Interdependent domains controlling the enzymatic activity of mitogen-activated protein kinase kinase 1.
The activation of human mitogen-activated protein kinase kinase 1 (MKK1) is achieved by phosphorylation at Ser218 and Ser222 within a regulatory loop. Partial activation was achieved by replacing these residues with aspartic/glutamic acid. Higher activity was obtained by introducing four acidic residue substitutions in the regulatory loop, indicating that acidic residues in the loop stabilize an active configuration by the introduction of negative charge. Activation of MKK1 is also achieved by deleting residues 44-51, N-terminal to the consensus catalytic core. Although substitution of residues within this segment by alanine does not affect activity, introduction of proline residues elevates kinase activity, indicating that activation results from perturbation of secondary structure within residues 44-51. Pseudosubstrate inhibition, a commonly observed mechanism of kinase regulation, is not operative in this process. Both the acidic substitutions and the N-terminal deletion increase Vmax, V/K(m),ERK2, and V/K(m),ATP, as is also observed following phosphorylation of wild-type MKK1. A synergistic enhancement of these steady-state rate parameters occurs upon combining the mutations, suggesting that conformational changes induced by mutagenesis together mimic those seen upon phosphorylation.
Mansour
S J
SJ
Department of Molecular, Cellular, and Developmental Biology, Howard Hughes Medical Institute, University of Colorado, Boulder 80309, USA.
Candia
J M
JM
Matsuura
J E
JE
Manning
M C
MC
Ahn
N G
NG
D000595:Amino Acid Sequence; D004789:Enzyme Activation; D006801:Humans; D048369:MAP Kinase Kinase 1; D020929:Mitogen-Activated Protein Kinase Kinases; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D017384:Sequence Deletion
D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D048369:MAP Kinase Kinase 1; C482125:MAP2K1 protein, human; D020929:Mitogen-Activated Protein Kinase Kinases