Possible role for protein kinase B in the anti-apoptotic effect of prolactin in rat Nb2 lymphoma cells

Prolactin (PRL) is a mitogen for a number of cell types and its action as a survival factor has recently been demonstrated in Nb2 lymphoma cells. Howe ver, the intracellular signalling pathways by which PRL promotes the surviv al of Nb2 cells is unknown. In previous studies, we have shown that PRL cau sed the activation of phosphatidylinositol S-kinase (PI3-kinase) and its as sociation with tyrosine phosphorylated fyn. Protein kinase B (PKB), a serin e/threonine kinase, is now known to be a downstream component of the PI3-ki nase pathway. The aim of the present study was to examine the effect of PRL on the activation of PKB and to find out whether this has any role on the PRL-induced survival of Nb2 cells. Our studies have revealed the phosphoryl ation and activation of PKB in PRL-stimulated Nb2 cells. We have also obser ved, using confocal microscopy, translocation of PKB to the membrane of Nb2 cells in response to PRL. These effects were blocked by the PI3-kinase inh ibitor, LY294002 (10 mug/ml). Apoptosis was induced by the: general protein kinase inhibitor, staurospori ne (STS; 0.1-1 muM), the synthetic glucocorticoid, dexamethasone (Dex; 100 nM) or ionising radiation by exposing Nb2 cells to X-irradiation (IR; 10 Gy ). PRL had no effect on STS-induced apoptosis. On the other hand, PRL. (100 ng/ml) inhibited apoptosis induced by Dex or IR; this effect of PRL was re versed by the addition of LY294002 (10 mug/ml). Furthermore, Western blot a nalysis using phosphospecific PKB antibody on lysates from PRL-treated Nb2 cells showed that phosphorylation of PKB in response to PRL was inhibited b y STS (0.5 muM), but not by Dex (100 nM). These results suggest that the PI3-kinase/PKB pathway may mediate the anti- apoptotic effect of PRL in Nb2 cells.