Cortactin is a filamentous actin (F-actin)-binding protein that regulates cytoskeletal dynamics by activating the Arp2/3 complex; it binds to F-actin by means of six N-terminal "cortactin repeats". Gene amplification of 11q13 and consequent overexpression of cortactin in several human cancers is associated with lymph node metastasis. Overexpression as well as tyrosine phosphorylation of cortactin has been reported to enhance cell migration, invasion, and metastasis. Here we report the identification of two alternative splice variants (SV1 and SV2) that affect the cortactin repeats: SV1-cortactin lacks the 6th repeat (exon 11), whereas SV2-cortactin lacks the 5th and 6th repeats (exons 10 and 11). SV-1 cortactin is found co-expressed with wild type (wt)-cortactin in all tissues and cell lines examined, whereas the SV2 isoform is much less abundant. SV1-cortactin binds F-actin and promotes Arp2/3-mediated actin polymerization equally well as wt-cortactin, whereas SV2-cortactin shows reduced F-actin binding and polymerization. Alternative splicing of cortactin does not affect its subcellular localization or growth factor-induced tyrosine phosphorylation. However, cells that overexpress SV1-or SV2-cortactin show significantly reduced cell migration when compared with wt-cortactin-overexpressing cells. Thus, in addition to overexpression and tyrosine phosphorylation, alternative splicing of the F-actin binding domain of cortactin is a new mechanism by which cortactin influences cell migration.

We have identified a novel gene, EMSI, that is consistently amplified and overexpressed in human carcinomas with an amplification of the chromosome llq13 region. Comparisons of the EMSl sequences with those present in the GenBank databases revealed a high identity with chicken cortactin. Southern and western blot analyses confirm the high sequence conservation during evolution. An antiserum specific for human cortactin, showed in gene transfer experiments that both human p80 and p85 isoforms are encoded by the EMSl cDNA. Further comparisons demonstrated an high sequence and structural homology with HSI that is implicated in signal transduction in lymphoid cells only. Expression of EMSllcortactin mRNA was restricted to tumor cell lines derived from non-lymphoid origin. Cortactin contains ( i ) a filamentous actin binding tandem repeat domain, (ii) a proline-rich SH3-binding and (iii) a SH3 domain that is common in proteins involved in signal transduction. Our data suggest that human EMS11 cortactin has a function in signal transmission between cell-matrix contact sites and the cytoskeleton and, as such, its overexpression due to 1 lq13 amplification might effect adhesive properties of human carcinomas.

The EMS1 gene, located at the chromosome 11q13 region, is the human homologue of p80/p85 cortactin, a chicken pp60 src tyrosine kinase substrate. In cells derived from breast carcinomas and squamous carcinomas of the head and neck, DNA amplification of this region results in overexpression of cortactin. Overexpression is accompanied by a partial redistribution of cortactin from the cytoplasm into cell-matrix contact sites. To investigate whether overexpression only is sufficient for this redistribution, we performed biochemical analysis of human cortactin derived from carcinoma cell lines with either normal levels (UMSCC8) or with excessive levels of cortactin due to chromosome 11q13 amplification (UMSCC2). Pulse-chase experiments performed with UMSCC2 cells revealed that p85 originated from p80 by post-translational modifications. However, the conversion of p80 into p85 was hardly observed in UMSCC8 cells, indicating a different processing of the two isoforms in cells with a normal expression level of cortactin. Western blot analysis showed that treatment of UMSCC2 cells with cycloheximide, serum, epidermal growth factor, or vanadate resulted in the disappearance of the p80 form and conversion into p85. Conversion of p80 into p85 was accompanied by a redistribution of cortactin from cytoplasm to cell-matrix contact sites. In UMSCC8 cells, these treatments had no effect on the p80/p85 ratio, and cortactin remained in the cytoplasm. Conversion into p85 therefore is correlated with a relocalization of cortactin to the cell periphery. In addition, p85 from epidermal growth factor-or vanadate-treated UMSCC2 cells showed a significant enhancement in phosphorylation compared with p85 in UMSCC8 cells. Our findings demonstrate that in carcinoma cells with 11q13 amplification not only overexpression but also post-translational modifications of cortactin coincides with the redistribution from the cytoplasm into cell-matrix contact sites.

Background: In human carcinomas, overexpression of cortactin correlates with poor prognosis. Cortactin is an F-actin-binding protein involved in cytoskeletal rearrangements and cell migration by promoting actin-related protein (Arp)2/3 mediated actin polymerization. It shares a high amino acid sequence and structural similarity to hematopoietic lineage cell-specific protein 1 (HS1) although their functions differ considerable. In this manuscript we describe the genomic organization of these two genes in a variety of species by a combination of cloning and database searches. Based on our analysis, we predict the genesis of the actin-binding repeat domain during evolution.

Background: In human breast cancers, amplification of chromosome 11q13 correlates with lymph node metastasis and increased mortality. To date, two genes located within this amplicon, CCND1 and EMS1, were considered to act as oncogenes, because overexpression of both proteins, respectively cyclin D1 and cortactin, correlated well with 11q13 amplification. Cyclin D1 is involved in cell cycle regulation and the F-actin-binding protein cortactin in cytoskeletal dynamics and cell migration. To study the role of cortactin in mammary gland tumorigenesis, we examined mouse mammary tumor virus (MMTV)-cortactin transgenic mice and MMTVcortactin/-MMTV-cyclin D1 bitransgenic mice.