| Autor: |
Emrick MA; Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309, USA., Hoofnagle AN, Miller AS, Ten Eyck LF, Ahn NG |
| Jazyk: |
angličtina |
| Zdroj: |
The Journal of biological chemistry [J Biol Chem] 2001 Dec 07; Vol. 276 (49), pp. 46469-79. Date of Electronic Publication: 2001 Oct 08. |
| DOI: |
10.1074/jbc.M107708200 |
| Abstrakt: |
Constitutively active mutant forms of signaling enzymes provide insight into mechanisms of activation as well as useful molecular tools for probing downstream targets. In this study, point mutations in ERK2 at conserved residues L73P and S151D were identified that individually led to 8-12-fold increased specific activity and in combination reached 50-fold, indicating synergistic interactions between these residues. Examination by mass spectrometry, phosphatase sensitivity, and Western blotting revealed that the mutations enhanced ERK2 activity by facilitating intramolecular autophosphorylation predominantly at Tyr-185 and to a lesser extent at Thr-183 and that phosphorylation at both sites is required for activation. A set of short molecular dynamics simulations were carried out using different random seeds to sample locally accessible configurations. Simulations of the active mutant showed potential hydrogen bonding interactions between the phosphoryl acceptor and catalytic nucleophile, which could account for enhanced intramolecular autophosphorylation. In intact cells, the ERK2 mutants were functionally active in phosphorylating Elk-1 and RSK1 and activating the c-fos promoter. This activity was only partially reduced upon treatment of cells with the MKK1/2 inhibitor, U0126, indicating that in vivo the mechanism of ERK2 activation occurs substantially through autophosphorylation and partially through phosphorylation by MKK1/2. |
| Databáze: |
MEDLINE |
| Externí odkaz: |
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