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.Cells undergo rearrangement of the cortical cytoskeleton, a submembranous actin filament (F-actin)-based network, during a variety of cellular processes including differentiation, proliferation, migration, and oncogenic transformation (9, 10, 43, 67). The cortical cytoskeleton not only controls cell morphology but is also involved in transmitting signals between the plasma membrane and intracellular compartments (7,8,38). A large body of evidence indicates that small GTP-binding proteins, tyrosine kinases, and serine/threonine kinases play a pivotal role in regulating the dynamic structure of the cortical cytoskeleton (30, 33). The molecular mechanisms by which these enzymes regulate cortical actin polymerization and reorganization are currently unclear. Identification of actin-associated targets of these enzymes is important for unveiling signaling pathways correlated with the cortical F-actin remodeling.Cortactin, an F-actin-binding substrate for the nonreceptor tyrosine kinase pp60 src (39,78), is distinguished by the presence of several potential signaling sequence motifs (63,77,79). The N-terminal half of the protein contains six and a half tandem repeats of a 37-amino-acid sequence. The repeat region is required and sufficient for efficient association with F-actin as assessed by in vitro cosedimentation assays (78). The role of this region in mediating the interaction with...

Insulin receptor substrate of 53 kDa links postsynaptic shank to PSD‐95

“…Naisbitt et al 1999). IRSp53.1 and GKAP/SAPAPs would therefore act in concert to provide a stable double bridge between the PSD-95 proteins, which anchor transmitter receptors, and shanks, which are more deeply buried within the PSD (Valtschanoff and Weinberg 2001) and link the whole complex to cytoskeletal associated proteins such as cortactin (Du et al 1998) and fodrin (Böckers et al 2001). An important difference between IRSp53.1 and GKAP/ SAPAPs, however, is that the interaction of IRSp53.1 with shank1 is regulated by the small G-protein cdc42 (Soltau et al 2002).…”

“…The function of the PSD appears to be to physically link postsynaptic receptors to signalling molecules, and to provide stable attachment of the receptors to the actin-based cytoskeleton of the dendritic spine. Shank proteins (shank1-3, also known as SSTRIP, ProSAP, synamon or CortBP) constitute another group of postsynaptic scaffolding molecules which link transmitter receptors (Kreienkamp et al 2000;Naisbitt et al 1999;Yao et al 1999;Zitzer et al 1999) to actin binding proteins (Du et al 1998;Boeckers et al 2001;Okamoto et al 2001). Overexpression of shank1 in neurones leads to enhanced maturation of dendritic spines .…”

Signaling mechanisms that regulate actin‐based motility processes in the nervous system

“…Another potential mechanism for Arp2/3 to be activated in neurons involves the actin binding protein cortactin. Cortactin is found in the lamellipodia and ruffles of growth cones (Du et al 1998). In non-neuronal cells, cortactin binds and activates Arp2/3 via its N-terminal domain, causing both formation and stabilization of actin branches (Uruno et al 2001;Weaver et al 2001).…”

Cortactin: coupling membrane dynamics to cortical actin assembly

“…Northern blot analysis indicates that cortactin is expressed in nearly all mammalian tissues (Miglarese et al, 1994;Du et al, 1998). One notable exception is in hematopoietic cells, where the related protein HS1 is expressed in place of cortactin (Kitamura et al, 1989).…”

“…Boxes indicate the positions of conserved proline residues. Sequences are from Du et al (1998) Takahisa et al (1996) (ZO-1), Ohoka and Takai (1998) (CBP90), and Cook et al (1994) (Dynamin) speci®c ZAP-70 kinase, containing two amino-terminal SH2 domains and a carboxyl-terminal catalytic domain (Chan et al, 1992;Muller et al, 1994). Syk is activated following ®brinogen ligation to a IIB b 3 integrin, and requires the intact cytoplasmic domains of both integrin subunits .…”