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, β1 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.

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.

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 EFhand 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.

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.

-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 -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 À1 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.

Insulin-like Growth Factor-1 signaling has a well-described function in facilitating tumourigenesis and promoting tumour growth. Attempts to suppress IGF signals at the level of the IGF-1 Receptor have been disappointing, with kinase inhibitors and blocking antibodies generally showing poor efficacy in clinical trials. To address the mechanisms for this lack of efficacy we sought to identify proteins that modulate the cytotoxic response to IGF-1R tyrosine kinase inhibition using a functional siRNA screen. We identified the non-receptor tyrosine FES-related (FER) kinase as a mediator of sensitivity to the IGF-1R tyrosine kinase inhibitor in MCF-7 cells. We found that FER and the IGF-1R co-locate and can be co-immunoprecipitated from different cell types. Ectopic expression of FER strongly enhanced IGF-1R expression and phosphorylation on the atuophosphorylation sites at 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 the AKT or MAPK signaling pathways in breast cancer cell lines. The IGF-1R, β1 Integrin, FER and its substrate cortactin were all observed to be co-located 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 ALK inhibitor 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 and resistance to IGF-1R inhibition. Citation Format: Joanna Stanicka, Leonie Rieger, Orla T. Cox, Sandra O'Shea, Michael Coleman, Ciara O'Flanagan, Barbara Addario, Nuala McCabe, Richard Kennedy, Rosemary O'Connor. The FES-related tyrosine kinase associates with and activates the insulin-like growth factor 1 receptor at sites of cell adhesion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1794.

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