A Golgi-Localized ERK

Science's STKE  28 Sep 2004:
Vol. 2004, Issue 252, pp. tw344
DOI: 10.1126/stke.2522004tw344

Extracellular signal-regulated kinases (ERKs) are members of the mitogen-activated protein kinase (MAPK) family best known for being activated by phosphorylation in response to various stimuli, after which these proteins can accumulate in the nucleus and regulate gene expression by phosphorylation of nuclear targets. Bind et al. revisited the ERK3 member of this family, which has been reported to have undergone substantial divergence between the rat and the mouse and human homologs in terms of protein size and localization. Resequencing of the rat gene revealed a missing G. The protein predicted by this revised sequence is essentially identical to the mouse and human forms, and the size of the predicted protein was confirmed by in vitro translation and immunoblot analysis with an antibody against the C-terminal region. The previously reported form was truncated and did not include this C-terminal region. More interestingly, the authors used this C-terminally directed antibody to show localization of ERK3 to the Golgi and endoplasmic reticulum-Golgi intermediate compartment (ERGIC). When transfected cells expressing a green fluorescent protein (GFP)- and FLAG-tagged version of ERK3 (GFP-FL-ERK3) were analyzed, the protein was detected in the Golgi and ERGIC compartments and in the nucleus with the C-terminal antibody. In contrast, an antibody that recognizes a region within the N-terminal catalytic domain only labeled GFP-FL-ERK3 in the nucleus. Furthermore, truncation of the C terminus produced a form that was only detected in the speckled nuclear pattern, suggesting that a motif in the C terminus may be responsible for the Golgi and ERGIC localization and that cleavage of the C terminus may be a mechanism for regulation of ERK3 localization. Indeed, ERK3 has the sequence KHLN in its C-terminal domain, which is predicted to be an ER retention and retrieval motif, and mutation of the lysine resulted in an increase in the proportion of cells exhibiting a completely nuclear localization. The abundance of ERK3 in the nucleus varied with the cell cycle, based on the analysis of synchronized cultured cells, such that ERK3 was shifted from the Golgi and ERGIC compartments to the nucleus after G1. After completion of mitosis, ERK3 was predominantly localized to the Golgi and ERGIC. In the presence of proteasome inhibitors, a fragment under 80 kD was detected with the C-terminal antibody, consistent with the possibility that C-terminal cleavage contributes to ERK3 regulation. Deletion of the region containing two potential caspase-like cleavage motifs in the C-terminal region of ERK3 resulted in loss of cleavage products and a decrease in the proportion of cells in which ERK3 was localized to the nucleus. Thus, ERK3 may be retained in internal membranes and cleaved through a cell cycle-regulated mechanism to release a truncated form that can translocate to the nucleus.

E. Bind, Y. Kleyner, D. Skowronska-Krawczyk, E. Bien, B. D. Dynlacht, I. Sánchez, A novel mechanism for mitogen-activated protein kinase localization. Mol. Biol. Cell 15, 4457-4466 (2004). [Abstract] [Full Text]