, Recombinant Proteins, Sequence Homology, Amino Acid, Signal Transduction, Substrate Specificity, Surface Plasmon Resonance, Time Factors, src Homology Domains
J. Biol. Chem.
Date: Jun. 17, 2005
Download Curated Data For This Publication
134574
Switch View:
  • Interactions 11