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.
Solomaha
Elena
E
Department of Neurobiology, Pharamacology, and Physiology, University of Chicago, IL 60637, USA.
Szeto
Frances L
FL
Yousef
Mohammed A
MA
Palfrey
H Clive
HC
D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry; D033942:Adaptor Proteins, Vesicular Transport / Q000737:chemistry; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D000081247:CSK Tyrosine-Protein Kinase; D051360:Cyclin-Dependent Kinase 5; D018844:Cyclin-Dependent Kinases / Q000737:chemistry / Q000378:metabolism; D034284:Dynamin I / Q000737:chemistry; D034285:Dynamin II / Q000737:chemistry; D034281:Dynamins / Q000737:chemistry*; D004705:Endocytosis; D051380:GRB2 Adaptor Protein; D005982:Glutathione Transferase / Q000378:metabolism; D007700:Kinetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D011392:Proline / Q000737:chemistry*; D011485:Protein Binding; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000737:chemistry; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D013379:Substrate Specificity; D020349:Surface Plasmon Resonance; D013997:Time Factors; D018909:src Homology Domains; D019061:src-Family Kinases
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D051380:GRB2 Adaptor Protein; C495950:Grb2 protein, rat; D009419:Nerve Tissue Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; C115919:intersectin 1; C075637:amphiphysin; D011392:Proline; D005982:Glutathione Transferase; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; D051360:Cyclin-Dependent Kinase 5; C495907:Cdk5 protein, rat; D018844:Cyclin-Dependent Kinases; D034284:Dynamin I; D034285:Dynamin II; D034281:Dynamins