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Structural characterization of the PIT-1/ETS-1 interaction: PIT-1 phosphorylation regulates PIT-1/ETS-1 binding.

ABSTRACT: The POU-domain transcription factor Pit-1 and Ets-1, a member of the ETS family of transcription factors, can associate in solution and synergistically activate the prolactin promoter by binding to a composite response element in the prolactin promoter. We mapped the minimal region of Ets-1 required for the interaction with the Pit-1 POU-homeodomain. Here, we describe a detailed NMR study of the interaction between the POU-homeodomain of Pit-1 and the minimal interacting region of Ets-1. By using heteronuclear single quantum coherence titration experiments, we were able to map exact residues on the POU-homeodomain that are involved in the interaction with this minimal Ets-1 interaction domain. By using our NMR data, we generated point mutants in the POU-homeodomain and tested their effect on the interaction with Ets-1. Our results show that phosphorylation of Pit-1 can regulate the interaction with Ets-1.

SUBMITTER: Augustijn KD

PROVIDER: S-EPMC130516 | biostudies-literature | 2002 Oct

REPOSITORIES: biostudies-literature

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Structural characterization of the PIT-1/ETS-1 interaction: PIT-1 phosphorylation regulates PIT-1/ETS-1 binding.

Augustijn Kevin D KD Duval Dawn L DL Wechselberger Rainer R Kaptein Rob R Gutierrez-Hartmann Arthur A van der Vliet Peter C PC

Proceedings of the National Academy of Sciences of the United States of America 20020919 20

The POU-domain transcription factor Pit-1 and Ets-1, a member of the ETS family of transcription factors, can associate in solution and synergistically activate the prolactin promoter by binding to a composite response element in the prolactin promoter. We mapped the minimal region of Ets-1 required for the interaction with the Pit-1 POU-homeodomain. Here, we describe a detailed NMR study of the interaction between the POU-homeodomain of Pit-1 and the minimal interacting region of Ets-1. By usin ...[more]

PMID: 12242337

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Project description:ERK2, a major effector of the BRAF oncogene, is a promiscuous protein kinase that has a strong preference for phosphorylating substrates on Ser-Pro or Thr-Pro motifs. As part of a program to understand the fundamental basis for ERK2 substrate recognition and catalysis, we have studied the mechanism by which ERK2 phosphorylates the transcription factor Ets-1 at Thr-38. A feature of the mechanism in the forward direction is a partially rate-limiting product release step (koff = 59 +/- 6 s(-1)), which is significant because to approach maximum efficiency substrates for ERK2 may evolve to ensure that ADP dissociation is rate-limiting. To improve our understanding of the mechanism of product release, the binding of the products to ERK2 was assessed and the reaction was examined in the reverse direction. These studies demonstrated that phospho-Ets-1 (p-Ets) binds >20-fold more tightly to ERK2 than ADP (Kd = 7.3 and 165 microM, respectively) and revealed that the products exhibit little interaction energetically while bound to ERK2 and that they can dissociate ERK2 in a random order. The overall equilibrium for the reaction in solution (Keq = 250 M(-1)) was found to be similar to that with the substrate bound to the enzyme (Kint = 525 M(-1)). To determine what limits koff, several pre-steady-state experiments were performed. A catalytic trapping approach furnished a rate constant (k-ADPa) of 61 +/- 12 s(-1) for the dissociation of ADP from the abortive ternary complex, ERK2.Ets.ADP. To examine p-Ets dissociation, the binding of a fluorescent derivative (p-Ets-F), which binds ERK2 with an affinity similar to that of p-Ets, was examined by stopped-flow kinetics. Using this approach, p-Ets-F was found to bind through a single-step mechanism, with the following parameters: k-p-Ets-F = 121 +/- 3.8 s(-1), and kp-Ets-F = (9.4 +/- 0.3) X 10(6) M(-1) s(-1). Similar results were found in the presence of a saturating ADP concentration. These data suggest that koff may be limited by the dissociation of both products and are consistent with the notion that Ets-1 has evolved to be an efficient substrate for ERK2, where ADP release is, at least, partially rate-limiting. A molecular mechanics model of the complex formed between ERK2 and residues 28-138 of Ets-1 provides insight into the role of substrate docking interactions.

Dataset Information

Structural characterization of the PIT-1/ETS-1 interaction: PIT-1 phosphorylation regulates PIT-1/ETS-1 binding.

Publications

Structural characterization of the PIT-1/ETS-1 interaction: PIT-1 phosphorylation regulates PIT-1/ETS-1 binding.

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