Engineering extrinsic disorder to control protein activity in living cells.

Optogenetic and chemogenetic control of proteins has revealed otherwise inaccessible facets of signaling dynamics. Here, we use light- or ligand-sensitive domains to modulate the structural disorder of diverse proteins, thereby generating robust allosteric switches. Sensory domains were inserted into nonconserved, surface-exposed loops that were tight and identified computationally as allosterically coupled to active sites. Allosteric switches introduced into motility signaling proteins (kinases, guanosine triphosphatases, and guanine exchange factors) controlled conversion between conformations closely resembling natural active and inactive states, as well as modulated the morphodynamics of living cells. Our results illustrate a broadly applicable approach to design physiological protein switches.

Radiation-induced lipid peroxidation activates src kinase and triggers nuclear EGFR transport.

PURPOSE: Elucidation of the molecular mechanism of radiation-induced activation of src kinase, which initiates EGFR internalization and nuclear transport. MATERIAL AND METHODS: Radiation-induced src activation was investigated in the bronchial carcinoma cell line A549. Proteins were Western blotted and quantified by the help of specific antibodies. Residual DNA-damage was quantified with gammaH(2)AX-foci analysis. Radiation-induced lipid peroxidation was prevented by acetyl-cysteine. RESULTS: The radiation-induced src activation and EGFR stabilization could be mimicked by addition of hydroxy-nonenal (HNE), one of the major lipid peroxidation products. Radiation-generated HNE is bound to EGFR and src and correlated with complex formation between both following radiation. Treatment with HNE activated src and stimulated radiation-associated EGFR and caveolin 1 phosphorylations resulting in increased nuclear transport of EGFR. Consequently, radiation-induced phosphorylation and activation of DNA-PK were increased. This phosphorylation was associated with improved removal of residual damage 24h after irradiation. Inhibition of radiation-induced HNE generation by acetyl-cysteine blocked radiation-induced src activation and EGFR phosphorylation. CONCLUSIONS: HNE generated in response to radiation exposure activates src kinase and is involved in regulation of radiation-stimulated DNA-repair processes.

Radiation-induced HIF-1alpha cell survival pathway is inhibited by soy isoflavones in prostate cancer cells.

We previously showed that treatment of prostate cancer cells with soy isoflavones and radiation resulted in greater cell killing in vitro, and caused downregulation of NF-kappaB and APE1/Ref-1. APE1/Ref-1 functions as a redox activator of transcription factors, including NF-kappaB and HIF-1alpha. These molecules are upregulated by radiation and implicated in radioresistance of cancer cells. We extended our studies to investigate the role of HIF-1alpha survival pathway and its upstream Src and STAT3 molecules in isoflavones and radiation interaction. Radiation induced phosphorylation of Src and STAT3 leading to induction of HIF-1alpha. Genistein, daidzein or a mixture of soy isoflavones did not activate this pathway. These data were observed both in PC-3 (AR-) and C4-2B (AR+) androgen-independent cell lines. Pretreatment with isoflavones inhibited Src/STAT3/HIF-1alpha activation by radiation and nuclear translocation of HIF-1alpha. These findings correlated with decreased expression of APE1/Ref-1 and DNA binding activity of HIF-1alpha and NF-kappaB. In APE1/Ref-1 cDNA transfected cells, radiation caused a greater increase in HIF-1alpha and NF-kappaB activities but this effect was inhibited by pretreatment with soy prior to radiation. Transfection experiments indicate that APE1/Ref-1 inhibition by isoflavones impairs the radiation-induced transcription activity of NF-kappaB and HIF-1alpha. This mechanism could result in the inhibition of genes essential for tumor growth and angiogenesis, as demonstrated by inhibition of VEGF production and HUVECs tube formation. Our novel findings suggest that the increased responsiveness to radiation mediated by soy isoflavones could be due to pleiotropic effects of isoflavones blocking cell survival pathways induced by radiation including Src/STAT3/HIF-1alpha, APE1/Ref-1 and NF-kappaB.

Low-power laser irradiation activates Src tyrosine kinase through reactive oxygen species-mediated signaling pathway.

