FES-related tyrosine kinase activates the insulin-like growth factor-1 receptor at sites of cell adhesion.

IGF-1 receptor (IGF-1R) and integrin cooperative signaling promotes cancer cell survival, proliferation, and motility, but whether this influences cancer progression and therapy responses is largely unknown. Here we investigated the non-receptor tyrosine adhesion kinase FES-related (FER), following its identification as a potential mediator of sensitivity to IGF-1R kinase inhibition in a functional siRNA screen. We found that FER and the IGF-1R co-locate in cells and can be co-immunoprecipitated. Ectopic FER expression strongly enhanced IGF-1R expression and phosphorylation on tyrosines 950 and 1131. FER phosphorylated these sites in an IGF-1R kinase-independent manner and also enhanced IGF-1-mediated phosphorylation of SHC, and activation of either AKT or MAPK-signaling pathways in different cells. The IGF-1R, ß1 Integrin, FER, and its substrate cortactin were all observed to co-locate in cell adhesion complexes, the disruption of which reduced IGF-1R expression and activity. High FER expression correlates with phosphorylation of SHC in breast cancer cell lines and with a poor prognosis in patient cohorts. FER and SHC phosphorylation and IGF-1R expression could be suppressed with a known anaplastic lymphoma kinase inhibitor (AP26113) that shows high specificity for FER kinase. Overall, we conclude that FER enhances IGF-1R expression, phosphorylation, and signaling to promote cooperative growth and adhesion signaling that may facilitate cancer progression.

Characterisation of Schizosaccharomyces pombe α-actinin.

The actin cytoskeleton plays a fundamental role in eukaryotic cells. Its reorganization is regulated by a plethora of actin-modulating proteins, such as a-actinin. In higher organisms, α-actinin is characterized by the presence of three distinct structural domains: an N-terminal actin-binding domain and a C-terminal region with EF-hand motif separated by a central rod domain with four spectrin repeats. Sequence analysis has revealed that the central rod domain of α-actinin from the fission yeast Schizosaccharomyces pombe consists of only two spectrin repeats. To obtain a firmer understanding of the structure and function of this unconventional α-actinin, we have cloned and characterized each structural domain. Our results show that this a-actinin isoform is capable of forming dimers and that the rod domain is required for this. However, its actin-binding and cross-linking activity appears less efficient compared to conventional α-actinins. The solved crystal structure of the actin-binding domain indicates that the closed state is stabilised by hydrogen bonds and a salt bridge not present in other α-actinins, which may reduce the affinity for actin.

Crystallization and preliminary X-ray analysis of the Entamoeba histolytica α-actinin-2 rod domain.

α-Actinins form antiparallel homodimers that are able to cross-link actin filaments. The protein contains three domains: an N-terminal actin-binding domain followed by a central rod domain and a calmodulin-like EF-hand domain at the C-terminus. Here, crystallization of the rod domain of Entamoeba histolytica α-actinin-2 is reported; it crystallized in space group P2(1)2(1)2(1), with unit-cell parameters a = 47.8, b = 79.1, c = 141.8 Å. A Matthews coefficient V(M) of 2.6 Å(3) Da(-1) suggests that there are two molecules and 52.5% solvent content in the asymmetric unit. A complete native data set extending to a d-spacing of 2.8 Å was collected on beamline I911-2 at MAX-lab, Sweden.

The domain structure of Entamoeba α-actinin2.

Entamoeba histolytica, a major agent of human amoebiasis, expresses two distinct forms of α-actinin, a ubiquitous actin-binding protein that is present in most eukaryotic organisms. In contrast to all metazoan α-actinins, in both isoforms the intervening rod domain that connects the N-terminal actin-binding domain with the C-terminal EF-hands is much shorter. It is suggested that these α-actinins may be involved in amoeboid motility and phagocytosis, so we cloned and characterised each domain of one of these α-actinins to better understand their functional role. The results clearly showed that the domains have properties very similar to those of conventional α-actinins.

Characterization of Entamoeba histolytica alpha-actinin2.

We have cloned and characterized a second alpha-actinin isoform in Entamoeba histolytica. This protein, alpha-actinin2, has a N-terminal actin-binding domain, a C-terminal calcium-binding domain and an intervening rod domain containing two spectrin repeats. The protein binds and cross-links actin filaments in a calcium-dependent manner. Therefore alpha-actinin2 is a genuine alpha-actinin except for the shorter rod domain compared to the rod domain of isoforms of higher organisms. A alpha-actinin-like protein has previous been implicated in the adherence to the host cell and infection. It is therefore possible that alpha-actinin2 is involved in mechanism of infection, and in particular in reorganisation of the parasite's cytoskeleton that follows on adherence. E. histolytica alpha-actinin2 represents one of the first members of the spectrin superfamily where well defined spectrin repeats are found. The isolation and characterization of this second alpha-actinin isoform is valuable not only into the study of E. histolytica infection mechanisms, but also for understanding the evolution processes of the spectrin superfamily.