In neurons, the MAP-kinases ERK-1 and ERK-2 are activated under a variety of stimulation conditions that include depolarization, synaptic activation, and growth factors such as BDNF acting through Trk-receptors (Segal et al. 1996
). The patterns of genes expression, however, for each of the various types of stimuli are likely very different (Choudhuri et al. 2002
; Nedivi et al. 1993
). It is possible, and indeed likely, that activation of additional kinases in stimulus-specific manners would result in the differential responses. Although this explanation may work well for distinguishing the different responses to depolarization versus growth factors or neuromodulators, for example, it is less clear how synaptically-induced depolarization is distinguished from depolarization due to hyperkalemic conditions; most calcium-stimulated enzymes are activated under both conditions (Bito et al. 1996
). One way to accomplish a signal cut-off feature, to serve in cases of pathological depolarization, would be to have protein phosphatases closely associated with the active kinase. Associations between MAP-kinases, MAP-kinase-kinases, and appropriate MAP-kinase phosphatases have been well described in yeast (Martin et al. 2005
), and rely on scaffolding molecules to bring the specific molecules together (Whitmarsh et al. 1998
). Consistent with such an association with MAP-kinase phosphatases, our study shows that a complex-associated ERK1 can behave differently from its monomeric counterpart in that it can be dephosphorylated under conditions where the monomeric ERK is not. It remains unknown, however, which MAP-kinase phosphatase would be associated with PERC-160 and why it would be more active under conditions of potassium depolarization. Curiously, the dephosphorylation of CREB only occurs after NMDA receptor activation and not
with potassium depolarization (Sala et al. 2000
Scaffolding functional groups of enzymes together has been increasingly appreciated as a means by which enzymes can be regulated as groups (Ferrell 2000
). In yeast, regulation of the scaffolding molecule itself can enhance or inhibit certain cellular responses depending on protein expression levels; one system demonstrating this behavior prominently features a MAP-kinase (Whitmarsh et al. 1998
). Another function of scaffolding molecules is to localize the complex near its site of action, be it nuclear or cytoplasmic. In the case of PERC-160, the complex is clearly restricted to nuclei, and it is likely too big to exit through nuclear pores. Thus, crosslinking the proteins together could be a way the cell retains ERK in the nucleus without neosynthesis of nuclear retention proteins (Lenormand et al. 1998
). We also find another p-ERK and 14-3-3 reactive band at 70 kDa in the cytosol, but it remains undetermined whether this represents an incomplete version of PERC-160, or whether it serves a unique cytosolic function. Importantly, scaffolding can serve to increase the speed of signaling, a critical feature if ERKs are to regulate genes such as arc
, which can be detected as early as 2 minutes after stimulation (Guzowski et al. 1999
; Waltereit et al. 2001
). This association would certainly not be without trade-offs, however, in that scaffolding ERK to its nuclear substrates and/or upstream activators in such a 1:1:1 ratio would restrict any amplification processes (Kolch 2005
) (). Nevertheless, scaffolding enzyme cascades, particularly those containing ERK, has been established as playing an important role in localizing and regulating enzyme function.
Fig. 7 Proposed ERK-containing complexes in neuronal nuclei. ERK1 and 14-3-3 are contained in PERC-160, with phosphorylated MEK (p-MEK) and phosphorylated p90RSK possibly associated (not confirmed). Phospho-epitopes are most likely to be exposed, with the majority (more ...)
14-3-3 is a small, acidic group of proteins that serves a diverse group of functions. This protein family can either positively modulate enzyme activity, in the cases of serotonin N-acetyltransferase (Obsil et al. 2001
) and Protein Kinase C (Van Der Hoeven et al. 2000
), or negatively modulate activity, such as with CaM-kinase kinase (Davare et al. 2004
). Specifically in the ERK pathways, 14-3-3 does not impact the activity of MEK (Shimizu et al. 1994
), but it has been reported to stimulate the enzyme upstream from MEK, Raf (Freed et al. 1994
) (Irie et al. 1994
) and associates with other upstream kinases in the pathway (Yamamori et al. 1995
). Therefore 14-3-3 is not an unlikely candidate for association with ERK1 in a stable complex. Consistent with this is our finding that along with ERK1, 14-3-3 reactive bands can be electroeluted from PERC-160. The 14-3-3 could only have come from PERC-160 in that the high molecular weight complex was cut out of the gel, with the proteins subsequently removed from by electroelution.
