Analysis of Raf-1 activation in response to TCR activation and costimulation in murine T-lymphocytes: Effect of age

Stimulation of the ERK (MAPK) pathway in T-lymphhocytes contributes to cell activation and IL-2 production. The ERK pathway is initiated by the activa tion of the serine/threonine kinase Raf-l in a Ras-dependent manner. Raf-l activates the dual-specific kinase MEK, which in turn activates ERK, To see if aging leads to an alteration of Raf-l kinase activity we performed in v itro kinase assays on Raf-l isolated from CD4(+) T-cells from young and old mice. We found an age-related impairment in the kinase activity of Raf-l i n T-cells stimulated by a combination of antibodies to the CD3 epsilon chai n of the T-cell receptor and CD4, Aging Zed to a two- to fourfold decline i n Raf-l activity (depending on the stimulation time) without a change in th e kinetics of enzyme activation. We also found that Raf-l activation by CD3 /CD4 costimulation is lower in memory cells than in naive cells from mice o f the same age, However, aging also leads to a decline in Raf-l activity in the naive subset of CD4(+) T-cells, suggesting that two mechanisms lead to the age related decline in Raf-l function. Finally, me found that antibodi es to the costimulatory molecule CD28 trigger Raf-l activation and enhance anti-CD3-mediated Raf-l activation but cannot restore Raf-l activation leve ls from old T-cells to those seen in young mice. Our data suggest that age- dependent declines in T-cell ERK function are caused by alterations in the signals that activate Raf-l and that age-dependent defects in T-cell cytoki ne production and proliferation may be caused at least in part by defects i n signals that activate Raf-l. (C) 1998 Academic Press.