A single amino-acid change in ERK1/2 makes the enzyme susceptible to PP1 derivatives.
We generated extracellular signal-regulated kinase 1/2 (ERK1/2) mutants by introducing a single amino-acid substitution in subdomain V of the catalytic domain and then examined the susceptibility of these mutants to PP1 derivatives originally designed as Src inhibitors. Substituting smaller amino acids (alanine [Ala (A)] or glycine [Gly (G)]) for glutamine [Gln (Q)] in subdomain V drastically increased the susceptibility of ERK1/2 to 1-naphthyl PP1 (1NA-PP1). Wild-type ERK1/2 was resistant to 1NA-PP1 inhibition. ERK1(Q122A) and ERK2(Q103A) were inhibited by 1NA-PP1 at IC(50) values of 1.7 +/- 0.13 and 2.1 +/- 0.18 microM, respectively. ERK1(Q122G) and ERK2(Q103G) were inhibited by 1NA-PP1 with IC(50) values of 3.6 +/- 0.26 and 18 +/- 2.2 microM, respectively. Other derivatives of PP1 (1-naphthylmethyl PP1 and 2-naphthylmethyl PP1) did not significantly inhibit ERK1/2 and its various mutants. In addition, these ERK1/2 mutants were activated by TPA when they were expressed in mammalian cells. These results suggest that the Gln residue of subdomain V is important in determining the susceptibility of ERK1/2 to 1NA-PP1 without significant changes in their enzymatic characteristics.
Endo
Shogo
S
Unit for Molecular Neurobiology of Learning and Memory, Initial Research Project, Okinawa Institute of Science and Technology, Uruma, Japan. sendo@irp.oist.jp
Satoh
Yasushi
Y
Shah
Kavita
K
Takishima
Kunio
K
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D004789:Enzyme Activation; D019950:Mitogen-Activated Protein Kinase 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011720:Pyrazoles / Q000737:chemistry* / Q000378:metabolism*; D011743:Pyrimidines / Q000737:chemistry* / Q000378:metabolism*; D013329:Structure-Activity Relationship
C108123:4-amino-5-(4-methylphenyl)-7-(tert-butyl)pyrazolo(3,4-d)pyrimidine; D011720:Pyrazoles; D011743:Pyrimidines; D019950:Mitogen-Activated Protein Kinase 1