Previous studies have suggested that the actin-binding protein cortactin may mediate aspects of cell signaling associated with the cortical cytoskeleton. The present study provides evidence that cortactin forms an SH3 domain-dependent protein complex with a novel 180-kDa protein. This protein represents the first identified natural ligand for the cortactin SH3 domain and is thus referred to as CortBP1. Sequence analysis of CortBP1 revealed the presence of two readily identifiable sequence motifs in the C-terminal region: a sequence virtually identical to a consensus sequence defined for the cortactin SH3 domain-binding peptides (66
) and a sequence similar to recently identified SAM domains (58
). The expression pattern of CortBP1 is restricted, being expressed predominantly in brain tissue. The stable interaction of cortactin and CortBP1 was demonstrated by coimmunoprecipitation of cortactin with CortBP1 in PC12 cell lysates. In addition, CortBP1, overexpressed in fibroblasts, colocalizes with cortactin and cortical F-actin within lamellipodia and membrane ruffles in normal cells and within podosome-like structures in c-Src overexpressors. Colocalization of endogenous cortactin and CortBP1 was also observed in growth cones of differentiating rat hippocampal neurons. These data indicate that endogenous CortBP1 and cortactin reside within the same subcellular compartment and suggest a possible involvement of cortactin and CortBP1 in the dynamic actin reorganization during neuritogenesis.
Data obtained by yeast two-hybrid and in vitro interaction analysis indicate that the SH3 domain of cortactin is responsible for mediating the observed interaction with CortBP1. Mutation of the highly conserved tryptophan residue to lysine in the cortactin SH3 domain efficiently blocked the interaction of full-length cortactin with CortBP1, as evidenced by the inability of GST-CortBP1 to pull down mutant cortactin from cell lysates. These data, coupled with the observation that mutant SH3 domain fusions fail to bind CortBP1 in the yeast two-hybrid assay, indicate the dependence of the interaction with CortBP1 on the structural integrity of the cortactin SH3 domain. The critical role of the cortactin SH3 domain for interacting with CortBP1 is further supported by the presence of the ppI motif in CortBP1, which displays a precise match with the consensus sequence (Fig. D) for cortactin SH3 domain-binding peptides. The sequence of this consensus motif, identified by screening a biased X6
peptide library with GST-cortactin SH3 fusion proteins, is related to class II ligands for SH3 domains (66
). Interestingly, the cortactin SH3 domain does not show detectable interaction with peptides preferred by SH3 domains of other proteins including Src, Yes, Abl, Crk, Grb2, and phospholipase Cγ (66
). The apparent specificity of the cortactin SH3 domain may derive from two unusual residue selections in cortactin SH3 domain-binding peptides. First, whereas an aliphatic residue is present at the −1 position (Fig. D) in most class II ligands (27
), a positively charged residue (lysine or arginine) is present at the analogous position in cortactin SH3 domain-binding peptides. Second, the cortactin SH3 domain-binding peptides show a strict preference for lysine over arginine at the 5 position (Fig. D). We have examined the contribution of the CortBP1 ppI motif to interaction with cortactin by mutational analysis. Mutation of proline 948 and proline 950 to alanine within the ppI motif (e.g., …KPA948
PKP…) reduced the ability of CortBP1 to bind the cortactin SH3 domain in two-hybrid interaction analysis and with endogenous cortactin in in vitro GST pull-down experiments (data not shown). While these results underscore the importance of the CortBP1 ppI motif in binding the cortactin SH3 domain, we cannot rule out the contribution of other CortBP1 sequences in mediating the stable interactions with the cortactin SH3 domain.
The C-terminal 67 amino acids of CortBP1 display extensive similarity to SAM domains, recently identified protein modules present in diverse eukaryotic organisms from yeasts to humans (60
). SAM-containing proteins have been implicated in developmental regulation and signal transduction (60
). For example, SAM-containing proteins are essential for pheromone-induced sexual differentiation in yeast (Byr2p, Ste11p, and Ste4p [16
]) and for regulating anterior-posterior patterning in Drosophila
oocytes (polyhomeotic protein and Bicaudal-C [18
]). Two yeast SAM domain-containing proteins, Boi1p and Boi2p, are associated with cytoskeleton and play an important role in the maintenance of cell polarity during bud formation (6
). While the molecular function of SAM domains remains largely unclear, recent studies of several SAM-containing proteins suggest a possible role of SAM domains in mediating protein-protein interactions (3
). Given the apparent association of CortBP1 with cortactin–F-actin complexes, it will be interesting to further investigate whether the CortBP1 SAM domain is responsible for recruiting other signaling proteins to the dynamic cortical cytoskeleton in growth cones.
