Cortactin is a Src substrate that interacts with F-actin and can stimulate actin polymerization by direct interaction with the Arp2/3 complex. Complete loss-of-function mutants of the single Drosophila Cortactin gene have been isolated. Mutants are viable and fertile, showing that Cortactin is not an essential gene. However, Cortactin mutants show distinct defects during oogenesis. During oogenesis, Cortactin protein is enriched at the F-actin rich ring canals in the germ line, and in migrating border cells. In Cortactin mutants, the ring canals are smaller than normal. A similar phenotype has been observed in Src64 mutants and in mutants for genes encoding Arp2/3 complex components, supporting that these protein products act together to control specific processes in vivo. Cortactin mutants also show impaired border cell migration. This invasive cell migration is guided by Drosophila EGFR and PDGF/VEGF receptor (PVR). Accumulation of Cortactin protein is positively regulated by PVR. Also, overexpression of Cortactin can by itself induce F-actin accumulation and ectopic filopodia formation in epithelial cells. Evidence is presented that Cortactin is one of the factors acting downstream of PVR and Src to stimulate F-actin accumulation. Cortactin is a minor contributor in this regulation, consistent with the Cortactin gene not being essential for development (Somogyi, 2004).

Cortactin was originally identified as a major substrate of the Src tyrosine kinase (Wu, 1991). The name Cortactin reflects that the protein binds to F-actin and that it localizes to the cell cortex, including membrane ruffles and lamellipodia (Wu, 1993). These features, plus the presence of an SH3 domain and proline-rich regions in the Cortactin protein, suggest that Cortactin might link signaling events to the actin cytoskeleton. Phosphorylation of Cortactin stimulated by Src modulates its activity in vivo (Huang, 1997). Cortactin phosphorylation and subcellular localization are also affected by receptor tyrosine kinases (RTKs) (Maa, 1992 and Zhan, 1993). The cortactin gene was also identified as EMS1, a putative oncogene encoding one of the transcripts amplified in certain human carcinomas (Schuuring, 1993). Directed overexpression of EMS1/cortactin has been shown to increase the motility of and invasion of fibroblasts (Patel, 1998) and the metastatic potential of breast cancer cells (Li, 2001). Cortactin is also enriched in 'invadopodia' from invasive tumor cells, cellular protrusions associated with degradation of extracellular matrix (Bowden, 1999). Together, these studies suggest that Cortactin may play a role in promoting cell motility of cancer cells, as well as of normal cells in response to growth factor stimulation (Somogyi, 2004 and references therein).

Recent studies have shown that Cortactin can directly influence actin polymerization. The Arp2/3 complex is an important nucleator of actin polymerization. It stimulates actin polymerization while binding to actin filaments and can thereby induce formation of a branched actin network. The Arp2/3 complex consists of multiple proteins and is very conserved through evolution, from yeast to man. Cortactin can bind to and activate the Arp2/3 complex as well as stabilize the resulting F-actin network (Uruno, 2001 and Weaver, 2001). Cortactin may act synergistically with proteins of the WASP family, that are potent regulators of Arp2/3 activity (Weaver, 2002). In addition to WASP and N-WASP, the WASP family includes the SCAR/WAVE proteins. The WASP family of proteins is regulated by GTPases of the Rho subfamily, namely CDC42 and Rac, WASP by direct binding (Higgs, 2001), and SCAR/WAVE by dissociation of an inhibitory complex (Eden, 2002). Cortactin can also associate with the adaptor protein WIP (WASP interacting protein), which binds monomeric actin and thus may aid Cortactin in actin nucleation (Kinley, 2003). Arp2/3 and its regulators are required for extensions of lamellipodia and other actin-rich structures, but also for intracellular trafficking events such as endocytosis. Cortactin has been suggested to link endocytosis to Arp2/3 mediated actin polymerization in mammalian cells. Cortactin is associated with dynamin2 and with vesicles (McNiven, 2000). Consistent with this type of function, inhibition of Cortactin inhibits receptor mediated endocytosis (Cao, 2003) (Somogyi, 2004 and references therein).

