It is now clear MAPK signalling is considerably more intricate than originally believed. Once considered to occur only at the plasma membrane, signalling is now known to originate from endomembranes including the Golgi, ER and endosomes, as well as from specific plasma membrane compartments. The cellular domain from which MAPK signals is an important factor in determining how the cell responds to a particular stimulus. This localisation is partly under the regulation of post-translational modifications, such as farnesylation. However, it is becoming evident that molecular scaffolds are integral to spatio-temporal regulation of signalling.

The combination of spatial, temporal and amplitude regulation, coupled with the large number of external factors known to activate the MAPKs, provides a glimpse of a complex network of pathways and how cells are able to interpret diverse signals to appropriately respond to their environment.

While many of the MAPK components have been targeted for therapy, as yet this strategy has failed to yield effective pharmacotherapeutic agents. The lack of efficacy may be due, at least in part, to the lack of selectivity when inhibiting a key signalling kinase, such as MEK, Raf or p38. Because these kinases regulate myriad cellular functions, inhibiting their activity is likely to affect multiple processes, some not linked to the pathophysiology of the disease being targeted. Therefore, new approaches are being explored to enhance specificity. Different strategies could be adopted to modulate signalling from a specific compartment. An indirect approach would be to inhibit proteins that alter the subcellular localisation of MAPKs. This concept has been shown in principle with the development of FTIs, which prevent membrane localisation of Ras. The efficacy is limited by the relative non-specificity of FTIs, which prevents Ras from anchoring into any membrane. However, as we gain further insight into the mechanisms controlling the location of the MAPKs, more specific targets may be identified. For example, inhibition of as yet unidentified chaperone proteins might prevent Ras from localising to the Golgi, but not affect localisation of Ras to the plasma membrane.

A second approach would be to develop small molecules that are, themselves, localised to specific cellular compartments. For example, by restricting a small molecule inhibitor of B-Raf to the endosome, B-Raf signalling from that compartment would be antagonised, while signalling from other compartments, such as the plasma membrane, would remain unaffected. This concept has been established in principle, with the development of fluorescent probes that localise to specific subcellular compartments and organelles (Zorov et al. 2004). This idea is further supported by the development of a specific peptide inhibitor of calmodulin, which was selectively localised to the nucleus or plasma membrane. Antagonism of cal-modulin function in discrete subcellular domains differentially modulates cellular responses (Li et al. 2003; Mataraza et al. 2007). It is therefore theoretically possible to target inhibitors of MAPKs to specific cellular domains.

An alternative strategy to modulate compartmentalised MAPK signalling is to manipulate the function of protein scaffolds. These molecules regulate many aspects of the MAPK cascade and may offer an opportunity to provide the specificity required for effective therapy. Several MAPK scaffolds have been identified, and it is likely that other, as yet unidentified, molecules exist. Therefore, scaffolds provide a reservoir of potential targets, each of which controls highly specific aspects of MAPK function.

Despite the intense effort directed by scientists in both academia and industry, the progress in developing effective therapies for malignant or inflammatory disease by targeting MAPK signalling has been disappointing. While preclinical findings have been promising, toxicity and/or a poor therapeutic response has prevented widespread adoption of MAPK inhibitors as pharmacotherapeutic agents. We look forward to advances in the understanding of MAPK signalling, including the role played by molecular scaffolds, and how this translates into the development of specific, targeted pharmacologic agents.

Acknowledgements We thank Rob Krikorian for assistance with preparation of the manuscript. Work in the authors' laboratory is funded by grants from the National Institutes of Health.

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