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  • ACK can be localized to clathrin coated vesicles

    2024-04-18

    ACK1 can be localized to clathrin-coated vesicles [1]. The association of SNX9 with synaptojanin-1 would suggest the two cooperate during endocytosis at the synapse by coupling the sorting nexin to changes in phospholipid composition. ACK1 in turn negatively regulates the association of SNX9 with targets probably via phosphorylation of Y56 (as shown for the Drosophila protein). While the specific role of ACK1 in clathrin-coated vesicle-mediated endocytosis remains to be established, the observation that ACK1 is associated with vesicles relatively depleted in clathrin [1] suggests that an association with partially uncoated vesicles, an event occurring prior to docking or fusion with endosomes.
    Acknowledgement
    Protein kinases have been shown to play very important roles in many signal transduction pathways. Pharmaceutical and biotechnology companies have invested extensively in the discovery and development of small molecule inhibitors targeting protein kinases for various human diseases such as cancer and inflammation , . For example, deregulation due to chromosomal translocation of the non-receptor protein kinase Abl leads to chronic myelogenic leukemia (CML). The Abl inhibitor Gleevec (STI571, Novartis) was the first U.S. Food and Drug Administration (FDA)-approved small molecule drug targeting protein kinases. Subsequently the FDA approved two epidermal growth factor receptor (EGFR) kinase inhibitors, Tarceva (OSI774, OSI Pharmaceuticals/Genentech) and Iressa (ZD1839, AstraZeneca) for the treatment of non-small cell lung cancer. Recently, the number of FDA-approved kinase inhibitors grew significantly and now includes the RAF, VEGFR-2, and PDFR-β inhibitor Nexavar (sorafenib, codeveloped by Onyx and Bayer) and the multiple kinase inhibitors Sutent (SU11248, Pfizer) and Sprycel (dasatinib, Bristol–Myers Squibb). These successes demonstrate that protein kinases are attractive drug targets. Furthermore, sequencing of the human genome revealed that more than 500 protein kinases were encoded, making this gene family one of the largest in the genome . Together, these discoveries have continued to fuel great interest in protein kinases as small molecule drug targets across the pharmaceutical and biotechnology industries, and many more kinase inhibitors are in clinical development , . Activated Cdc42-associated kinase 1 (ACK1) is a non-receptor tyrosine kinase. It initially was identified as a binder to the GTP-bound Cdc42 with consequent inhibition of GTPase activity and, hence, was suggested as an AB1010 of Cdc42 . ACK1 contains an N-terminal tyrosine kinase domain, followed by an Src homology 3 domain (SH3), a Cdc42/Rac interactive binding domain (CRIB), and a proline-rich domain (PR) (A). An ACK isoform termed ACK2 was identified in a bovine complementary DNA (cDNA) library , but according to the literature and database searches, other species, including mouse and human, have only one ACK gene and protein (ACK1). Several reports presented evidence for an important role of ACK in the transduction of Ras/cdc42 signals in several cellular processes such as stimulating the activities of guanine nucleotide exchange factors Ras-GRF1 and Dbl , cell spreading and motility , , , v-Ha-Ras-induced transformation , receptor degradation , , and cell survival , . Recently, we reported that the ACK1 gene is amplified in several types of tumors and that ACK1 is involved in the process of metastasis in vitro and in vivo. In cancer cell lines of epithelial origin, its overexpression enhances cellular motility, invasiveness, and the ability to metastasize to the lung, resulting in increased mortality . Therefore, ACK1 is a very interesting target for small molecule inhibitors for various oncology indications. To enable the testing of highly potent compounds (picomolar ) in lead optimization projects, one often needs to develop a highly sensitive assay requiring only very low concentrations of enzymes. It is preferable that the assay be nonradioactive and minimally affected by interference from color quenching or fluorescent compounds. Historically, this has generally been achieved by incorporating a plate-washing step into the assay protocol. However, nonradioactive assay formats involving plate washing, such as enzyme-linked immunosorbent assay (ELISA) and dissociation and enhancement lanthanide fluoroimmunoassay (DELFIA), commonly give a high coefficient of variance (CV) (for reviews of different assay formats for protein kinases, see Refs. , ). Furthermore, as reported by Yokoyama and Miller , the specific activity of ACK1 is very low; ACK1 has a turnover number of only 0.24/min, and the of the peptide used by this group was 570 μM. The low catalytic efficiency (480min M) is not high enough to enable conventional assays (e.g., fluorescence polarization, homogeneous time-resolved fluorescence [HTRF], coupled assay, scintillation proximity assay [SPA], flash plate) for ACK1. Here we report the development of an ultrasensitive BV-tag-based electrochemiluminescence (BV ECL, which is different from the conventional horseradish peroxidase-based ECL) assay using the BV-tag technology from BioVeris. The high sensitivity of this nonradioactive format allows accurate measurement of the values of small molecule inhibitors of ACK1 in the picomolar range. This assay format can be applied to other protein tyrosine kinases given that the BV-tag-labeled monoclonal antibody (mAb) is a generic phosphotyrosine mAb that recognizes all products of tyrosine kinase reactions. Furthermore, this method can also be used for protein Ser/Thr protein kinases when phospho-specific antibodies against the phosphorylated products are available.