A Cortactin protein has been identified in Drosophila (Katsube, 1998), but not in yeast. It is possible that other proteins substitute for Cortactin in yeast. It is also possible that the function of Cortactin is specific to higher eukaryotes that, contrary to yeast, use tyrosine kinases for cell-cell communication and cell regulation. There are multiple RTKs in Drosophila that control cell growth, differentiation and morphogenesis including EGFR, insulin receptor and PDGF/VEGF receptor. The Drosophila genome also encodes a number of non-receptor tyrosine kinases such as Src. There are two Src genes, Src42 and Src64. Src42 may act downstream of RTKs to modify their signaling. Src64 is specifically required for proper morphogenesis of actin-rich structures in the female germ line, called ring canals. Drosophila Cortactin protein is localized to the cell cortex and shows protein-protein interactions similar to that of mammalian Cortactin (Katsube, 1998). To understand the function of Cortactin in vivo, complete loss-of-function mutants of Drosophila Cortactin have been generated. Cortactin mutants are fully viable and are fertile, but have subtle defects during oogenesis. This analysis suggests that Cortactin acts downstream of RTKs and Src in vivo. The analysis also indicates that Cortactin is only a minor mediator of the effects of tyrosine kinases on the actin cytoskeleton (Somogyi, 2004 and references therein).

Cortactin was originally isolated as a substrate of the Src kinase. Further biochemical characterization of mammalian Cortactin indicates that it could directly affect actin polymerization by stimulating activity of the Arp2/3 complex in addition to stabilizing the F-actin network. Analysis of mammalian cells has also indicated that phosphorylation by Src is critical for Cortactin activity in affecting cell behavior such as cell migration. In fact, a non-phosphorylatable form can act as a dominant negative protein (Huang, 1998). Phosphorylation by Src can also affect the ability of Cortactin to crosslink F-actin (Huang, 1997), but phosphorylation may not directly affect Arp2/3 interaction (Weaver, 2001 and Uruno, 2001). Genetic analyses in Drosophila indicate that Src, Cortactin and Arp2/3 components control at least some of the same processes in vivo, rather than Src and Arp2/3 being associated with different aspects of Cortactin function. For example, in the germ line of the ovary, mutations in Src64, Cortactin or components of the Arp2/3 complex all affect the actin-rich structures called ring canals in a similar way. This supports the notion that Src, Cortactin and Arp2/3 act together in vivo. However, the interactions may be quite indirect. Perhaps Src induced phosphorylation of Cortactin influence its localization or stability in vivo, which in turn affects the ability of Cortactin to stimulate Arp2/3 (Somogyi, 2004).

The effects of Cortactin in border cells and other follicle cells allow its relationship to a specific RTK, namely PVR, to be investiged. This is of some interest as Cortactin has been implicated both in stimulation of actin polymerization per se, and in endocytosis. The two functions could reflect the same actin-regulatory biochemical function of Cortactin. PVR signaling stimulates actin polymerization in follicle cells and down-regulation of PVR via endocytosis might limit this effect. Genetic analysis indicates that Cortactin plays a positive role downstream of PVR in stimulating actin polymerization, rather than a negative role by limiting receptor signaling. That Cortactin also contributes in a minor way to stimulating receptor endocytosis, in these cells or in other cells, cannot be ruled out. However, genes encoding other proteins that directly control endocytosis such as alpha-adaptin or Hepatocyte growth factor regulated tyrosine kinase substrate are essential in Drosophila. Some mutants have phenotypes that reflect upregulation of specific signaling pathways indicating that endocytosis is important for limiting activity of these signaling pathways in vivo. One of the useful features of Drosophila as a model system is that distinctive defects are produced upon mis-regulation of different signaling pathways. However, no such visible phenotypes were seen in the Cortactin mutants (Somogyi, 2004).

It is perhaps surprising that Cortactin in not an essential gene. Many modulators of essential processes in the cell such as dynamics of the cytoskeleton, cell adhesion or cell signaling are very well conserved in higher eukaryotes. They may add to the robustness and fidelity of the regulation, but they may only be essential to the organism if their absence completely changes the behavior or fate of specific, important cells. In mammals, there are often multiple closely related genes and simple redundancy between these gene products may explain an absence of phenotypes in knockout mice. In Drosophila, this type of simple redundancy is less frequent. For example, there is no evidence for another Cortactin gene in the sequenced Drosophila genome. However, more distantly related genes may have overlapping functions. Subtle phenotypes may also reflect that one process can be regulated in multiple ways. Combining multiple mutations can then be used to genetically help define which genes and pathways overlap in function (Somogyi, 2004).

GENE STRUCTURE

cDNA clone length - 4067

Bases in 5' UTR - 732

Exons - 4

Bases in 3' UTR - 5420

PROTEIN STRUCTURE

Amino Acids - 559

Structural Domains

The gene coding for Drosophila Cortactin was cloned as a Polychaetoid interacting protein using the yeast two hybrid technique. Drosophila Cortactin is a 559-amino acid protein highly expressed in embryos, larvae, and pupae but relatively underexpressed in adult flies. An SH3 domain of approximately 60 amino acids dominates the C-terminal region of Cortactin; no other significant structural motif is found. The SH3 domain is known to bind to a PXXP motif often found in proline-rich regions (Katsube, 1998).

date revised: 4 April 2004

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