Interleukin 2 and a lactogen regulate proliferation and protein phosphorylation in Nb2 cells

Abstract

Cell proliferation and protein phosphorylation in response to activation of lactogenic and interleukin 2 (IL-2) receptors were studied in Nb2 cells, a rat T-lymphocyte cell line. Human growth hormone (hGH) and rat IL-2 stimulated Nb2-cell proliferation to approximately the same degree, and the actions of both mitogens were potentiated by phorbol 12-myristate 13-acetate (PMA). A monoclonal antibody specific for the rat IL-2 receptor inhibited the mitogenic actions of rat IL-2, but not those of hGH. Exposure of Nb2 cells to either mitogen for 2-3 h increased phosphorylation of an 18,600-Da protein and decreased phosphorylation of a 15,600-Da protein. PMA also inhibited phosphorylation of the latter protein, but, by itself, PMA did not stimulate phosphorylation of the 18,600-Da protein. Overall, the results suggest that hGH and IL-2 act through separate receptors to stimulate proliferation of Nb2 cells, and that some of the actions of both mitogens may be mediated, in part, through regulation of protein phosphorylation.

Implication of alpha4 phosphoprotein and the rapamycin-sensitive mammalian target-of-rapamycin pathway in prolactin receptor signalling

“…Nb2 cells were labelled with [ 32 P]-orthophosphate (TPA PRL) as described (Rayhel et al 1988, Fulton et al 1999, with modifications. Briefly, quiescent Nb2 cells (10 10 6 per treatment) were pelleted at 200 g for 5 min at room temperature, washed twice with phosphate-free RPMI 1640 (Life Technologies, Burlington, Ontario, Canada) and resuspended in 5 ml of the same medium for a 1-h incubation at 37 C. Cells were incubated with [ 32 P]-orthophosphate (0·1 mCi/ml) for 70 min at 37 C. TPA (20-80 nM) PRL (10 ng/ml) was added and inactive phorbol 4 -PHR (40 nM) was used as negative control.…”

Growth and protein phosphorylation in the Nb2 lymphoma: Effect of prolactin, cAMP, and agents that activate adenylate cyclase

“…It is generally believed that many of the actions of cAMP in eukaryotic cells are mediated through protein kinases; accordingly, Nb2 cell proteins phosphorylated in response to 8-Br-CAMP and PRL were analyzed by two-dimensional gel electrophoresis and autoradiography. Cells were exposed to test agents for 4 h, because previous studies [9,14] have demonstrated changes in protein phosphorylation after exposure of Nb2 cells to PRL for 3 4 h. In the absence of 8-Br-cAMP, PRL decreased incorporation of radioactive phosphate into four proteins with PIS of approximately 5.7 and MWs ranging from 3 1,000 to 48,000 (Fig. 2).…”

“…The second dimension was sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (4% stacking gel, 5-20% linear-gradient separating gel) as described by Laemmli [ 181. The gels were dried and autoradiographed as described previously [9]. Pre-stained protein markers (Bio-Rad) were used for molecular weight (MW) determinations.…”

Sequential effects of interleukin 2-diphtheria toxin fusion protein on T-cell activation.

“…Binding of IL-2 to its high-affinity receptor rapidly activates intracellular processes that lead to gene activation and de novo gene transcription, DNA synthesis, and cell cycle progression (3)(4)(5)(6). Even though IL-2-toxin rapidly inhibits protein synthesis in IL-2R-bearing phytohemagglutinin (PHA)-activated T cells, we now note that this chimeric toxin also transiently stimulates DNA synthesis (e.g., thymidine incorporation).…”

“…The mature form of IL-2-toxin has a deduced molecular mass of 68 kDa, and the fusion protein retains at least some functional attributes of both its diphtheria toxin and IL-2 components. The fusion protein (i) binds with high affinity to the multimeric IL-2 receptor (IL-2R), (ii) is internalized by IL-2R-mediated endocytosis, and (iii) mediates the ADP-ribosylation of elongation factor 2 in the target-cell cytosol (2).Binding of IL-2 to its high-affinity receptor rapidly activates intracellular processes that lead to gene activation and de novo gene transcription, DNA synthesis, and cell cycle progression (3)(4)(5)(6). Even though IL-2-toxin rapidly inhibits protein synthesis in IL-2R-bearing phytohemagglutinin (PHA)-activated T cells, we now note that this chimeric toxin also transiently stimulates DNA synthesis (e.g., thymidine incorporation).…”