Inactive p42 MAP Kinase at a concentration of 2 µg/20 µl reaction can be phosphorylated by an up-stream kinase in an in vitro kinase assay with 1X Kinase Buffer #9802 and 200 µM ATP #9804. After a 30-minute assay at 30°C, phosphorylation can be detected by Western blot with Phospho-p44/42 MAP Kinase (Thr202/Tyr204) Antibody #9101.
Inactive p42 MAP Kinase (Erk2) serves as a useful substrate for MEK (MAPK/ERK kinase), which will phosphorylate it at Thr183 and Tyr185 (1). It is expressed as a recombinant full-length untagged protein, and carries a Lys52Arg mutation in the ATP-binding region, rendering it kinase-inactive regardless of its phosphorylation state (2).
Mitogen-activated protein kinases (MAPKs) are a widely conserved family of serine/threonine protein kinases involved in many cellular programs, such as cell proliferation, differentiation, motility, and death. The p44/42 MAPK (Erk1/2) signaling pathway can be activated in response to a diverse range of extracellular stimuli including mitogens, growth factors, and cytokines (1-3), and research investigators consider it an important target in the diagnosis and treatment of cancer (4). Upon stimulation, a sequential three-part protein kinase cascade is initiated, consisting of a MAP kinase kinase kinase (MAPKKK or MAP3K), a MAP kinase kinase (MAPKK or MAP2K), and a MAP kinase (MAPK). Multiple p44/42 MAP3Ks have been identified, including members of the Raf family, as well as Mos and Tpl2/COT. MEK1 and MEK2 are the primary MAPKKs in this pathway (5,6). MEK1 and MEK2 activate p44 and p42 through phosphorylation of activation loop residues Thr202/Tyr204 and Thr185/Tyr187, respectively. Several downstream targets of p44/42 have been identified, including p90RSK (7) and the transcription factor Elk-1 (8,9). p44/42 are negatively regulated by a family of dual-specificity (Thr/Tyr) MAPK phosphatases, known as DUSPs or MKPs (10), along with MEK inhibitors, such as U0126 and PD98059.
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