The mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway regulates many cellular processes, including proliferation, differentiation, transcription, and cellular motility (28
). MAPKs are activated via a kinase cascade that results in their dual phosphorylation on tyrosine and threonine and their consequent activation (6
). Although a great deal is known about the biochemical steps involved in the transduction of signals through this pathway, considerably less is known about how these signals are converted into specific biological responses. Understanding this process of signal implementation will require identification of the in vivo substrates of the MAPKs. Although over 150 possible ERK substrates have been reported (49
), in very few cases has it been demonstrated that these proteins are in vivo substrates and that MAPK is directly responsible for the phosphorylation.
In the inactive state the ERKs are anchored in the cytoplasm by their association with the MAPK/ERK kinases (MEKs) and several other proteins. Upon activation, the ERKs are released from their anchor and migrate to the nucleus, where they phosphorylate several transcription factors (such as Ets factors) and play an important role in transcriptional regulation (5
). Nuclear localization of ERKs can be brought about by either passive diffusion of monomers or active transport (1
). Interaction with nucleoporins such as Nup153 and Nup214 has also been suggested to play a role in localization of ERK2 to the nucleus (26
), but virtually nothing is known about the ERK partners and substrates that might play a role in this process or in aspects of posttranscriptional gene regulation.
Once at their sites of action, ERKs recognize and phosphorylate serine or threonine residues in the sequence context of S/TP or PXS/TP (17
). However, the specificity of the interaction and phosphorylation comes also from docking motifs on the substrates. Two different kinds of domains or motifs have been identified on candidate ERK substrates. The first is a KIM (k
otif, also known as a D domain) which consists of a stretch of basic amino acids surrounded by aliphatic hydrophobic residues (leucines, isoleucines, or valines) (3
). The second is a DEF domain (d
ocking site for E
XF) consisting of two phenylalanine residues separated by one residue, followed by a proline (FXFP), although the proline residue is not essential (11
To understand the functions of the pathway, identifying and characterizing the direct in vivo targets of ERK phosphorylation and analyzing the molecular basis for substrate specificity are of primary importance. In a previous report (10
), we identified candidate ERK substrates in cell lysates using a method developed by Shokat and colleagues, in which a structural “pocket” is engineered into protein kinases so that they can utilize ATP orthologs that have bulky substituents (37
). In addition to the known ERK2 substrate Rsk2, two novel substrates, E3 ubiquitin ligase EDD (E
3 identified by d
isplay) and nuclear pore complex protein Tpr (t
egion), were found to associate with and be phosphorylated by the ERK2 pocket mutant.
Tpr was originally identified by its fusion (short fragments of Tpr) to various proto-oncogenes such as met
). Tpr localizes to the nuclear basket of the nuclear pore complex and is also found in the nucleus in the form of discrete foci (14
). The functions of Tpr are poorly understood, but several lines of evidence indicate that it is involved in the process of nuclear export. Tpr has been shown to have a role in the nuclear export of proteins containing a leucine-rich nuclear export signal (14
) and in the nuclear export of Huntingtin, a protein with no obvious nuclear export signal (8
). Ectopic expression of mammalian Tpr has also been reported to result in accumulation of poly(A)+
RNA in the nucleus (2
In this report, we characterize Tpr-ERK2 interactions and phosphorylation of Tpr by ERK2 in vitro and in vivo. We identify structural elements in Tpr and ERK2 important for Tpr and ERK association. ERK2 interacts with Tpr through positive cooperative interactions of DEF and the ERK phosphorylation sites. This is in contrast to the other ERK substrates identified with the “pocket mutant” technique, which display decreased binding following phosphorylation. Because phosphorylation of Tpr by activated ERK stabilizes their interaction, we hypothesize that this phosphorylation is not part of a signal amplification cascade but rather positions activated ERK to perform a continuing function in the nuclear pore. We also show that depletion of Tpr results in decreased nuclear accumulation of activated ERK2, suggesting a role for Tpr in modulating ERK2 translocation into the nucleus.