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 performed in vitro kinase 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 CD3epsilon 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 naïve cells from mice of the same age. However, aging also leads to a decline in Raf-1 activity in the naïve 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.
Kirk
C J
CJ
Graduate Program in Cellular and Molecular Biology, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA.
Miller
R A
RA
D000375:Aging / Q000276:immunology* / Q000378:metabolism*; D000818:Animals; D018106:CD28 Antigens / Q000378:metabolism; D017252:CD3 Complex / Q000378:metabolism; D015704:CD4 Antigens / Q000378:metabolism; D015496:CD4-Positive T-Lymphocytes / Q000201:enzymology / Q000276:immunology; D016207:Cytokines / Q000096:biosynthesis; D004789:Enzyme Activation; D007156:Immunologic Memory; D008213:Lymphocyte Activation; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism*; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism*; D013601:T-Lymphocytes / Q000201:enzymology* / Q000276:immunology*
D018106:CD28 Antigens; D017252:CD3 Complex; D015704:CD4 Antigens; D016207:Cytokines; D011948:Receptors, Antigen, T-Cell; D019908:Proto-Oncogene Proteins c-raf