Peter Parker : Protein Phosphorylation

The Laboratory’s interests are in mapping, monitoring and intervening in the signalling network that controls tumourassociated properties including growth, survival and migration/invasion. Altered functions of key nodes within this network are known to be drivers of aberrant behaviour, and many targeted therapeutics are consequently directed at these nodes. In particular, our work is focussed around the PKC superfamily. Members of this serine/threonine protein kinase family are variously implicated in growth, survival and migration/invasion. We are engaged in understanding how specific family members exert their control, how they themselves fit into the signalling network, what value intervention can have and, more broadly, what generic lessons might be learned from their study.

Migration and polarisation

PKN3 has been reported to be over-expressed in prostate tumours and to be involved in invasive behaviour. In addressing the general penetrance of PKN requirements, we have assessed models that display distinct patterns of PKN1-3 expression. In the 5637 bladder tumour cell model, PKN2 is the dominant expressed isoform and it also dominates PKN-requirements in migration and invasion. Use of PKN1/2/3 chimera has demonstrated that it is the regulatory domain wiring that drives specificity of action in this model.

A second series of migratory studies centres around the aPKCs. We have shown previously that an aPKC-Exocyst complex is involved in triggering ERK activation at the leading edge. We have shown, using rapalogue recruitment of an activated MEK allele to the leading edge, that ERK comprises a partially sufficient element driving migration downstream of aPKCs. We have retained this recruitment ‘trick’ to map the ERK substrates that reside at the leading edge and are responsible for controlling focal adhesion turnover. aPKC isotypes have also been clearly implicated in the polarisation of epithelial cells; a characteristic lost on transformation. We have exploited the migratory and polarised cell models to assess aPKC interventions, to derive substrates/biomarkers of aPKC action and to probe aPKC function when polarisation is lost following transformation. We continue to collaborate with Banafshe Larijani (Cell Biophysics Laboratory) to develop tumour biomarkers.

Finally, in relation to the subcellular compartmentation of pro-migratory signals, we have continued our collaboration with Dr Stephanie Kermorgant (QMUL) to understand where and how distinct events downstream of HGF-activated cMet are triggered. This has uncovered distinct, spatially restricted processes governed by activated cMet residing in different compartments. In assessing the movement of cMet between these compartments it was found that the kinesin motor inhibitor ATA, blocks cMet activity via an allosteric site on the receptor.

Cell division

Prior studies from the lab have identified a role for PKCε in controlling the completion of cytokinesis. A key feature of PKCε engagement is its accumulation at the furrow when selectively inhibited. The recruitment of PKCε and its domain requirements have been under investigation as a means of determining binding partner(s) at this site. Linked to this analysis, we have identified earlier cell cycle events that predispose cells to a requirement for PKCε. The underlying mechanisms associated with theseearlier events are currently being investigated, but appear to be associated with a delay/checkpoint for which PKCε is required. We are particularly interested in determining how the sensitivity or resistance to PKCe loss of function maps to this control point, and whether aberrations of cell cycle control in transformed cells provides a therapeutic index for PKCε intervention.

Structure, function and intervention

We continue to collaborate with Neil McDonald (Structural Biology Laboratory) on the structural analysis of PKC isoforms. This relates both to the aPKC-directed drug discovery programme with Cancer Research Technology and to the highresolution dissection of targeting mechanisms for substrates. The aPKC kinase domains structureswith bound inhibitor have permitted our development of a drug insensitive form of aPKCι, which we have employed extensively in substrate/biomarker and transcriptional signature screens. This has identified multiple candidate substrates that we are assessing specifically as biomarkers (pharmacodynamic and/or predictive markers). Amongst these are putative substrates that we believe will provide mechanistic insights into the migratory, polarised and transformed models described above. While these studies have relied on a designated ‘tool’ compound, the drug development programme itself has yielded very potent and selective aPKC inhibitors. We anticipate that the proximal targets and transcriptional signature we identify will feed in to this drug development programme. One aspect of aPKC function that has emerged from the structural programme, is the means by which aPKC phosphorylates one of its key polarity targets. The docking of this protein outside the substrate binding pocket, has been found to be essential for its aPKC-dependent phosphorylation in cells.

In respect of PKC family priming events, we have identified a general process associated with the TORC2 complex that appears to determine the
targeting of AGC kinases by mTOR (Figure 1). It transpires that PKC interacts with the CRIM domain of Sin1. Interestingly, inducible expression of a ΔCRIM Sin1 construct can be exploited to dissect the role of TORC2 in this, and other, AGC kinase pathways. ΔCRIM Sin1 assembly into endogenous TORC2 complexes disrupts CRIM domain recruitment and phosphorylation of target kinases is lost. We are exploiting this behaviour to assess the consequences of selectively switching TORC2 off in various contexts.