Source: Recombinant human produced GST-tagged in E. coli
Purity: > 80% by SDS-PAGE, apparent Mr ~ 95 kDa
Supplied: 100 ng in 50 μl 50 mM Tris-HCl pH 7.0 buffer containing 14 mM β-mercaptoethanol, 1 mM benzamidine, 0.1 mM phenylmethanesulfonyl fluoride, 1 mM EDTA, 0.03% Brij 35 and 10% glycerol.
Activity: 20 units/mg with MEK as substrate. One unit of Raf-1 is the amount of the enzyme that generates one unit of MAP kinase per minute. One unit of MAP kinase is the amount that incorporates 1 nmol of phosphate into myelin basic protein (MBP, (GLO126-010, GLO126-025) per min at 30° C, pH 7.0. Maintain preparations in aliquots at -70° C. Avoid repeated thawing.
Synonyms: c-Raf; Raf1; c-Raf-1
Background: Raf-1 is a protein serine/threonine kinase which plays a central role in the m itogenic response of cells to growth factors, cytokines, and many oncogenes. The kinase has been implicated as a pivotal regulator of endothelial cell survival during angiogenesis. It m ay also have a role in antineoplastic drug resistance. Inhibition of phosphorylation of Raf-1 by Akt sensitizes human ovarian cancer cells to paclitaxel, a natural product which has significant anti-tumor activity particularly in advanced ovarian and breast carcinomas.
Raf-1 is activated through both Ras-dependent and Ras-independent mechanisms. Once activated, Raf-1 phosphorylates and activates the dual specificity protein kinases MEK1 and MEK2 which in turn phosphorylate and activate the serine/threonine specific MAP kinases, ERK1 and ERK2. Activated ERKs are pleiotropic effectors and play an important role in the control of diverse cellular processes.
The protein kinase activity of Raf-1 is strongly influenced by two key tyrosine residues upstream of the ATP binding site (Y340 and Y341). Dephosphorylation of Ser259 by protein phosphatase 2A (GLO130-GLO132) contributes to Raf-1 activation by supporting its membrane accumulation rather than by increasing the specific activity of the kinase. Phosphorylation of Raf-1 by PAK1 (GLO100-001, GLO100-005, GLO100-010) and Src regulates Raf-1 autoinhibition. PAK1 primes MEK1 for phosphorylation by Raf-1 kinase during cross-cascade activation of the ERK pathway.