Low-power laser therapy in medicine is widespread but the mechanisms are not fully understood. It has been suggested that low-power laser irradiation (LPLI) could induce photochemical reaction and activate several intracellular signaling pathways. Reactive oxygen species (ROS) are considered to be the key secondary messengers produced by LPLI. Here, we studied the signaling pathway mediated by ROS upon the stimulation of LPLI. Src tyrosine kinases are well-known targets of ROS and can be activated by oxidative events. Using a Src reporter based on fluorescence resonance energy transfer (FRET) and confocal laser scanning microscope, we visualized the dynamic Src activation in Hela cells immediately after LPLI. Moreover, Src activation by LPLI was in a dose-dependent manner. The increase of Src phosphorylation at Tyr416 was detected by Western blotting. In the presence of vitamin C, catalase alone, or the combination of catalase and superoxide dismutase (SOD), the activation of Src by LPLI is significantly abolished. In contrast, Gö6983 loading, a PKC inhibitor, did not affect this response. Treatment of Hela cells with exogenous H(2)O(2) also resulted in a concentration-dependent activation of Src. These results demonstrated that it was ROS that mediated Src activation by LPLI. Cellular viability assay revealed that laser irradiation of low doses (

Light-mediated liberation of enzymatic activity: "small molecule" caged protein equivalents.

Light-activatable ("caged") proteins have been used to correlate, with exquisite temporal and spatial control, intracellular biochemical action with global cellular behavior. However, the chemical or genetic construction of caged proteins is nontrivial, with subsequent laborious introduction into living cells, potentially problematic competition with natural endogenous counterparts, and challenging intracellular incorporation at levels equivalent to the natural enzymes. We describe the design, synthesis, and characterization of small molecular equivalents of a caged Src kinase. These compounds are easy to prepare and function by inhibiting the action of the natural unmodified enzyme.

Lyn regulates the cell death response to ultraviolet radiation through c-Jun N terminal kinase-dependent Fas ligand activation.

The Src-related tyrosine kinase, Lyn, plays an important role in mediating the cell cycle arrest and cell death response to genotoxic agents such as ionizing radiation. In this report we provide evidence to show that the catalytic function of Lyn is required for ultraviolet radiation (UV)- and methyl methanesulfonate (MMS)- but not for cisplatin (CDDP)- or ionizing radiation (IR)-induced cell death. Consequently, fibroblasts deficient in Lyn function were protected against cell death induction by UV and MMS, but showed normal cell death to IR and CDDP treatment. In Lyn(-/-) cells, UV-induced activation of stress-responsive kinases, Erk1/2 and p38, was normal; however, JNK activation was diminished. In addition, FasL induction by UV was also diminished in these cells. Reintroduction of wild-type Lyn restored JNK activation, FasL induction, and sensitivity to UV and MMS. A role for FasL in the cell death induction by Lyn-JNK signaling is indicated by the inhibition of cell death response by FasL neutralizing antibody. Together, the results support the presence of the Lyn-JNK signaling pathway that mediates the cell death response to UV and MMS treatment through FasL induction.

Role of hydroxyl radicals in radiation-induced activation of lyn tyrosine kinase in human B-cell precursors.

Here we show that exposure of human B-cell precursors to gamma-rays stimulates the enzymatic activity of the Src protooncogene family protein tyrosine kinase LYN. LYN activation in irradiated cells is not triggered by DNA damage or a nuclear signal since gamma-rays effectively stimulated LYN kinase in enucleated B-cell precursors as well. LYN activation in irradiated cells was abrogated by presence of the OH* radical scavenger dimethylsulfoxide and exposure of intact or enucleated B-cell precursors to chemically generated OH* radicals instead of gamma-rays also triggered LYN kinase activation and enhanced tyrosine phosphorylation of multiple electrophoretically distinct protein substrates. Thus, OH* radicals appear to be both mandatory and sufficient for radiation-induced LYN kinase activation in irradiated B-cell precursors. We further present evidence which indicates that OH* radicals activate LYN by a novel mechanism which involves disruption of inactive LYN-LYN homodimers and monomerization of the LYN kinase after proteolytic degradation of a putative LYN-associated adapter protein through a cytoplasmic TPCK-sensitive chymotrypsin-like protease following its oxidation. LYN kinase plays a pivotal role in initiation of signal cascades that affect the proliferation, differentiation, and survival of B-cell precursors. Our results prompt the hypothesis that a growth regulatory balance might be altered in human B-cell precursors by radiation-induced stimulation of LYN kinase.