Characterization of an ERK-binding domain in microphthalmia-associated transcription factor and differential inhibition of ERK2-mediated substrate phosphorylation.
Efficient and specific signaling by mitogen-activated protein kinases (MAPKs) is enhanced by docking sites found on many MAPK substrates and regulators. Here we show that the MAPKs ERK1 and ERK2 form a stable complex (Kd approximately 6 microm) with their substrate the microphthalmia-associated transcription factor (MITF). Complex formation requires a domain of MITF of approximately 100 residues that is nearby, but C-terminal to, the MAPK phosphorylation site at Ser73. MITF derivatives lacking this ERK-binding domain do not bind ERK2 and are phosphorylated less efficiently by ERK2. The ERK-binding domain of MITF bears no obvious resemblance to previously characterized MAPK docking motifs; in particular, it does not contain a consensus D-site. Consistent with this, ERK2-MITF binding does not require the integrity of the CD/sevenmaker region of ERK2. Furthermore, D-site peptides, which are able to potently inhibit ERK2-mediated phosphorylation of the Elk-1 transcription factor (IC50= 3 microm), are relatively poor inhibitors of ERK2-mediated phosphorylation of MITF, exhibiting >15-fold selectivity for inhibition of Elk-1 versus MITF. These observations demonstrate substrate-selective kinase inhibition: the possibility that small molecules that target docking interactions may be used to selectively inhibit the phosphorylation of a subset of the substrates of a kinase.
Molina
Douglas M
DM
Department of Developmental and Cell Biology, University of California, Irvine, California 92697, USA.
Grewal
Seema
S
Bardwell
Lee
L
D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D017931:DNA Primers; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D051739:Microphthalmia-Associated Transcription Factor / Q000737:chemistry / Q000378:metabolism*; D019950:Mitogen-Activated Protein Kinase 1 / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D013379:Substrate Specificity
D017931:DNA Primers; D004791:Enzyme Inhibitors; D051739:Microphthalmia-Associated Transcription Factor; D019950:Mitogen-Activated Protein Kinase 1