In contrast to the cortactin gene, which is expressed in a wide variety of tissues, the expression pattern of the CortBP1 gene appears restricted. Northern blot analysis has shown that the major species of CortBP1 transcript (~8 kb) is present exclusively in brain tissue and a less abundant species (~9.5 kb) is present in brain, kidney, lung, and liver tissues. Whereas the CortBP1 protein is readily detected in brain tissues by Western blot analysis, we have failed to detect p180 CortBP1 in kidney, lung, and liver tissues. The difficulty of detecting the CortBP1 protein in these tissues may be due to the lower sensitivity of the Western blot analysis. The restricted expression pattern of CortBP1 suggests that the cortactin-CortBP1 interaction may have physiological significance in neurons and that in other cell types cortactin may interact with either CortBP1-like protein(s) or another tissue-specific binding partner(s).
Previous studies have shown that cortactin displays an intense staining within lamellipodia as well as a punctate staining within the cytoplasm in adherent cells cultured in the presence of serum (77
). Recent studies from our laboratory (73
) have shown that the distribution of cortactin between the cortical cytoskeleton and cytoplasmic structures is regulated. Sequestration of cortactin to the cytoplasmic pool increases upon serum starvation. Translocation of cortactin into lamellipodia and membrane ruffles is dramatically enhanced by activation of Rac1, a small GTP-binding protein of the Rho family. The cortical cytoskeleton-targeting signal is located within the N-terminal half of cortactin, whereas sequences within the C-terminal half appear to be dispensable for cortical actin localization (74
). Based on these data and the documented function of SH3 domains in recruiting their ligands to certain subcellular compartments (15
), we propose that the cortactin SH3 domain plays a role in targeting its binding partner(s) to the cortical cytoskeleton. In support of this, we have shown here that a significant portion of overexpressed CortBP1 colocalizes with cortactin and F-actin in lamellipodia and membrane ruffles of cultured murine fibroblast cells. The colocalization of CortBP1 with cortactin and F-actin was also observed in podosome-like structures in 10T1/2 cells overexpressing c-Src. These observations strongly suggest that cortactin mediates formation of multiprotein complexes within the cortical cytoskeleton, by the concomitant interaction with cortical F-actin via its N-terminal repeats and with other cellular proteins via the SH3 domain at its C terminus. Thus, cortactin would serve as a cortical actin-specific docking protein for binding partners such as CortBP1.
We have further shown in this study that endogenous cortactin and CortBP1 colocalize within the cortical F-actin-containing subcellular compartment in primary cultures of differentiating rat neurons. Indirect immunofluorescence analysis suggests that both cortactin and CortBP1 are components of growth cones in cultured neurons isolated from embryonic 18-day rat hippocampus. These cells, when placed in culture, extend neurites mimicking their developmental morphology in vivo (22
). The immunostaining of CortBP1 within growth cones appeared to be specific, since preimmune serum and antigen-blocked antibody failed to stain growth cones. The cortactin staining is essentially confined to growth cones, similar to the staining pattern of cortactin observed within growth cones of cultured Xenopus
spinal cord neurons (57
). Both CortBP1 staining and cortactin staining in growth cones of rat hippocampal neurons are primarily localized within the central portion of growth cones, very similar to the previously described staining pattern of F-actin in these cells (31
). Additionally, costaining experiments support the colocalization of cortactin and F-actin in growth cones of the rat hippocampal neurons (data not shown). Previous studies have suggested that the F-actin remodeling within neuronal growth cones, in response to extracellular attraction or repulsion cues, plays an important role in regulating directional neurite extension (7
). Many proteins, including members of the Src family of tyrosine kinases (36
); actin-binding proteins such as profilin (26
), gelsolin (68
), myosin-V (72
), and neurabin (52
); and membrane-associated protein GAP-43 (2
), have been identified as components of growth cones. Of note, the recently identified neural tissue-specific F-actin-binding protein neurabin has been implicated in neurite formation (52
). The localization of cortactin and CortBP1 within growth cones, together with the brain-specific expression of CortBP1, suggests that the cortactin-CortBP1 interaction may also be involved in signaling pathways associated with the formation and migration of growth cones during neurite outgrowth.
A database search for CortBP1 homologs in other organisms reveals two human brain cDNA fragments in the expressed sequence tag database (accession no. m86079 and h41098 [1
]) and one human DNA segment in the sequence-tagged site database (accession no. z51760 [21
]). Both display high DNA sequence identity (81, 90, and 88%, respectively) to rat CortBP1 cDNA. The similarity between rat CortBP1 and the predicted open reading frames of these DNA sequences spans the N-terminal region (amino acids 294 to 405, 27 to 171, and 1 to 57, respectively). The existence of a putative human CortBP1 homolog is an indication of the conservation of CortBP1 function in other organisms and an involvement of CortBP1 in common biological processes in diverse organisms.