Characterization of an ERK-binding Domain in Microphthalmia-associated Transcription Factor and Differential Inhibition of ERK2-mediated Substrate Phosphorylation

Douglas M Molina,Seema Grewal,Lee Bardwell

doi:10.1074/jbc.m510590200

Douglas M Molina, Seema Grewal + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m510590200

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Abstract

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.

Highlights

Many extracellular signals are transmitted to downstream targets by an evolutionarily conserved protein kinase cascade designated the mitogen-activated protein kinase (MAPK)2 cascade
microphthalmia-associated transcription factor (MITF) Binds ERK2 and with High Affinity—To assess the nature of the interaction between human ERK2 and its target transcription factor MITF, rat ERK2 was fused at its N terminus to Schistosoma japonicum glutathione S-transferase (GST), and the resulting fusion protein was expressed in bacteria and purified by adsorption to glutathione-Sepharose beads
GST-ERK2 beads were incubated with radiolabeled human MEK2; we have previously shown that ERK2-MEK2 binding can be detected using this assay [48]

Summary

EXPERIMENTAL PROCEDURES

Plasmids for in Vitro Transcription/Translation—The following mammalian genes were used in this study: human MITF (transcript variant 4, GenBankTM/EBI accession number NM_000248), human MEK1 (MAP2K1, accession number NM_002755), MEK2 (MAP2K2, NM_030662), MKK6 (MAP2K6, NM_002758), and p38␣ (MAPK14, L35253), and rat ERK2 (M64300). Plasmids for Transfection into Mammalian Cell Lines—To construct pcDNA-MITF-V5, the full-length MITF coding sequence was amplified by high fidelity PCR (using the cDNA Image clone as template), digested with EcoRI and ApaI and ligated into the corresponding sites of the pcDNA3.1-V5 vector (Invitrogen). Kinase reactions (20 ␮l) for ERK2 phosphorylation of MITF contained kinaseassay buffer (50 mM Tris/HCl, pH 7.5, 10 mM MgCl2, 1 mM EGTA, 2 mM dithiothreitol), 50 ␮M ATP, 1 ␮Ci of [␥32-P]ATP, 1 ␮M MITF (a fusion protein consisting of residues 35–198 or 35–98), and 10 units (ϳ1 ng) of active mouse ERK2 (New England Biolabs). Forty-eight hours post-transfection cells from two 35-mm wells were lysed into 200 ␮l of lysis buffer (50 mM HEPES, 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 10% glycerol, 1% Triton X-100, 0.2 mM NaVO4, 50 mM glycerophosphate, protease inhibitor mixture (Sigma), pH 7.6) and centrifuged at 14,000 ϫ g for 15 min at 4 °C. Immunofluorescence Microscopy—The protocol for immunostaining has been described previously [56]

RESULTS

GST fusion

DISCUSSION