Role of Tlk1/pK90 in the cellular radiation response

One feasible option to improve the outcome of radiotherapy in the treatment of cancer lies in pharmacologic interventions, which increase the radiosensitivity of tumor cells or reduce the radiation response in normal tissue cells. The intrinsic cellular radiosensitivity depends to a great deal on intra- and extra-cellular signals, which are transmitted and integrated by signal transduction cascades. Therefore the identification of radiobiologically relevant signal transduction cascades is an important step in identifying potential target proteins for a therapeutic interventions. Many the know signal transduction cascades involve several protein kinases as well as protein phosphatases to transduct the signals responsible for the cellular radiation response. By using an in-gel-renaturation assay we have identified a protein kinase activity in the human promyelocytic leukemia line HL-60, the activity of which is downregulated within minutes after exposure of the cells to ionizing radiation. This activity appears at a molecular weight of about 90 kDa. Therefore we named this kinase pK90. Further investigations showed that pK90 is a nuclear serienhreonine kinase, the activity of which is modulated by phosphorylation and which is found intracellular exclusively in a 450 kDa multiprotein complex. Using liquid chromatography we partially purified pK90 and performed mass spectrometric protein analysis. We found pK90 to be identical with the kinase Tlk1. At the same time another group found that Tlk1 is is a downstream target of Chk1, and that the modulation of the Tlk1 activity after irradiation requires functional ATM and NBS1 proteins. It is therefore the aim of the proposed project to characterize the role oh Tlk1/pK90 in the cellular response to ionizing radiation. It is intended to elucidate the radiobiological role of Tlk1/pK90 by exogenously modifying the activity of the kinase through various strategies like constitutive and regulated overexpression, siRNA techniques and the construction of a transdominant negative mutant Tlk1. In addition it is intended to identify new substrates of Tlk1/pK90, identify the other proteins present in the 450 kDa complex and to look for phosphatases involved in the modulation of Tlk1/pK90 activity. Taken together this project will characterize Tlk1/pK90 to a level where it is possible to judge whether the enzyme offers in interesting and feasible target for therapeutic intervention aiming at a modification of the radiosensitivity of tumors or normal tissues.

One feasible option to improve the outcome of radiotherapy in the treatment of cancer lies in pharmacologic interventions, which increase the radiosensitivity of tumor cells or reduce the radiation response in normal tissue cells. The intrinsic cellular radiosensitivity depends to a great deal on intra- and extra-cellular signals, which are transmitted and integrated by signal transduction cascades. Therefore the identification of radiobiologically relevant signal transduction cascades is an important step in identifying potential target proteins for a therapeutic interventions. Many the know signal transduction cascades involve several protein kinases as well as protein phosphatases to transduct the signals responsible for the cellular radiation response. By using an in-gel-renaturation assay we have identified a protein kinase activity in the human promyelocytic leukemia line HL-60, the activity of which is downregulated within minutes after exposure of the cells to ionizing radiation. This activity appears at a molecular weight of about 90 kDa. Therefore we named this kinase pK90. Further investigations showed that pK90 is a nuclear serienhreonine kinase, the activity of which is modulated by phosphorylation and which is found intracellular exclusively in a 450 kDa multiprotein complex. Using liquid chromatography we partially purified pK90 and performed mass spectrometric protein analysis. We found pK90 to be identical with the kinase Tlk1. At the same time another group found that Tlk1 is is a downstream target of Chk1, and that the modulation of the Tlk1 activity after irradiation requires functional ATM and NBS1 proteins. It is therefore the aim of the proposed project to characterize the role oh Tlk1/pK90 in the cellular response to ionizing radiation. It is intended to elucidate the radiobiological role of Tlk1/pK90 by exogenously modifying the activity of the kinase through various strategies like constitutive and regulated overexpression, siRNA techniques and the construction of a transdominant negative mutant Tlk1. In addition it is intended to identify new substrates of Tlk1/pK90, identify the other proteins present in the 450 kDa complex and to look for phosphatases involved in the modulation of Tlk1/pK90 activity. Taken together this project will characterize Tlk1/pK90 to a level where it is possible to judge whether the enzyme offers in interesting and feasible target for therapeutic intervention aiming at a modification of the radiosensitivity of tumors or normal tissues.