Temperature sensitivity of cyclic-adenosine-3':5'-monophosphate-binding proteins, activity of protein kinases and the regulation of cell growth

Abstract

A clone of neuroblastoma cells has been selected for its ability to survive and multiply at 40 degrees C. This temperature-resistant clone, like clones of neuroblastoma cells selected for resistance to dibutyryladenosine 3':5'-monophosphate (Bt2-Ado-3':5'-P) showed an increased tumorogenicity in animals and an increased saturation density at 37 degrees C. The Ado-3':5'-P-binding proteins and Ado-3':5'-P-dependent protein kinases from the temperature-resistant and non-resistant cells have been partially purified by chromatography on a DEAE-cellulose column. The Ado-3':5'-P-binding proteins from temperature-resistant cells were more sensitive to temperature than the binding proteins from non-resistant cells. After incubation of binding proteins from resistant cells at 37 degrees C, the specific activity of Ado-3':5'-P-binding to proteins was decreased about 50% and the apparent association constant (Ka) for Ado-3':5-p-binding was decreased from 7.4 X 10(7)M-1 to 4.4 x 10(7)M-1. There was no such decrease with binding proteins from non-resistant cells. A decrease in the activity of binding proteins from the temperature-resistant cells, but not of those from non-resistant cells, was also found when the proteins were stored at 2 degrees C. Treatment with 2-mercaptoethanol made binding proteins from the resistant cells less temperature-sensitive. In the absence of added Ado-3:5-P the protein kinase activity from the temperature-resistant cells was about 50% of the activity from non-resistant cells. Kinase activity was increased by addition of Ado-3:5-P and there was a greater increase with kinases from resistant cells. The maximum protein kinase activity was found in the presence of 10muM Ado-3':5'-P for the temperature-resistant cells and 0.1 muM Ado-3':5'-P for the non-resistant cells. The results indicate that the temperature sensitivity of Ado-3':5'-P-binding proteins, and the activity of protein kinase from cells selected for resistance to high temperature, are similar to those of cells selected for resistance to Bt2-Ado-3':5'-P. It is suggested that the temperature sensitivity of Ado-3':5'-P-binding proteins and the activity of Ado-3':5'-P-dependent protein kinases are involved in the regulation of malignancy and of cell growth at different temperatures.