Transcriptional co-activators YAP/TAZ: Potential therapeutic targets for metastatic breast cancer.

Breast cancer is the most commonly diagnosed cancer among women. Although routine and targeted therapies have improved the survival rate, there are still considerable challenges in the treatment of breast cancer. Metastasis is the leading cause of death in patients diagnosed with breast cancer. Yes-associated protein (YAP) and/or PDZ binding motif (TAZ) are usually abnormally activated in breast cancer leading to a variety of effects on tumour promotion, such as epithelial-mesenchymal transition, cancer stem cell production and drug-resistance. The abnormal activation of YAP/TAZ can affect metastasis-related processes and promote cancer progression and metastasis by interacting with some metastasis-related factors and pathways. In this article, we summarise the evidence that YAP/TAZ regulates breast cancer metastasis, its post-translational modification mechanisms, and the latest advances in the treatment of YAP/TAZ-related breast cancer metastasis, besides providing a new strategy of YAP/TAZ-based treatment of human breast cancer.

Identification of type I and type II inhibitors of c-Yes kinase using in silico and experimental techniques.

c-Yes kinase is considered as one of the attractive targets for anti-cancer drug design. The DFG (Asp-Phe-Gly) motif present in most of the kinases will adopt active and inactive conformations, known as DFG-in and DFG-out and their inhibitors are classified into type I and type II, respectively. In the present study, two screening protocols were followed for identification of c-Yes kinase inhibitors. (i) Structure-based virtual screening (SBVS) and (ii) Structure-based (SB) and Pharmacophore-based (PB) tandem screening. In SBVS, the c-Yes kinase structure was obtained from homology modeling and seven ensembles with different active site scaffolds through molecular dynamics (MD) simulations. For SB-PB tandem screening, we modeled ligand bound active and inactive conformations. Physicochemical properties of inhibitors of Src kinase family and c-Yes kinase were used to prepare target focused libraries for screenings. Our screening procedure along with docking showed 520 probable hits in SBVS and tandem screening (120 and 400, respectively). Out of 5000 compounds identified from different computational methods, 2410 were examined using kinase inhibition assays. It includes 266 compounds (5.32%) identified from our method. We observed that 14 compounds (12%) are identified by the present method out of 168 that showed > 30% inhibition. Among them, three compounds are novel, unique, and showed good inhibition. Further, we have studied the binding of these compounds at the DFG-in and DFG-out conformations and reported the probable class (type I or type II). Hence, we suggest that these compounds could be novel drug leads for regulation of colorectal cancer.

Identification of potential inhibitors based on compound proposal contest: Tyrosine-protein kinase Yes as a target.

A search of broader range of chemical space is important for drug discovery. Different methods of computer-aided drug discovery (CADD) are known to propose compounds in different chemical spaces as hit molecules for the same target protein. This study aimed at using multiple CADD methods through open innovation to achieve a level of hit molecule diversity that is not achievable with any particular single method. We held a compound proposal contest, in which multiple research groups participated and predicted inhibitors of tyrosine-protein kinase Yes. This showed whether collective knowledge based on individual approaches helped to obtain hit compounds from a broad range of chemical space and whether the contest-based approach was effective.

Rhodium(II) metallopeptide catalyst design enables fine control in selective functionalization of natural SH3 domains.

Chemically modified proteins are increasingly important for use in fundamental biophysical studies, chemical biology, therapeutic protein development, and biomaterials. However, chemical methods typically produce heterogeneous labeling and cannot approach the exquisite selectivity of enzymatic reactions. While bioengineered methods are sometimes an option, selective reactions of natural proteins remain an unsolved problem. Here we show that rhodium(II) metallopeptides combine molecular recognition with promiscuous catalytic activity to allow covalent decoration of natural SH3 domains, depending on choice of catalyst but independent of the specific residue present. A metallopeptide catalyst succeeds in modifying a single SH3-containing kinase at endogenous concentrations in prostate cancer (PC-3) cell lysate.

Individual Src-family tyrosine kinases direct the degradation or protection of the clock protein Timeless via differential ubiquitylation.

