Structural Studies of MAP Kinase Cascade Components
MAPK cascade components have been the subject of structural analysis, advancing our understanding of how these enzymes are activated and how they interact. A surprising finding has been that unique inactive conformers are adopted by many of these kinases.
- PDF / 1,686,655 Bytes
- 15 Pages / 504 x 720 pts Page_size
- 85 Downloads / 191 Views
1. Introduction MAPK cascades are central transducers of information from the plasma membrane to intracellular targets and participate in the regulation of diverse cellular processes including proliferation, differentiation, transformation, cell death, and senescence (1, 2). Aberrant signaling through these cascades has been associated with human diseases including inflammation, cancer, and disease of the central nervous system (3–6). Many of these enzymes are being actively pursued as drug targets. p38alpha MAP kinase is under study for the treatment of rheumatoid arthritis, pain, dementia, and stroke (7–9). In the ERK1 and ERK2 pathway, kinases at all three levels are under consideration for treatment of various kinds of cancer and other diseases (3, 4, 10–12). Good recent reviews of the role of MAP kinases in disease and their potential as drug targets are available (3, 5).
Rony Seger (ed.), MAP Kinase Signaling Protocols: Second Edition, Methods in Molecular Biology, vol. 661, DOI 10.1007/978-1-60761-795-2_13, © Springer Science+Business Media, LLC 2010
223
224
Goldsmith et al.
MAPK pathways are comprised of three enzymes acting in concert, an MAP3K (for MAP kinase kinase kinase), an MAP2K (for MAP kinase kinase), and an MAPK (MAP kinase). Several distinct pathways have been identified, the best studied of which leads to activation of the MAPK ERK2, activation of the MAPK p38, and activation of the MAPK JNK (13). Thus, mechanisms must be in place to assure pathway specificity (14). One such mechanism is the use of docking interactions (mediated at loci outside the active site). MAP2Ks utilize docking interactions to bind both MAPKs (15) and MAP3Ks (16). MAPKs utilize docking interactions to bind substrates and phosphatases as well (reviewed, for example, in (17)). A second mechanism is the use of inactive conformers, as has been observed in the MAP2K MEK6 (18) (discussed below). Given the medical and biochemical interest in these proteins, it is perhaps not surprising that there are over 200 Protein Data Bank entries for MAPKs and their activators. These structures include contributions from academic labs, numerous drug industry labs, and structural genomics consortia. Here, we summarize the primary crystallographic studies to date (not including inhibitor complexes). To obtain these structures, most often the protein of interest has been crystallized and molecular replacement from the original structure of ERK2 (19) has been used to generate and refine electron density maps . In contrast, kinases at the MAP2K and MAP3K levels have proved to be unexpectedly unique and have required de novo phasing of crystallographic data. Incorporation of selenomethionine (SeMet) in place of methionine into proteins during expression has proved to be a relatively straightforward and robust way to modify the electron content of a crystal without changing the lattice constants or space group, thus making single anomalous dispersion phasing straightforward (20). Here, we offer protocols for expressing SeMet-containing MAP2
Data Loading...
