ArgBP2 (Arg-Binding Protein 2/SORBS2) is an adaptor protein involved in cytoskeleton associated signal transduction, thereby regulating cell migration and adhesion. These features are associated with its antitumoral role in pancreatic cancer cells. Tyrosine phosphorylation of ArgBP2, mediated by c-Abl kinase and counterbalanced by PTP-PEST phosphatase, regulates many of its interactions. However, the exact mechanisms of action and of regulation of ArgBP2 remain largely unknown. We found that ArgBP2 has the capacity to form oligomers which are destabilized by tyrosine phosphorylation. We could show that ArgBP2 oligomerization involves the binding of one of its SH3 domains to a specific proline rich cluster. ArgBP2 self-association increases its binding to some of its molecular partners and decreased its affinity for others. Hence, the phosphorylation/oligomerization state of ArgBP2 directly regulates its functions by modulating its adaptive capabilities. Importantly, using a human pancreatic cancer cell model (MiaPaCa-2 cells), we could validate that this property of ArgBP2 is critical for its cytoskeleton associated functions. In conclusions, we describe a new mechanism of regulation of ArgBP2 where tyrosine phosphorylation of the protein interfere with a SH3 mediated self-interaction, thereby controlling its panel of interacting partners and related functions.

Polycystin-2 (PC2), encoded by the PKD2 gene, is mutated in ~15% of autosomal dominant polycystic kidney disease. Filamins are actin-binding proteins implicated in scaffolding and membrane stabilization. Here we studied the effects of filamin on PC2 stability using filamin-deficient human melanoma M2, filamin-A (FLNA)-replete A7, HEK293 and IMCD cells together with FLNA siRNA/shRNA knockdown (KD). We found that the presence of FLNA is associated with higher total and plasma membrane PC2 protein expression. Western blotting analysis in combination with FLNA KD showed that FLNA in A7 cells represses PC2 degradation, prolonging the half-life from 2.3 to 4.4 hours. By co-immunoprecipitation and Far Western blotting we found that the FLNA C-terminus (FLNAC) reduces the FLNA-PC2 binding and PC2 expression, presumably through competing with FLNA for binding PC2. We further found that FLNA mediates PC2 binding with actin through forming complex PC2-FLNA-actin. FLNAC acted as a blocking peptide and disrupted the link of PC2 with actin through disrupting the PC2-FLNA-actin complex. Finally, we demonstrated that the physical interaction of PC2-FLNA is Ca-dependent. Taken together, our current study indicates that FLNA anchors PC2 to the actin cytoskeleton through complex PC2-FLNA-actin to reduce degradation and increase stability, and possibly regulate PC2 function in a Ca-dependent manner.

The importance of S100A4, a Ca(2+)-binding protein, in mediating tumour cell migration, both intracellularly and extracellularly, is well documented. Tissue transglutaminase (TG2) a Ca(2+)-dependent protein crosslinking enzyme, has also been shown to enhance cell migration. Here by using the well characterised non-metastatic rat mammary R37 cells (transfected with empty vector) and highly metastatic KP1 cells (R37 cells transfected with S100A4), we demonstrate that inhibition of TG2 either by TG2 inhibitors or transfection of cells with TG2 shRNA block S100A4-accelerated cell migration in the KP1cells and in R37 cells treated with exogenous S100A4. Cell migration was also blocked by the treatment with the non-cell permeabilizing TG2 inhibitor R294, in the human breast cancer cell line MDA-MB-231 (Clone 16, which has a high level of TG2 expression). Inhibition was paralleled by a decrease in S100A4 polymer formation. In vitro co-immunoprecipitation and Far Western blotting assays and cross-linking assays showed not only the direct interaction between TG2 and S100A4, but also confirmed S100A4 as a substrate for TG2. Using specific functional blocking antibodies, a targeting peptide and a recombinant protein as a competitive treatment, we revealed the involvement of syndecan-4 and α5β1 integrin co-signalling pathways linked by activation of PKCα in this TG2 and S100A4-mediated cell migration. We propose a mechanism for TG2-regulated S100A4-related mediated cell migration, which is dependent on TG2 crosslinking.

Tyrosine kinases drive the proliferation and survival of many human cancers. Thus profiling the global state of tyrosine phosphorylation of a tumor is likely to provide a wealth of information that can be used to classify tumors for prognosis and prediction. However, the comprehensive analysis of tyrosine phosphorylation of large numbers of human cancer specimens is technically challenging using current methods.

