Identification of a Novel Cortactin SH3 Domain-Binding Protein and Its Localization to Growth Cones of Cultured Neurons

ABSTRACT

Cortactin is an actin-binding protein that contains several potential signaling motifs including a Src homology 3 (SH3) domain at the distal C terminus. Translocation of cortactin to specific cortical actin structures and hyperphosphorylation of cortactin on tyrosine have been associated with the cortical cytoskeleton reorganization induced by a variety of cellular stimuli. The function of cortactin in these processes is largely unknown in part due to the lack of information about cellular binding partners for cortactin. Here we report the identification of a novel cortactin-binding protein of approximately 180 kDa by yeast two-hybrid interaction screening. The interaction of cortactin with this 180-kDa protein was confirmed by both in vitro and in vivo methods, and the SH3 domain of cortactin was found to direct this interaction. Since this protein represents the first reported natural ligand for the cortactin SH3 domain, we designated it CortBP1 for cortactin-binding protein 1. CortBP1 contains two recognizable sequence motifs within its C-terminal region, including a consensus sequence for cortactin SH3 domain-binding peptides and a sterile alpha motif. Northern and Western blot analysis indicated that CortBP1 is expressed predominately in brain tissue. Immunofluorescence studies revealed colocalization of CortBP1 with cortactin and cortical actin filaments in lamellipodia and membrane ruffles in fibroblasts expressing CortBP1. Colocalization of endogenous CortBP1 and cortactin was also observed in growth cones of developing hippocampal neurons, implicating CortBP1 and cortactin in cytoskeleton reorganization during neurite outgrowth.

ACKNOWLEDGMENTS

We thank G. Banker and H. Asmussen for in vitro-cultured rat hippocampal neurons, S. J. Parsons for 10T1/2neo and 5Hd47 cells, A. A. Lanahan for pPC86 and pPC97 vectors and the rat hippocampus cDNA library in pPC86, A. Hall for the pRK5myc vector, J. S. Morrow for the pcDNA3Flag2AB vector, and M. T. Harte for the GST-FAKcte expression vector. We also thank M. E. Cox for helpful discussions.

This work was supported by grants CA29243 and CA40042 from the DHHS-NCI and grant 4491 from the Council for Tobacco Research, Inc., to J. T. Parsons; S. A. Weed is supported by NIH postdoctoral fellowship 1 F32 CA75695-01; W.-C. Xiong is supported by NIH NRSA fellowship NS09918.

ADDENDUM IN PROOF

CortBP1 contains a single PDZ domain (amino acids 38 to 133) that has sequence similarity to the PDZ domains of PSD95, Disc large-1, and Z0-1.