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...
Sec3p is a component of the exocyst complex that tethers secretory vesicles to the plasma membrane at exocytic sites in preparation for fusion. Unlike all other exocyst structural genes, SEC3 is not essential for growth. Cells lacking Sec3p grow and secrete surprisingly well at 25 degrees C; however, late markers of secretion, such as the vesicle marker Sec4p and the exocyst subunit Sec8p, localize more diffusely within the bud. Furthermore, sec3Delta cells are strikingly round relative to wild-type cells and are unable to form pointed mating projections in response to alpha factor. These phenotypes support the proposed role of Sec3p as a spatial landmark for secretion. We also find that cells lacking Sec3p exhibit a dramatic defect in the inheritance of cortical ER into the bud, whereas the inheritance of mitochondria and Golgi is unaffected. Overexpression of Sec3p results in a prominent patch of the endoplasmic reticulum (ER) marker Sec61p-GFP at the bud tip. Cortical ER inheritance in yeast has been suggested to involve the capture of ER tubules at the bud tip. Sec3p may act in this process as a spatial landmark for cortical ER inheritance.
The endoplasmic reticulum (ER) consists of a polygonal array of interconnected tubules and sheets that spreads throughout the eukaryotic cell and is contiguous with the nuclear envelope. This elaborate structure is created and maintained by a constant remodeling process that involves the formation of new tubules, their cytoskeletal transport and homotypic fusion. Since the ER is a large, single-copy organelle, it must be actively segregated into daughter cells during cell division. Recent analysis in budding yeast indicates that ER inheritance involves the polarized transport of cytoplasmic ER tubules into newly formed buds along actin cables by a type V myosin. The tubules then become anchored to a site at the bud tip and this requires the Sec3p subunit of the exocyst complex. The ER is then propagated along the cortex of the bud to yield a cortical ER structure similar to that of the mother cell. In animal cells, the ER moves predominantly along microtubules, whereas actin fibers serve a complementary role. It is not yet clear to what extent the other components controlling ER distribution in yeast might be conserved in animal cells.
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