Timeless was originally identified in Drosophila as an essential component of circadian cycle regulation, where its function is tightly controlled at the protein level by tyrosine phosphorylation and subsequent degradation. In mammals, Timeless has also been implicated in circadian rhythms as well as cell cycle control and embryonic development. Here we report that mammalian Timeless is an SH3 domain-binding protein and substrate for several members of the Src protein-tyrosine kinase family, including Fyn, Hck, c-Src and c-Yes. Co-expression of Tim with Fyn or Hck was followed by ubiquitylation and subsequent degradation in human 293T cells. While c-Src and c-Yes also promoted Tim ubiquitylation, in this case ubiquitylation correlated with Tim protein accumulation rather than degradation. Both c-Src and c-Yes selectively promoted modification of Tim through Lys63-linked polyubiquitin, which may explain the differential effects on Tim protein turnover. These data show distinct and opposing roles for individual Src-family members in the regulation of Tim protein levels, suggesting a unique mechanism for the regulation of Tim function in mammals.

Non-destructive inhibition of metallofullerenol Gd@C(82)(OH)(22) on WW domain: implication on signal transduction pathway.

Endohedral metallofullerenol Gd@C(82)(OH)(22) has recently been shown to effectively inhibit tumor growth; however, its potential adverse bioeffects remain to be understood before its wider applications. Here, we present our study on the interaction between Gd@C(82)(OH)(22) and WW domain, a representative protein domain involved in signaling and regulatory pathway, using all-atom explicit solvent molecular dynamics simulations. We find that Gd@C(82)(OH)(22) has an intrinsic binding preference to the binding groove, particularly the key signature residues Y28 and W39. In its binding competition with the native ligand PRM, Gd@C(82)(OH)(22) is shown to easily win the competition over PRM in occupying the active site, implying that Gd@C(82)(OH)(22) can impose a potential inhibitory effect on the WW domain. Further analyses with binding free energy landscapes reveal that Gd@C(82)(OH)(22) can not only directly block the binding site of the WW domain, but also effectively distract the PRM from its native binding pocket.

Role of the Yes and Csk tyrosine kinases in the development of a pathological state in the human retina.

Amplification and a cloning of fragments of genes of human retina tyrosine kinases, the nucleotide sequences of which feature a high homology to the gene families of the Yes and Csk tyrosine kinases, and a cloning of the complete coding sequence of the cDNA of the Csk tyrosine kinase gene of the human lymphocytes have been carried out. It has been established that this sequence contains 1,624 bp and encodes a protein that, with a 99% homology, corresponds to the human tyrosine kinase. A comparative analysis of the nucleotide sequences of the full-size cDNA of the Csk tyrosine kinase of the lymphocytes of healthy donors and of patients with an eye choroidal melanoma has shown that a risk of development of an eye choroidal melanoma can be estimated by the frequency of occurrence of a mutant allele in the 10th exon.

Differential trafficking of Src, Lyn, Yes and Fyn is specified by the state of palmitoylation in the SH4 domain.

Src-family tyrosine kinases (SFKs), which participate in a variety of signal transduction events, are known to localize to the cytoplasmic face of the plasma membrane through lipid modification. Recently, we showed that Lyn, an SFK member, is exocytosed to the plasma membrane via the Golgi region along the secretory pathway. We show here that SFK trafficking is specified by the palmitoylation state. Yes is also a monopalmitoylated SFK and is biosynthetically transported from the Golgi pool of caveolin to the plasma membrane. This pathway can be inhibited in the trans-Golgi network (TGN)-to-cell surface delivery by temperature block at 19 degrees C or dominant-negative Rab11 GTPase. A large fraction of Fyn, a dually palmitoylated SFK, is directly targeted to the plasma membrane irrespective of temperature block of TGN exit. Fyn(C6S), which lacks the second palmitoylation site, is able to traffic in the same way as Lyn and Yes. Moreover, construction of Yes(S6C) and chimeric Lyn or Yes with the Fyn N-terminus further substantiates the importance of the dual palmitoylation site for plasma membrane targeting. Taken together with our recent finding that Src, a nonpalmitoylated SFK, is rapidly exchanged between the plasma membrane and late endosomes/lysosomes, these results suggest that SFK trafficking is specified by the palmitoylation state in the SH4 domain.

The cysteine-cluster motif of c-Yes, Lyn and FAK as a suppressive module for the kinases.