The apparent presence of 14-3-3 in the 70 kDa band in both cytosolic and nuclear fractions could represent an incomplete version of the complex or one that is localizing phospho-ERK to non-nuclear targets in addition to nuclear ones. Because this 14-3-3- and pERK-immunoreactive band was observed inconsistently, however, no conclusions on its function can be made. It is possible that variations in conditions during nuclear purification could have resulted in its leakage from the nucleus. Further work will be required to characterize this possible clue to the composition PERC-160 complex.
For the most part, PERC-160 remains stable through a variety of harsh treatments including boiling in SDS, guanidine, or urea. It is curious, therefore, why immunoreactivity for phospho-ERK at its correct molecular weight (44/42 kDa) increases upon boiling of nuclear fractions, while decreasing slightly in cytosolic fractions (). This was not apparent when the non-phospho-antibodies were used (). The most likely explanation is that the apparent increase in phospho-ERK in the nuclear fraction is due to a small amount phospho-ERK (but not unphosphorylated ERK) falling apart from otherwise stable high molecular weight complexes. Interestingly, this was not accompanied by a decrease in the 160 kDa band, and so one must therefore postulate that the increase in phospho-p44/42 ERK is coming from either an unlabeled source, or else from PERC-160, with PERC-160 being replenished by some higher complex falling apart. Decreases in both the phospho- and non-phospho staining for p44/42 ERK in cytosolic fractions may be due to degradation of the protein under such harsh conditions.
How is it that this ERK1-containing complex is otherwise so stable? A possibility is that transglutaminase acts to crosslink the components of the complex, which makes the complex extremely resistant to denaturation. We show that transglutaminase associates with p-ERK and/or PERC-160 in that it co-immunoprecipitates with p-ERK. Further, an antibody against transglutaminase-mediated isopeptide crosslinks clearly recognizes the PERC-160 band in nuclear preparations. Moreover, one study looking at the distribution of such immunoreactivity for isopeptide crosslinks in brain found some structures in neuronal nuclei in hippocampus (Maggio et al. 2001
). That transglutaminase can be active in nuclei in response to calcium has been described (Lesort et al. 1998
), and transglutaminase associates with importin molecules (Peng et al. 1999
), indicating a possible regulated nuclear function. To date, though, the only nuclear proteins demonstrated to be modified by transglutaminase in response to physiological stimulation are histones (starfish egg fertilization, (Nunomura et al. 2003
)). Transglutaminase is activated under high calcium or low GTP conditions (Lorand et al. 2003
), indicating that it may be activated during times of cellular stress, and nuclear localization of transglutaminase attenuates apoptosis (Milakovic et al. 2004
). Thus it is unclear whether such a transglutaminase-stabilized complex would be formed in a physiological homeostatic role, or inadvertently in a pathological role. These results might have provided us with a mechanism for how the late phase of CREB phosphorylation is attributed to ERK activation without continued stimulation (Impey et al. 1998
; Wu et al. 2001
), but this explanation is unlikely because PERC-160 appears to be dephosphorylated with a time-course similar to that of monomeric ERK in slices. A similar complex containing ERK2 may instead be responsible.
What could be the purpose of having a complex stabilized so permanently? Protection from dephosphorylation or proteolysis is not likely, as PERC-160 is as sensitive to phosphatase and protease activity as p-ERK (data not shown). Also, because levels of the complex do not appear to form immediately after neuronal stimulation, it could be that transglutaminase-stabilized complexes form over much longer time frames; this may be one way that a cell could adjust its nuclear signaling to average neuronal activity over hours or days. Alternatively, the large complex may allow ERK and MEK to be primed for activity in a form too large for export out of the nucleus: MEK has a nuclear export sequence (Fukuda et al. 1996
), and it is thought the unphosphorylated ERK must associate with MEK in order to leave the nucleus (Adachi et al. 2000
). A preformed complex in the nucleus could also have the purpose of facilitating a rapid ERK signaling in response to action potentials by positioning it with its substrates like p90RSK (Adams et al. 2005
; Zhao et al. 2005
). As dimers of phosphorylated ERK1 have the highest levels of ERK activity (Philipova et al. 2005
), the purpose of the complex may also be related specifically to maintain peak activity. Interestingly, stable complexes containing recombinant ERK1 dimers could be generated by incubation with extracts from sea urchin embryos but not from HeLa cells (Philipova et al. 2005
). We propose that 14-3-3 and/or transglutaminase were present in the sea urchin extract.
ERK1 and ERK2 are very similar enzymes, but there is much to be learned about the different roles of the two; further research focusing on PERC-160 may aid in finding how they respond differentially to different types of neuronal stimuli.