FUNCTIONAL MAPPING OF THE N-TERMINAL REGULATORY DOMAIN IN THE HUMAN RAF-1 PROTEIN-KINASE

Raf-1 is a serine/threonine kinase poised at a key relay point in mito genic signal transduction pathways hom the cell surface to the nucleus . Activation of the transforming potential of Raf-1 has been associate d with N-terminal truncation and/or fusion to other proteins, suggesti ng that the Raf-1 N-terminal half harbors a negative regulatory domain , Seven internal deletion mutants that together scan the entire N-term inal half of human Raf-1 protein were generated to map functional regi ons in this regulatory domain. Effects of the deletion mutations on ki nase activity of Raf-1 were evaluated using a baculovirus/insect cell overexpression system and an in vitro kinase assay with the known phys iological substrate of Raf-1, mitogen-activated protein kinase kinase. Deletion of amino acids 276-323 in the unique sequence between conser ved regions 2 and 3 leads to modest elevation of Raf-1 basal kinase ac tivity, whereas deletion of amino acids 133-180 in conserved region 1 results in diminished kinase activity. Surprisingly, none of the Raf-1 N-terminal deletion mutants, including a truncated version that is tr ansforming in rodent fibroblasts, exhibits greatly increased levels of basal kinase activity. In addition, while activation of Raf-1 kinase by Ras requires sequences in conserved region 1, only the C-terminal h alf containing the kinase domain of Raf-1 is required for activation b y Src, These findings demonstrate that N-terminal deletions in Raf-1 d o not necessarily result in constitutively elevated basal kinase activ ity and that the N-terminal regulatory domain is completely dispensabl e for Raf-1 activation by Src.