Src interacts with dynamin and synapsin in neuronal cells

Abstract

The nonreceptor tyrosine kinase Src is expressed at a high level in cells that are specialized for regulated secretion, such as the neuron, and is concentrated on secretory vesicles or at the site of exocytosis. To investigate the possibility that Src may play a role in regulating membrane traffic, we searched for neuronal proteins that will interact with Src. The SH3 domain of Src, but not that of the splice variant N-Src, bound to three proteins from mouse synaptosomes or PC12 cells: dynamin, synapsin Ia, and synapsin Ib. Dynamin and the synapsins coprecipitated with Src from PC12 cell extracts, and they colocalized with a subset of Src in the PC12 cell by immunofluorescence. Neither dynamin nor the synapsins were phosphorylated by Src, suggesting that the interaction of these proteins serves to direct the kinase activity of Src toward other proteins in the vesicle population. In immunoprecipitates containing Src and dynamin, the clathrin adaptor protein alpha-adaptin was also found. The association of Src and synapsin suggests a role for Src in the life cycle of the synaptic vesicle. The identification of a complex containing Src, dynamin, and alpha-adaptin indicates that Src may play a more general role in membrane traffic as well.

Figure 1

Mouse synaptosome proteins that interact with the SH3 domain of Src. (A) Mouse brain synaptosomes were lysed, resolved by electrophoresis in a polyacrylamide gel, and used in an overlay assay with the indicated GST fusion proteins (GST, GST alone; SH3, GSTSrcSH3; NSH3, GSTNSrcSH3). (B) The same preparation was Western blotted with antibodies to dynamin and synapsins Ia and Ib.

Figure 2

Immunofluorescence patterns of Src, dynamin, and the synapsins in PC12 cells. PC12 cells were induced to differentiate with nerve growth factor and were prepared for immunofluorescence with antibodies to the following proteins: Src (A and B), dynamin (C and D), synapsin I (E and F), the transferrin receptor (G), and pyruvate kinase (H). (Final magnification for A, C, and E is ×100; for B, D, and F it is ×250; and for G and H it is ×50.)

Figure 3

Coprecipitation of dynamin and synapsin with Src from PC12 cells. (A and B) PC12 cells were lysed and prepared for immunoprecipitation with normal rabbit serum (NRS) and antibodies to Src and dynamin. Whole cell lysate (WCL) and the immunoprecipitates were Western blotted for dynamin or the synapsins as indicated. (C) Lysates from PC12 cells, fibroblasts from src−/− mice, and 3T3 cells overexpressing Src were immunoprecipitated with NRS and a Src antibody, and Western blotted for dynamin.

Figure 4

α-Adaptin coprecipitates with Src from PC12 cells. PC12 cells were lysed and prepared by immunoprecipitation with NRS or Src antibody. Whole cell lysate (WCL) and the immunoprecipitates were Western blotted with antibodies to α-adaptin (A) and γ-adaptin (B).

Figure 5

Association of dynamin with other Src family members. PC12 cells were lysed and prepared for immunoprecipitation with NRS and antibodies to Src, Fyn, and Yes. The immunoprecipitates were Western blotted with an antibody to dynamin (A) and with a pan-Src-family antiserum (B).