Our finding that ERK3 resides in the ERGIC and the Golgi was unexpected because MAPKs are thought to be soluble cytoplasmic enzymes. Because ERK3 lacks a trans-membrane domain, its association with these membranous organelles must rely on relatively stable interactions with other molecules in these compartments. We have explored the contribution of a carboxy-terminal dilysine motif (KHLN) and find that it stabilizes but is not sufficient for the association of the full-length form of ERK3 with the Golgi. Importantly, this motif is not present in any other member of the MAPK family, including the highly related p63 MAPK, which lacks the carboxy-terminal KHLN motif that is unique to ERK3.
Remarkably, an examination of the subcellular localization of GFP-ERK3 in synchronized cells reveals that nuclear entry increases as cells progress through S phase, concomitant with diminished Golgi partitioning. Reentry into the subsequent cell cycle was accompanied by a dramatic reversal as ERK3 localization shifts from the nucleus back to the Golgi. These studies further revealed a nuclear form of the protein that was carboxy-terminally truncated, suggesting that proteolytic cleavage releases ERK3 from the Golgi. Thus, ERK3, like other members of the MAPK family, translocates to the nucleus, but it does so through a wholly unique mechanism not shared by other ERKs. This interesting mechanism of regulating the nuclear translocation of ERK3 via a temporal sequestration in the Golgi raises the question as to the purpose for this precise localization. It is conceivable that ERK3 could have two different sets of substrates, one located in the cytoplasm and the other in the nucleus. In support of this hypothesis, we have found that both forms of this enzyme are produced and are active in cells stably expressing an epitope-tagged version of the protein in in vitro kinase assays in which myelin basic protein, histone H1, or H3 (unpublished data) was used as substrates. Residency in the Golgi may therefore be part of a mechanism that ensures that ERK3 does not have access to nuclear substrates at an improper time.
The presence of ERK3 in the Golgi further raises the question as to whether this enzyme has any role in the biology of this organelle. Malholtra and colleagues have implicated an unidentified MAPK in the fragmentation of the Golgi before mitosis (Acharya et al, 1998
; Colanzi et al, 2003
). Moreover, we find that both the PLK and cdc2/B cyclin, two enzymes also reported to participate in this process, robustly phosphorylate ERK3 in vitro (data not shown). Thus, although the role of ERK3 in Golgi biology has not been the focus of this report, the answer to this question will be of great interest to cell biologists and is the subject of an ongoing investigation. Finally, our study of ERK3 corroborates previous reports that there is a nuclear form of ERK3 and establishes for the first time that it is generated via proteolysis of a region within the carboxy-terminal part of the protein somewhere between amino acids 380–600. Thus, we are left with the very interesting question of the identity of the ERK3 specific proteolytic activity. In this report, we have uncovered several important clues as to the nature of the ERK3 protease. Our experiments indicate that the exit of ERK3 from the Golgi is principally mediated by an acidic patch spanned by amino acids 383–398. Second, ERK3 cleavage occurs in cycling cells. Thus, the ERK3 protease must be active in growing cells and may demonstrate some preference for short stretches of acidic residues. These requirements have led us to begin to consider caspases and/or the proteasome. Caspases prefer to cleave carboxy-terminal to acidic residues, and have been best studied in cells undergoing apoptosis. However, cell permeable caspase inhibitors have been shown to inhibit the growth of proliferating T cells (Fischer et al., 2003
) Moreover, negative regulators of the cell cycle such as Wee1 and CDC27 are cleaved by caspases (Fischer et al., 2003
). Finally, caspase 2 has been found to associate with the Golgi and to promote cleavage of golgin 160 during apoptosis thereby promoting disintegration of the Golgi (Mancini et al., 2000
). Thus, it is conceivable that a nonapoptotic caspase activity could be the ERK3 protease.
Our detection of ERK3 cleavage in growing cells with active proteasomes, the stabilization of ERK3 protein levels by proteasome inhibitors, and our analysis of the 383–98 deletion mutant also have led us to consider whether the ERK3 protease might be the postacidic cleavage activity specific to the β1 subunits of the 20S proteasome (Kisselev and Goldberg, 2001
). This preference of the proteasome for cleavage after short stretches of acidic residues has, however, been studied exclusively with peptide substrates and has not been demonstrated with a protein. The proteasome is a particularly attractive candidate for the ERK3 protease because a number of proteins, including the transcription factor nuclear factor-κB (NF-κB) have been shown to be undergo limited proteolytic processing by this protease. Indeed, the nuclear translocation of NF-κB is dependent on the proteasomal generation of the 55-kDa form of the IκB subunit from a 110-kDa precursor (Palombella et al., 1994
). A direct test of whether the proteasome can generate a carboxy-terminally truncated protein similar in size to the nuclear form of ERK3 from the full-length protein will require the reconstitution of ERK3 site-specific proteolysis in vitro by using purified proteasomes. This very interesting experiment is, however, well beyond the scope of this report.
We propose here that our biochemical data, together with our microscopic studies, are consistent with site-specific cleavage of the full-length form of ERK3, resulting in the nuclear translocation of the cleaved protein. In addition we find that the subcellular localization of ERK3 also is temporally regulated in cycling cells. Many of the details of this rather elaborate mechanism governing the intracellular location of ERK3 are not currently understood, but given that ERK3 is an MAPK, it is likely that phosphorylation may play a role. In conclusion, our study of ERK3 provides the first example of a mechanism linking a proteolytic activity to the nuclear translocation of a member of the MAPK family.