Aging Aging

Raf-1 Protein Kinase Activity 856Raf-1 Protein Kinase Activityin T Cells from Aged MiceChristopher J. Kirk and Richard A. Miller1. IntroductionMost of our models of signal transduction through the T-cell receptor (TCR)involve components and pathways first described in T-cell clones and T-celllymphomas such as the Jurkat cell line (1). These studies, while providing valuableinsights, are not always reliable guides to the analogous biochemicalevents in cells freshly isolated from live donors (2). Thus, studies of the effectsof aging on T-cell activation must frequently begin with a detailed study of theresponses of cells from young individuals. Studies of aging effects involve additionalchallenges, including the difficulty of purifying sufficient numbers ofcells from specific subsets, and allowing for the inherent variability amongdonors of any age.The mitogen-activated protein kinase (MAPK) pathway involves the sequentialactivation of three kinases—Raf-1, mitogen-activated protein kinase(MEK), and extracellular-signal-regulated kinase (ERK)—and plays an importantrole in T-cell activation (3). Here we describe an in vitro kinase assay forRaf-1, which utilizes Raf-1 specific antibodies and a recombinant substrate, toassess age-related differences in Raf-1 activation in mouse splenic CD4 + T-cellsstimulated through the TCR. The problems involved in analyzing Raf-1 activitylevels in freshly isolated T-cells are similar to those that are likely to befaced in the study of age-related alterations in the signal transduction of othercell types.1.1. Isolation of Primary LymphocytesT-cell populations are made up of many distinct subsets, each with differentactivation requirements, some of which change systematically with age. Thus,From: Methods in Molecular Medicine, Vol. 38: Aging Methods and ProtocolsEdited by: Y. A. Barnett and C. R. Barnett © Humana Press Inc., Totowa, NJ85