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-lymphocytes contributes to cell activation and IL-2 production. The ERK pathway is initiated by the activation of the serine/threonine kinase Raf-1 in a Ras-dependent manner. Raf-1 activates the dual-specific kinase MEK, which in turn activates ERK. To see if aging leads to an alteration of Raf-1 kinase activity we performedin vitrokinase assays on Raf-1 isolated from CD4+T-cells from young and old mice. We found an age-related impairment in the kinase activity of Raf-1 in T-cells stimulated by a combination of antibodies to the CD3ϵ chain of the T-cell receptor and CD4. Aging led to a two- to fourfold decline in Raf-1 activity (depending on the stimulation time) without a change in the kinetics of enzyme activation. We also found that Raf-1 activation by CD3/CD4 costimulation is lower in memory cells than in naı̈ve cells from mice of the same age. However, aging also leads to a decline in Raf-1 activity in the naı̈ve subset of CD4+T-cells, suggesting that two mechanisms lead to the age related decline in Raf-1 function. Finally, we found that antibodies to the costimulatory molecule CD28 trigger Raf-1 activation and enhance anti-CD3-mediated Raf-1 activation but cannot restore Raf-1 activation levels 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-1 and that age-dependent defects in T-cell cytokine production and proliferation may be caused at least in part by defects in signals that activate Raf-1.