Defining a spatially restricted aPKC/ERK phosphoproteome


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Nick Peel1, Carine Rosse1, Katrina Boeckeler1, Peter Parker1
1CR-UK London Research Institute, London, UK

Background

The formation and turnover of extracellular matrix adhesions is vital for efficient cell migration and invasion. The dynamic behaviour of these adhesions is controlled by various localised signalling events. In migrating NRK cells an atypical protein kinase C (aPKC), exocyst and kibra complex translocates to the leading edge to drive activation of the MAP kinases ERK1/2 and phosphorylation of the focal adhesion adapter protein paxillin. The spatially restricted signalling of ERK1/2 downstream of aPKC is required for regulating cell migration and appears to control disassembly of adhesions at the leading edge.

Method

To isolate targets of this pathway, a bioinformatic candidate search approach has been used to screen the adhesome for proteins containing canonical ERK phosphorylation motifs. Immunofluorescence, siRNA knockdown and phosphorylation motif analysis have been exploited to characterise putative ERK1/2 substrates that are candidates in the local control of focal adhesion turnover. One such target, parvin alpha, is described here.

Results

Morphological changes are observed in parvin alpha containing focal adhesions following PKC or MEK inhibition and paxillin containing focal adhesions appear enlarged following parvin alpha knockdown by siRNA. These changes in adhesion architecture are comparable to the effects seen following aPKC knockdown/inhibition. Leading edge reactivation of ERK in a background of aPKC inhibition has demonstrated a rescue of parvin alpha phosphorylation at a conserved N-terminal serine residue. This has lead to analysis of two potential ERK phosphorylation sites, which conform to the canonical ERK motif. Antisera to these have been developed to enable assessment of the localised behaviour of parvin alpha upon activation of the aPKC/ERK cascade.

Conclusion

The application of bioinformatic screening has proved a useful approach for identifying players in this adhesion pathway and parvin alpha represents one of those whose distribution to focal adhesions and posttranslational modification is susceptible to aPKC/ERK intervention.