This figure shows the scales that we work with in the lab

Neil McDonald : Polarity signalling assemblies at the plasma membrane

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Protein kinase C iota (PKCι) is a serine-threonine kinase that affects many cellular processes, including growth, proliferation, and motility.

Close-up of the CRT0066854 inhibitor binding-site within the PKCι nucleotide-binding pocket

Figure 1A: Close-up of the CRT0066854 inhibitor binding-site within the PKCι nucleotide-binding pocket (cyan). Inhibitor is shown in orange and dashed lines indicate hydrogen-bonds between protein and inhibitor. A bound water molecule and an iodide ion are shown as spheres. B. Schematic of the PKCι kinase domain structure bound to CRT0066854 highlighting the location of the RIPR motif which is required for the engagement of specialised PKCι substrates such as LLGL2.

PKCι associates with two discrete polarity complexes; one containing the polarity proteins Par-3 and Par-6 (PAR complex) and the other contains Crumbs, Stardust and PatJ (the Crbs complex). Both complexes are found in vertebrates and invertebrates where they are crucial for maintaining apical-basal polarity.

Loss of cell polarity is often associated with aggressive epithelial cell cancers, and PKCι is frequently found in abnormally high abundance in epithelial cancers. PKCι is therefore a validated oncogene and potential drug target.

This year we reported the discovery and mode of action of potent PKCi-selective chemical inhibitors (Kjaer et al., 2013; Biochem J. 451(2): 329-42).

These inhibitors were identified in collaboration with the Cancer Research Technology (CRT) Discovery Lab and the Protein Phosphorylation Group.

Prior to publication, few isoform-specific chemical biology tools were available to inhibit PKCι catalytic activity. We determined a crystal structure of PKCι bound to a representative compound, CRT0066854, revealing how it binds within the nucleotide cleft and displaces a crucial Asn-Phe-Asp motif found in many AGC kinases (Figure 1A).

CRT0066854 inhibits phosphorylation of the PKCι substrate LLGL2 in cell lines and exhibits phenotypic effects in a range of cell-based assays.

This compound has been sent out to numerous academic laboratories for use as a chemical biology tool to modulate PKCι/PKCζ activity in vitro and in vivo in a variety of cancer models. During the inspection of the PKCι-CRT0066854 inhibitor structure, we noticed an invariant motif on the surface of PKCi.

In collaboration with the Protein Phosphorylation Group, we explored a possible role for this RIPR motif in protein-protein interaction leading to the discovery that this motif is required to engage the PKCι substrate LLGL2 (Linch et al., 2013; Sci Signal. 6(293): ra82).

Surprisingly, PKCι mutants associated with human cancer were found that target this motif highlighting the importance of PKCι substrate recruitment.