We had previously identified the WW domain as a novel globular domain that is composed of 38 -40 semiconserved amino acids and is involved in mediating protein-protein interaction. The WW domain is shared by proteins of diverse functions including structural, regulatory, and signaling proteins in yeast, nematode, and mammals. Functionally it is similar to the Src homology 3 domain in that it binds polyproline ligands. By screening a 16-day mouse embryo expression library, we identified two putative ligands of the WW domain of Yes kinase-associated protein which we named WW domainbinding proteins 1 and 2. These proteins interacted with the WW domain via a short proline-rich motif with the consensus sequence of four consecutive prolines followed by a tyrosine. Herein, we report the cDNA cloning and characterization of the human orthologs of WW domain-binding proteins 1 and 2. The products encoded by these cDNA clones represent novel proteins with no known function. Furthermore, these proteins show no homology to each other except for a proline-rich motif. By fluorescence in situ hybridization on human metaphase chromosomes, we mapped the human genes for WW domain-binding proteins 1 and 2 to chromosomes 2p12 and 17q25, respectively. In addition, using sitedirected mutagenesis, we determined which residues in the WW domain of Yes kinase-associated protein are critical for binding. Finally, by synthesizing peptides in which the various positions of the four consecutive proline-tyrosine motif and the five surrounding residues were replaced by all possible amino acid residues, we further elucidated the binding requirements of this motif.The Src homology (SH) 1 2 and SH3 domains have assumed essential roles in furthering the understanding of how an extracellular signal is transmitted from the cellular membrane, through the cytoplasm, and finally into the nucleus where the signal is interpreted through the process of gene-specific transcription. The SH2 domain has been shown to interact specifically with sequences containing a phosphotyrosine residue, whereas the SH3 domain mediates binding to proline-rich sequences with the minimal consensus of PXXP (P represents proline, and X designates any amino acid) (1, 2). The SH2 and SH3 domains thus consist of a common binding core that recognizes phosphotyrosine-or proline-rich motifs, respectively, and which achieve binding specificity through unique flanking sequences (3-5). As a result, these domains determine which proteins can interact, and equally important, in what order the interaction occurs in the closely regulated pathways of signal transduction. Recently, two other important signaling modules were characterized: the pleckstrin homology domain and the protein interaction domain/phosphotyrosine binding domain (6 -10). These modular repeats represent true protein domains in that they constitute structurally distinct three-dimensional units that can properly fold and function in the context of other proteins or in isolation (11,12).We have previously identified a Yes ki...

Triple-negative breast cancer (TNBC) exhibits innate resistance to the EGFR inhibition despite high level expression of EGFR. Recently, we found that the proliferation of basal-like (BL) subtype TNBC cells is synergistically inhibited by combination of EGFR and PI3K/AKT inhibitors. On the contrary, TNBC cells of mesenchymal stem-like (MSL) subtype are resistant to these combinations. To identify potential synthetic lethal interaction of compounds for treatment of MSL subtype TNBC cells, we performed MTT screening of MDA-MB-231 cells with a small library of receptor tyrosine kinase inhibitors (RTKIs) in the presence of gefitinib, an EGFR inhibitor. We identified MET inhibitors as potent RTKIs that caused synthetic lethality in combination with gefitinib in MDA-MB-231 cells. We demonstrated that combination of a MET inhibitor SU11274 with various EGFR inhibitors resulted in synergistic suppression of cell viability (in MTT assay) and cell survival (in colony formation assay) of MSL subtype TNBC cells. We further demonstrated that SU11274 alone induced G2 arrest and gefitinib/SU11274 combination sustained the SU11274-induced G2 arrest in these cells. In addition, SU11274/gefitinib combination synergistically reduced the level of ribosomal protein S6 (RPS6) in MSL subtype TNBC cells. In addition, knockdown of RPS6 itself, in both HS578T and MDA-MB-231, markedly reduced the proliferation of these cells. Taken together, our data suggest that dual targeting of EGFR and MET inhibits the proliferation of MSL subtype TNBC cells through down-regulation of RPS6.

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Mammalian polo-like kinase 1 (Plk1) has been studied intensively as a key element in regulating diverse mitotic events during M-phase progression. Plk1 is spatially regulated through the targeting activity of the conserved polo-box domain (PBD) present in the C-terminal non-catalytic region. Over the years, studies have demonstrated that the PBD forms a phospho-epitope binding module and the PBD-dependent interaction is critical for proper subcellular localization of Plk1. The current prevailing model is that the PBD binds to a phospho-epitope generated by Cdc2 or other Pro-directed kinases. Here we discuss a recent finding that Plk1 also self-promotes its localization by generating its own PBD-docking site.

Pancreatic cancer remains an intractable cancer with a poor five-year survival rate, which requires new therapeutic modalities based on the biology of pancreatic oncogenesis. Nuclear factor E2 related factor-2 (NRF2), a key cytoprotective nuclear transcription factor, regulates antioxidant production, reduction, detoxification and drug efflux proteins. It also plays an essential role in cell homeostasis, cell proliferation and resistance to chemotherapy. We aimed to evaluate the possibility that modulation of NRF2 expression could be effective in the treatment of pancreatic cancer cells. We investigated whether the depletion of NRF2 by using small interfering RNAs (siRNAs) is effective in the expression of biomarkers of pancreatic cancer stemness such as aldehyde dehydrogenase 1 family, member A1 (ALDH1A1) and aldehyde dehydrogenase 3 family, member A1 (ALDH3A1). NRF2 knockdown markedly reduced the expression of NRF2 and glutamate-cysteine ligase catalytic subunit (GCLC) in cell lines established from pancreatic cancers. NRF2 silencing also decreased the ALDH1A1 and ALDH3A1 expression. Furthermore, this NRF2 depletion enhanced the antiproliferative effects of the chemotherapeutic agent, 5-fluorouracil (5-FU) in pancreatic cancer cells.