The characterization and regulation of a polyamine-responsive, cyclic nucleotide-independent protein kinase activity in the mouse mammary gland.

The existence of two cyclic nucleotide-independent protein kinases in the cytosolic extract of mouse mammary gland has been determined via DEAE-cellulose and Sephacryl column chromatography. Both enzymes phosphorylated casein in the absence of the exogenous cyclic nucleotides, cAMP and cGMP. One protein kinase was found to have a molecular weight of approx. 30 000, while the other was found to have a molecular weight in the range 150 000-250 000. The activity of the larger species was enhanced by polyamines and inhibited by heparin. This enzyme utilized both ATP and GTP as phosphate donors; the apparent Km values were 10 and 16 microM, respectively. The lower molecular weight protein kinase was not affected by either polyamines or heparin and utilized only ATP (Km = 8 microM) as the phosphate donor. The polyamine-responsive protein kinase activity in the mammary gland varied as a function of the reproductive development of the mouse. The activity was relatively low in the virgin and primiparous stages, increased during pregnancy and peaked during lactation. Studies using mammary organ culture indicated that the combination of insulin (5 micrograms/ml), cortisol (1 micrograms/ml) and prolactin (5 micrograms/ml) maintained the polyamine-responsive protein kinase activity that was present in noncultured tissue. In the absence of prolactin, however, the kinase activity was significantly lower than that observed in the three-hormone system. When dibutyryl cyclic AMP (0.5 mM) was added to the medium along with the three hormones, a significant decrease in enzyme activity was found. Slab gel electrophoresis and autoradiography showed that the majority of the phosphorylated endogenous substrates in the cytosolic fraction were caseins. The results of this study suggest that the polyamine-responsive protein kinase may play an important role in the growth and development of the mammary gland.