Cortactin, a filamentous actin (F-actin)-associated protein and prominent substrate of Src, is implicated in progression of breast tumours through gene amplification at chromosome 11q13. However, the function of cortactin remains obscure. Here we show that cortactin co-localizes with the Arp2/3 complex, a de novo actin nucleator, at dynamic particulate structures enriched with actin filaments. Cortactin binds directly to the Arp2/3 complex and activates it to promote nucleation of actin filaments. The interaction of cortactin with the Arp2/3 complex occurs at an amino-terminal domain that is rich in acidic amino acids. Mutations in a conserved amino-acid sequence of DDW abolish both the interaction with the Arp2/3 complex and complex activation. The N-terminal domain is not only essential but also sufficient to target cortactin to actin-enriched patches within cells. Interestingly, the ability of cortactin to activate the Arp2/3 complex depends on an activity for F-actin binding, which is almost 20-fold higher than that of the Arp2/3 complex. Our data indicate a new mechanism for activation of actin polymerization involving an enhanced interaction between the Arp2/3 complex and actin filaments.

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We recently reported the critical importance of Rac GTPase-dependent cortical actin rearrangement in the augmentation of pulmonary endothelial cell (EC) barrier function by sphingosine 1-phosphate (S1P). We now describe functional roles for the actin-binding proteins cortactin and EC myosin light chain kinase (MLCK) in mediating this response. Antisense down-regulation of cortactin protein expression significantly inhibits S1P-induced barrier enhancement in cultured human pulmonary artery EC as measured by transendothelial electrical resistance (TER). Immunofluorescence studies reveal rapid, Rac-dependent translocation of cortactin to the expanded cortical actin band following S1P challenge, where colocalization with EC MLCK occurs within 5 min. Adenoviral overexpression of a Rac dominant negative mutant attenuates TER elevation by S1P. S1P also induces a rapid increase in cortactin tyrosine phosphorylation (within 30 s) critical to subsequent barrier enhancement, since EC transfected with a tyrosinedeficient mutant cortactin exhibit a blunted TER response. Direct binding of EC MLCK to the cortactin Src homology 3 domain appears essential to S1P barrier regulation, since cortactin blocking peptide inhibits both S1P-induced MLC phosphorylation and peak S1P-induced TER values. These data support novel roles for the cytoskeletal proteins cortactin and EC MLCK in mediating lung vascular barrier augmentation evoked by S1P.The pulmonary endothelium is a functionally dynamic tissue that serves as a semipermeable barrier between circulating vascular contents and the interstitium and airspaces of the lung. The regulatory mechanisms involved in maintenance of this barrier are poorly understood; however, we recently reported that sphingosine 1-phosphate (S1P), 1 a potent phospholipid angiogenic factor released from activated platelets (1), produces significant endothelial cell (EC) barrier enhancement through Edg receptor ligation and Rac GTPase-dependent cortical actin rearrangement (2). Although the rapid, sustained, and dose-dependent increase in EC transmonolayer electrical resistance (TER) generated by S1P requires an intact actin cytoskeleton capable of undergoing dynamic rearrangement (2), the specific mediators and regulatory mechanisms that effect these actin cytoskeletal changes remain unclear.The 80/85-kDa actin-binding protein, cortactin, has been implicated in cortical actin rearrangement (3). Ideally suited for integrating multiple signals at sites of dynamic actin rearrangement, the amino acid structure of cortactin contains an N-terminal acidic region that stimulates actin polymerization by the Arp2-Arp3 complex (murine AA 1-90), a unique tandem repeat site for actin binding (AA 91-326), a Pro-and Tyr-rich area containing sites for p60 src phosphorylation (AA 401-495), and a C-terminal SH3 domain (AA 496 -546) (3). Cortactin stimulates and stabilizes Arp2-Arp3-mediated polymerization of branched actin filaments at peripheral sites of cytoskeletal rearrangement (4, 5), but regulation of cortactin's activity ...

Cortactin, a filamentous actin cross-linking protein and a substrate of Src protein tyrosine kinase, is phosphorylated at tyrosine residues upon stimulation by extracellular signals. We have previously demonstrated that the filamentous actin cross-linking activity of cortactin is attenuated by Src (Huang, C., Ni, Y., Gao, Y., Haudenschild, C. C., and Zhan, X. (1997) J. Biol. Chem. 272, 13911-13915).In vitro, tyrosine phosphorylation of cortactin occurs specifically within the region between the proline-rich sequence and the Src homology 3 domain. Among the nine tyrosine residues in this region, mutations at Tyr 421 , Tyr 466 , and Tyr 482 significantly reduced Src-meditated tyrosine phosphorylation both in vitro and in vivo. Ectopic expression of wild-type cortactin in ECV304, a spontaneously transformed human umbilical endothelial cell line, resulted in an enhanced cell migration. In contrast, overexpression of a cortactin mutant deficient in tyrosine phosphorylation impaired the migration of endothelial cells. These findings reveal an intracellular signaling mechanism whereby the motility of endothelial cells is regulated by a Src-mediated tyrosine phosphorylation of cortactin.Endothelial cells, which line the lumen of blood vessels, are versatile in morphology and undergo a rapid remodeling during vascularization. Whereas the mechanism for remodeling endothelial cells is not fully understood, the stimulation of the cytoskeletal reorganization that begins underneath the plasma membrane by extracellular signals, especially angiogenic factors, is likely to play an important role in physiological and pathological functions of endothelial cells such as angiogenesis (1).In a previous effort to characterize signaling molecules of fibroblast growth factor 1 (FGF-1), 1 a potent endothelial cell growth factor, we identified a phosphotyrosyl protein as cortactin, a substrate of Src protein tyrosine kinase (2). Tyrosine phosphorylation of cortactin induced by FGF-1 is elevated during the late G 1 phase (4 -6 h) in Balb 3T3 fibroblasts. The stimulation with FGF-1 also results in a transient association of Src with cortactin as well as in an association of FGF receptor 1 with Src (3), suggesting that the activation of Src is a mechanism for the tyrosine phosphorylation of cortactin induced by FGF-1. In addition to FGF-1, various other extracellular stimuli, which often result in a cell motility response, also stimulate the tyrosine phosphorylation of cortactin. These signals include epidermal growth factor (4), thrombin (5), collagen (6), integrin activation (7, 8), phagocytosis (9), and mechanical strain (10).The protein sequence of cortactin features a unique structure characterized by six and a half 37-amino acid tandem repeats and a Src homology 3 (SH3) domain at the carboxyl terminus. Between the SH3 and the repeat domains are an ␣-helical structure and a sequence rich in proline residues. This structural feature containing a repeat domain and a carboxyl-terminal SH3 domain resembles neufectin, a F-actin-associated pr...