Prolactin stimulates activation of c-jun N-terminal kinase (JNK)

In recent years the mitogen-activated protein (MAP) kinase family has expan ded to include both c-jun N-terminal kinases (JNKs), and the p38/HOG1 famil y in addition to the extracellular regulated kinase (ERK) family. These kin ases are activated by a variety of growth factors, as well as extra- and in tracellular insults such as osmotic stress, UV light, and chemotherapeutic agents. Stimulation of the PRL-dependent Nb2 cell line with PRL results in the rapid activation of JNK as determined by the glutathione-S-transferase (GST)-jun kinase assay. Activation was maximal 30 min after stimulation wit h 50 nM rat PRL (rPRL) and decreased after that time. Dose response studies indicated that concentrations as low as 10 nM rPRL resulted in maximal act ivation. The interleukin-3 (IL-3)-dependent myeloid progenitor cell line 32 Dcl3 was transfected with the long, Nb2, and short forms of the rat PRL rec eptor (rPRLR), as well as the long form of the human PRLR (hPRLR), The long and Nb2 forms of the PRLR were able to stimulate activation of JNK; howeve r, the short form of the rPRLR was not. This corresponds with the inability of the short form of the rPRLR to stimulate proliferation of 32Dcl3 cells. Activation of JNK in 32Dcl3 cells expressing the long form of the hPRLR wa s maximal at 30 min after stimulation with 100 nM ovine PRL (oPRL) and decl ined after that time. Dose response studies indicated that activation of JN K was maximal after 30 min at a concentration of 10 nM, and the amount of a ctivated JNK declined at the highest concentration of oPRL, 100 nM. Immunob lot analysis with an antibody that recognizes the activated (phosphorylated ) forms of JNK1 and JNK2 indicated that both JNK1 and JNK2 isoforms were ac tivated in 32D/hPRLR cells stimulated with oPRL. A recombinant human adenov irus expressing a kinase-inactive mutant of JNK1 (APF mutant) was used to d etermine the biological effect of blocking JNK activity in Nb2 cells. Expre ssion of the JNK1-APF mutant inhibited cellular proliferation and induced D NA fragmentation typical of cells undergoing apoptosis. These data suggest that activation of JNKs may be important in mitogenic signaling and/or supp ression of apoptosis in Nb2 cells.