Accumulating evidence suggests that protein tyrosine phosphorylation-based signaling pathways are under the regulation of reactive oxygen species. Although protein tyrosine phosphatases are directly regulated by reversible oxidation, it is not clear whether protein tyrosine kinases (PTKs) are also directly regulated by reduction/oxidation (redox). In this study we report a mechanism of direct oxidative inactivation specific for the PTKs in the Src and fibroblast growth factor receptor (FGFR) families, key enzymes in mammalian signal transduction. Src is fully active when reduced and retains 8–25% of the full activity toward various substrates when oxidized. This inactivation is caused by oxidation of a specific cysteine residue (Cys-277), which results in homodimerization of Src linked by a disulfide bridge. Cys-277 is located in the Gly loop in the catalytic domain. This cysteine residue is conserved only in 8 of the >90 PTKs in the human kinome, including 3 of the 10 Src family kinases and all 4 kinases of the FGFR family. FGFR1 is also reversibly regulated by redox because of this cysteine residue, whereas Csk, a PTK that lacks a cysteine residue at the corresponding position, is not similarly regulated. These results demonstrate a mechanism of direct redox regulation conserved in certain specific PTKs.

Protein tyrosine kinases (PTK) are key enzymes of mammalian signal transduction. For the fidelity of signal transduction, each PTK phosphorylates only one or a few proteins on specific Tyr residues. Substrate specificity is thought to be mediated by PTK–substrate docking interactions and recognition of the phosphorylation site sequence by the kinase active site. However, a substrate-docking site has not been determined on any PTK. C-terminal Src kinase (Csk) is a PTK that specifically phosphorylates Src family kinases on a C-terminal Tyr. In this study, by sequence alignment and site-specific mutagenesis, we located a substrate-docking site on Csk. Mutations in the docking site disabled Csk to phosphorylate, regulate, and complex with Src but only moderately affected its general kinase activity. A peptide mimicking the docking site potently inhibited (IC ₅₀ = 21 μM) Csk phosphorylation of Src but only moderately inhibited (IC ₅₀ = 422 μM) its general kinase activity. Determination of the substrate-docking site provides the structural basis of substrate specificity in Csk and a model for understanding substrate specificity in other PTKs.

Triple negative breast cancer (TNBC) is a heterogeneous group of breast cancers characterized by their lack of estrogen receptors, progesterone receptors, and the HER2 receptor. They are more aggressive than other breast cancer subtypes, with a higher mean tumor size, higher tumor grade, the worst five-year overall survival, and the highest rates of recurrence and metastasis. Developing targeted therapies for TNBC has been a major challenge due to its heterogeneity, and its treatment still largely relies on surgery, radiation therapy, and chemotherapy. In this review article, we review the efforts in developing targeted therapies for TNBC, discuss insights gained from these efforts, and highlight potential opportunities going forward. Accumulating evidence supports TNBCs as multi-driver cancers, in which multiple oncogenic drivers promote cell proliferation and survival. In such multi-driver cancers, targeted therapies would require drug combinations that simultaneously block multiple oncogenic drivers. A strategy designed to generate mechanism-based combination targeted therapies for TNBC is discussed.

Ponatinib is a multi-target protein tyrosine kinase inhibitor, and its effects on hepatocellular carcinoma cells have not been previously explored. In the present study, we investigated its effects on hepatocellular carcinoma cell growth and the underlying mechanisms. Toward SK-Hep-1 and SNU-423 cells, ponatinib induces apoptosis by upregulation of cleaved caspase-3 and -7 and promotes cell cycle arrest in the G1 phase by inhibiting CDK4/6/CyclinD1 complex and phosphorylation of retinoblastoma protein. It inhibits the growth-stimulating mitogen-activated protein (MAP) kinase pathway, the phosphorylation of Src on both negative and positive regulation sites, and Jak2 and Stat3 phosphorylation. Surprisingly, it also activates the PDK1, the protein kinase B (Akt), and the mechanistic target of rapamycin (mTOR) signaling pathway. Blocking mTOR signaling strongly sensitizes cells to inhibition by ponatinib and makes ponatinib a much more potent inhibitor of hepatocellular carcinoma cell proliferation. These findings demonstrate that ponatinib exerts both positive and negative effects on hepatocellular cell proliferation, and eliminating its growth-stimulating effects by drug combination or potentially by chemical medication can significantly improve its efficacy as an anti-cancer drug.

Csk and Src protein tyrosine kinases are structurally homologous but use opposite regulatory strategies. The isolated catalytic domain of Csk is intrinsically inactive and is activated by interactions with the regulatory Src homology 3 (SH3) and SH2 domains, while the isolated catalytic domain of Src is intrinsically active and is suppressed by interactions with the regulatory SH3 and SH2 domains. The structural basis for why one isolated catalytic domain is intrinsically active while the other is inactive is not clear. In this study, we identified structural elements in the N-terminal lobe of the catalytic domain that render the Src catalytic domain active. These structural elements include the alpha-helix C region, a beta turn between the beta4 and beta5 strands, and an Arg residue at the beginning of the catalytic domain. These three motifs interact with one another to activate the Src catalytic domain, but the equivalent motifs in Csk directly interact with the regulatory domains that are important for Csk activation. The Src motifs can be grafted to the Csk catalytic domain to obtain an active Csk catalytic domain. These results, together with available Src and Csk tertiary structures, reveal an important structural switch that determines the kinase activity of a catalytic domain and dictates the regulatory strategy of a kinase.