Storage proteins are the major protein synthesized in the fat body, released into hemolymph and re-sequestered into the fat body before pupation in most insect species. Storage proteins are important amino acid and nutrition resources during the non-feeding pupal period and play essential roles for the metamorphosis and oogenesis of insects. The sequestration of storage protein is a selective, specific receptor-mediated process. However, to date, the potential receptor mediating the sequestration of storage protein has not been determined in Bombyx mori. In this study, we expressed and purified the first ligand binding domain of Bombyx mori vitellogenin receptor (BmVgR), LBD1, and found LBD1 could bind with an unknown protein from the hemolymph of the ultimate silkworm larval instar via pull-down assay. This unknown protein was subsequently identified to be the female-specific storage protein SP1 by mass spectrometry. Furthermore, far western blotting assay, immunoprecipitation and isothermal titration calorimetry analysis demonstrated LBD1 specifically bound with the female-specific SP1, rather than another unisex storage protein SP2. The specific binding of LBD1 with SP1 was dependent on the presence of Ca2+ as it was essential for the proper conformation of LBD1. Deletion mutagenesis and ITC analysis revealed the first and third ligand binding repeats LBR1 and LBR3 were indispensable for the binding of LBD1 with SP1, and LBR2 and LBR4 also had a certain contribution to the specific binding. Our results implied BmVgR may mediate the sequestration of SP1 from hemolymph into the fat body during the larval-pupal transformation of Bombyx mori.

Cell adhesion is tightly regulated by specific molecular interactions and detachment from the extracellular matrix modifies proliferation and survival. HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) is a protein-lipid complex with tumoricidal activity that also triggers tumor cell detachment in vitro and in vivo, suggesting that molecular interactions defining detachment are perturbed in cancer cells. To identify such interactions, cell membrane extracts were used in Far-western blots and HAMLET was shown to bind α-actinins; major F-actin cross-linking proteins and focal adhesion constituents. Synthetic peptide mapping revealed that HAMLET binds to the N-terminal actin-binding domain as well as the integrin-binding domain of α-actinin-4. By co-immunoprecipitation of extracts from HAMLET-treated cancer cells, an interaction with α-actinin-1 and -4 was observed. Inhibition of α-actinin-1 and α-actinin-4 expression by siRNA transfection increased detachment, while α-actinin-4-GFP over-expression significantly delayed rounding up and detachment of tumor cells in response to HAMLET. In response to HAMLET, adherent tumor cells rounded up and detached, suggesting a loss of the actin cytoskeletal organization. These changes were accompanied by a reduction in β1 integrin staining and a decrease in FAK and ERK1/2 phosphorylation, consistent with a disruption of integrin-dependent cell adhesion signaling. Detachment per se did not increase cell death during the 22 hour experimental period, regardless of α-actinin-4 and α-actinin-1 expression levels but adherent cells with low α-actinin levels showed increased death in response to HAMLET. The results suggest that the interaction between HAMLET and α-actinins promotes tumor cell detachment. As α-actinins also associate with signaling molecules, cytoplasmic domains of transmembrane receptors and ion channels, additional α-actinin-dependent mechanisms are discussed.

Silk glands are specialized in the synthesis of several secretory proteins. Expression of genes encoding the silk proteins in Bombyx mori silk glands with strict territorial and developmental specificities is regulated by many transcription factors. In this study, we have characterized B. mori sage, which is closely related to sage in the fruitfly Drosophila melanogaster. It is termed Bmsage; it encodes transcription factor Bmsage, which belongs to the Mesp subfamily, containing a basic helix-loop-helix motif. Bmsage transcripts were detected specifically in the silk glands of B. mori larvae through RT-PCR analysis. Immunoblotting analysis confirmed the Bmsage protein existed exclusively in B. mori middle and posterior silk gland cells. Bmsage has a low level of expression in the 4th instar molting stages, which increases gradually in the 5th instar feeding stages and then declines from the wandering to the pupation stages. Quantitative PCR analysis suggested the expression level of Bmsage in a high silk strain was higher compared to a lower silk strain on day 3 of the larval 5th instar. Furthermore, far western blotting and co-immunoprecipitation assays showed the Bmsage protein interacted with the fork head transcription factor silk gland factor 1 (SGF1). An electrophoretic mobility shift assay showed the complex of Bmsage and SGF1 proteins bound to the A and B elements in the promoter of fibroin H-chain gene(fib-H), respectively. Luciferase reporter gene assays confirmed the complex of Bmsage and SGF1 proteins increased the expression of fib-H. Together, these results suggest Bmsage is involved in the regulation of the expression of fib-H by being together with SGF1 in B. mori PSG cells.