The Src family of non-receptor protein tyrosine kinases plays a critical role in the progression of human cancers so that the development of its specific inhibitors is important as a therapeutic tool. We previously reported that cysteine residues in the cysteine-cluster (CC) motif of v-Src were critical for the kinase inactivation by the SH-alkylating agents such as N-(9-acridinyl) maleimide (NAM), whereas other cysteine residues were dispensable. We found similar CC-motifs in other Src-family kinases and a non-Src-family kinase, FAK. In this study, we explored the function of the CC-motif in Yes, Lyn and FAK. While Src has four cysteines in the CC-motif, c-Yes and Lyn have three and two of the four cysteines, respectively. Two conserved cysteines of the Src family kinases, corresponding to Cys487 and Cys498 of Src, were essential for the resistance to the inactivation of the kinase activity by NAM, whereas the first cysteine of c-Yes, which is absent in Lyn, was less important. FAK has similar CC-motifs with two cysteines and both cysteines were again essential for the resistance to the inactivation of the kinase activity by NAM. Taken together, modification of cysteine residues of the CC-motif causes a repressor effect on the catalytic activity of the Src family kinases and FAK.

Crystallographic structure of the SH3 domain of the human c-Yes tyrosine kinase: loop flexibility and amyloid aggregation.

SH3 domains from the Src family of tyrosine kinases represent an interesting example of the delicate balance between promiscuity and specificity characteristic of proline-rich ligand recognition by SH3 domains. The development of inhibitors of therapeutic potential requires a good understanding of the molecular determinants of binding affinity and specificity and relies on the availability of high quality structural information. Here, we present the first high-resolution crystal structure of the SH3 domain of the c-Yes oncogen. Comparison with other SH3 domains from the Src family revealed significant deviations in the loop regions. In particular, the n-Src loop, highly flexible and partially disordered, is stabilized in an unusual conformation by the establishment of several intramolecular hydrogen bonds. Additionally, we present here the first report of amyloid aggregation by an SH3 domain from the Src family.

Involvement of the SH3 domain in Ca2+-mediated regulation of Src family kinases.

When cells are treated with Ca(2+) and Ca(2+)-ionophore, c-Src kinase activity increases, whereas c-Yes kinase activity decreases. This opposite modulation can be reproduced in an in vitro reconstitution assay and is dependent on Ca(2+) and on soluble factors present in cell lysates. Since c-Src and c-Yes share a high degree of homology, with the exception of their N-terminal "unique" domains, their activity was thought to be coordinately regulated. To assess the mechanism of regulation we generated stable cell lines expressing eight different constructs containing wild type c-Src and c-Yes, as well as swaps of the unique domain alone, unique and Src homology 3 (SH3) domains together and the SH3 domain alone. Swapping of the unique domains was not sufficient to reverse the regulation of the chimeric molecules. On the other hand, chimeras containing swaps of the unique plus the SH3 domains displayed reverse regulation, implicating both domains in the regulation of kinase activity by Ca(2+). To rule out the participation of the unique domain, we used chimeric molecules with swapped SH3 domains only and found that the SH3 domain is necessary and sufficient to confer Ca(2+)-mediated regulation of Src and Yes tyrosine kinases.

c-Yes response to growth factor activation.

Transmembrane receptors link the extracellular environment to the internal control elements of the cell. This signaling influences cell division, differentiation, survival, motility, adhesion, spreading and vesicular transport. Central to this signaling is the Src family of nonreceptor tyrosine kinases. The most studied kinase of this nine member family, c-Src, shares a similar structure, as well as a similar expression pattern to that of another Src family protein, c-Yes. Despite high conservation in sequence, molecular studies demonstrate that the functional domains of these kinases can contribute to specificity in signaling. At the cellular level, analysis of tight junction formation also serves as a model to differentiate c-Yes and c-Src signaling. Results suggest that c-Yes promotes formation of the tight junction by phosphorylating occludin, while c-Src signaling downregulates occludin formation in a Raf-1 dependent manner. In addition, pp62c-Yes knockout mice exhibit a specific physiological function phenotype that is distinct from c-src-/- mice. In these studies, c-yes-/- mice exhibit decreased transcytosis of pIgA from the blood to the bile, while c-src-/- mice exhibit deficits in osteoclasts function and bone resorption. Of particular interest in this review are receptor signals that specifically influence the actions of c-Yes. Growth factors that influence many Src family proteins include the PDGF-R, CSF-1 receptor and others. Since these receptors interact with various Src-family kinases, it is predicted that specific signaling is generated by differential recruitment to the cell membrane and/or differentiated interactions with substrates and binding partners. This review provides an overview of c-Yes interactions with specific receptor signaling pathways and how this interaction potentially influences the known physiological roles of c-Yes.