Aging Aging

Raf-1 Protein Kinase Activity 89requires either tyrosine or serine/threonine phosphorylation (11). The importanceof Raf-1 activity in T-cell activation and interleukin-2 (IL-2) productionhas been shown using Jurkat cells transfected with dominant negative forms ofRaf-1. In this system, inhibition of Raf-1 blocks IL-2 production through theTCR (12,13). It has also been shown in Jurkat cells that Raf-1 activationthrough the TCR involves a protein kinase C dependent step that may be uniqueto T-cells (14).As the first kinase activated in the MAPK pathway, Raf-1 phosphorylatesand activates the dual specificity kinases MEK1/2, which, in turn, activateERK1/2 (11). Early studies of Raf-1 in vitro kinase activity utilized either nonspecificsubstrates, such as histone H1, or Raf-1 autophosphorylation (11,15).Other measures of Raf-1 activity involved analyzing the retardation in mobilityof Raf-1 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), presumably due to activating phosphorylation. However, these assayslack the specificity of analyzing in vitro phosphorylation of a substrate knownto be phosphorylated by Raf-1 in vivo. Furthermore, as Raf-1 contains multiplesites of phosphorylation, including those that inhibit kinase function, the slowlymigrating form of Raf-1 in SDS-PAGE may contain a collection of differentiallyphosphorylated species of varying levels of activity (11).With the cloning of MEK into plasmids that allow for protein productionand purification from E. coli, a suitable substrate for in vitro Raf-1 kinaseassays is available (16). We used a plasmid encoding a MEK construct thatcontains a lysine to methionine substitution at residue 97 in the ATP bindingsite, and therefore lacks kinase activity (16). This kinase-inactivated MEK,which we call KIMEK, has been used as a substrate to determine the enzymaticactivity of Raf-1 in several cell systems, including Jurkat (7). In our hands,plasmid-derived KIMEK contains multiple species that migrate closely to thepredicted molecular weight for KIMEK of 50 kDa (Fig. 1), each of whichis phosphorylated by Raf-1 in in vitro kinase assays. These bands are likely torepresent degradation products of the KIMEK, and have been noted in otherpublications using this substrate (16,17). Another version of KIMEK is commerciallyavailable as a GST fusion protein from Upstate Biotechnology (LakePlacid, NY, USA).Raf-1 function is more difficult to quantify in freshly isolated lymphocytesthan in cell lines, in part because Raf-1 protein levels are lower in primary cellisolates. T-cells isolated from spleens are in a quiescent state and contain asmall cytosolic compartment as compared to a T-cell clone or Jurkat cell, whichare the size of T-cell blasts. In fact, Raf-1 protein levels are approximatelytenfold lower per cell in quiescent T-cells than in T-cell blasts (18), and wehave found that Raf-1 protein expression in Jurkat is approximately fivefoldgreater than in splenic CD4 + T-cells (Kirk and Miller, unpublished results).