Extracellular signal-regulated kinase 2 (ERK2) phosphorylation sites and docking domain on the nuclear pore complex protein Tpr cooperatively regulate ERK2-Tpr interaction
Authors: J Thomas Parsons
Marcin P Iwanicki
Divya Tiwari
W Richard Burack
Tomás Vomastek
Vinay Kumar Nandicoori
Michael J Weber
Devanand Kumar
Journal: Molecular and Cellular Biology (2008)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: MAPKs; ERK2; substrates; phosphorylation; binding
Affiliations: National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110 067, India., Department of Microbiology and Cancer Center, University of Virginia Health Sciences Center, P.O. Box 800734, Charlottesville, Virginia 22908-0734, University of Rochester Medical Center, 601 Elmwood Ave., Rochester, New York 14642-8626, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110 067, India

Abstract:  Identifying direct substrates of mitogen-activated protein kinases (MAPKs) and understanding how those substrates are selected is central to understanding how these ubiquitously activated enzymes generate diverse biological responses. In previous work, we identified several new candidate substrates for the MAPK ERK2 (extracellular signal-regulated kinase 2), including the nuclear pore complex protein Tpr (translocated promoter region). In this report, we identify sites on Tpr for ERK2 phosphorylation and binding and demonstrate their functional interaction. ERK2 phosphorylation and dimerization are necessary for ERK2-Tpr binding, and this occurs through a DEF (docking site for ERK2, FXF) domain on Tpr. Surprisingly, the DEF domain and the phosphorylation sites displayed positive cooperativity to promote ERK2 binding to Tpr, in contrast to substrates where phosphorylation reduces binding. Ectopic expression or depletion of Tpr resulted in decreased movement of activated ERK2 from the cytoplasm to the nucleus, implying a role for Tpr in ERK2 translocation. Collectively, the data provide direct evidence that a component of the nuclear pore complex is a bona fide substrate of ERK2 in vivo and that activated ERK2 stably associates with this substrate after phosphorylation, where it could play a continuing role in nuclear pore function. We propose that Tpr is both a substrate and a scaffold for activated ERKs. 

Retracted: No

PubPeer comments:
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 The two images have not been merged. A small but distinct gap was left between the two images. Please find attached a zoomed version of the image.![file] , 
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 The experiment was performed when I was a postdoctoral fellow in Prof. Michael J. Weber s lab at University of Virginia, Charlottesville back in 2003. This was a single experiment where IP (FLAG) and reverse IP (HA) were performed to evaluate interactions between FLAG-ERK2 and HA-TprC. The controls are the same. I apologise for not making this clear in the legend. Experiment demonstrates that FLAG-ERK2 and HA-TprC interact with each other only when ERK2 is activated with EGF. , 
 The response posted by the author is unsatisfactory. It does not matter when, where and in what capacity the experiment was carried out by the author. Only thing that matters is that it should be sound, rigorous, reproducible and away from the doubts of manipulation. There are clearly different parameters dealt with in the underlying experiment (different immunoprecipitation) and hence it was expected that the controls, even if same, should have been resolved on two separate gels membranes blots, alongside each test sample, per parameter. It appears that only one image of the lysates (controls) used in two panels to pass it on as a single thing. Even the backgrounds of the duplicate panels are identical. Had the authors repeated their experiment in several iterations, both the panels could have benefited from differently resolved control blots. It will be helpful if the authors upload their uncropped blots from this experiment and, would be really professional of them if they publish a clear protocol of this experiment plus their explanation as a correction at the journal concerned. ,