Beta-Like Importins Mediate the Nuclear Translocation of Mitogen-Activated Protein Kinases

Abstract

The rapid nuclear translocation of signaling proteins upon stimulation is important for the regulation of de novo gene expression. We have studied the stimulated nuclear shuttling of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinases (MAPKs) and found that they translocate into the nucleus in a Ran-dependent, but NLS-or NTS-independent, manner, unrelated to their catalytic activity. We show that this translocation involves three ␤-like importins, importins 3, 7, and 9 (Imp3/ 7/9). Knockdown of these importins inhibits the nuclear translocation of the MAPKs and, thereby, activation of their transcription factor targets. We further demonstrate that the translocation requires the stimulated formation of heterotrimers composed of Imp3/Imp7/MAPK or Imp3/Imp9/MAPK. JNK1/2 and p38␣/␤ bind to either Imp7 or Imp9 upon stimulated posttranslational modification of the two Imps, while Imp3 joins the complex after its stimulation-induced phosphorylation. Once formed, these heterotrimers move to the nuclear envelope, where importin 3 remains, while importins 7 and 9 escort the MAPKs into the nucleus. These results suggest that ␤-like importins are central mediators of stimulated nuclear translocation of signaling proteins and therefore add a central level of regulation to stimulated transcription. Rapid and massive nuclear translocation of signaling proteins is an important step in the induction of transcription upon extracellular stimulation (1). Despite the importance of this process, its molecular mechanism has only been elucidated for a few signaling components. The well-understood cases include signaling proteins such as NF-B (2) and extracellular signal-regulated kinase 5 (ERK5) (3), which utilize the classical nuclear localization signal (NLS)-mediated binding with importin-␣ (Imp␣) and Imp␤ (4). However, many other signaling proteins (Ͼ60) translocate to the nucleus shortly after stimulation by using distinct NLS-and Imp␣/␤-independent mechanisms. The rapid nature of the nuclear translocation and size of the shuttling proteins make it unlikely that their translocation is mediated by passive diffusion. Several other stimulus-dependent signaling mechanisms for such proteins that have been proposed include Imp-like activity by the shuttled protein itself (e.g., ␤-catenin [5]) or the use of shuttling proteins other than Imp␣/␤ (e.g., Smad4 [6]). We recently elucidated the molecular mechanism of ERK1/2 translocation, showing that it involves casein kinase 2 (CK2)-mediated phosphorylation of two Ser residues within a nuclear translocation signal (NTS) of ERK1/2 (7,8). This phosphorylation allows interaction with Imp7, which further facilitates ERK1/2 shuttling via nuclear pores. Aside from ERK1/2, NTS-like sequences have been seen in only a few other NLS-deficient translocating proteins (SMAD3, MEK1 [7], and Egr1 [9]), indicating that other proteins use a distinct mechanism for their translocation.The involvement of Imp7 in the nuclear translocation of ERK1/2 drew our attention to the group of ill-studi...

JNK Signaling: Regulation and Functions Based on Complex Protein-Protein Partnerships

“…Although a phosphorylation-based NLS has been proposed for ERK2 (137), the implicated site is not functional in JNK or the p38 kinases (138). The ERK2 NLS is also problematic due to structural reasons: the proposed site is rigidly folded and does not appear to be available for intramolecular autophosphorylation.…”

“…Regardless, nuclear translocation of both active and inactive MAPKs may also be mediated by several ␤-importins. For example, importin-3, importin-7, and importin-9 have all been implicated in mediating JNK nuclear entry (138). The use of these different importin systems could thus provide multiple nuclear entry modes for other MAPKs (142) as well as the JNKs.…”

The nuclear translocation of ERK1/2 as an anticancer target

“…In addition, we have recently shown that the mechanism of translocation of ERK1/2 is distinct from the other mitogenactivated protein kinases (MAPKs) JNK1/2 and p38a/b (ref. 23).…”

p38β Mitogen-Activated Protein Kinase Modulates Its Own Basal Activity by Autophosphorylation of the Activating Residue Thr180 and the Inhibitory Residues Thr241 and Ser261

“…S246 of ERK2 is the homologous residue of T241 of p38␤ and is generally very well conserved in all MAPKs (see below). However, in p38␤, T241 phosphorylation has no role in nuclear translocation (46) but rather has a role in the regulation of autophosphorylation activity. In addition to T241, we have identified two more previously unnoticed sites, S143 and S261.…”