Observing a home’s front door paints a picture of the people who live inside—children hurry out to catch the school bus every morning, groceries get delivered, and the family dog returns from his daily walk. Similarly, by looking at discrete regions on the surface of cancer cells, researchers find clues about what’s going on inside, which in turn, may reveal new therapeutic targets.
Take PAK4, for example. This protein kinase is concentrated at the site where cells contact each other: these cell-cell junctions contain a host of proteins that can promote cancer cell growth. Until now, however, cancer researchers have not identified which neighbouring proteins PAK4 might associate with, and why.
“PAK4 is known to promote survival of cancer cells, but no clear mechanism of action has emerged,” explained Yohendran Baskaran, a Senior Research Fellow at A*STAR’s Institute of Molecular and Cell Biology (IMCB). Baskaran, together with Felicia Pei-Ling Tay, Senior Research Officer, is part of a team led by Edward Manser, a Senior Principal Investigator at IMCB, that is using several molecular toolkits to unravel PAK4’s mysteries.
The researchers started with a hot lead: PAK4 activates proteins nearby via a biochemical process known as phosphorylation. “We wanted to understand what proteins PAK4 associates with, and then phosphorylates,” Baskaran said, adding that they had narrowed their search scope to a tiny region of approximately 20-nanometre radius at the cell-cell junction.
The scientists leveraged an ultrasensitive tagging method developed in Singapore called BioID. This technique allowed them to pin a molecular ‘beacon’ on PAK4 to identify only extremely close proteins. In addition, by adding isotope labelling they were able to rank the level of interaction. Their search turned up 27 close proximal proteins, of which one called Afadin appeared to play a prominent role—removing Afadin caused PAK4 to disappear from the cell membrane.
A deeper look at the Afadin-PAK4 connection revealed something more unexpected. “The striking dependence on Afadin indicates that PAK4 doesn’t work at the conventional sites of cell-cell adherens junctions,” said Manser. “Instead, the results suggest they are in a smaller 'control region' nearby linked to membrane proteins called nectins.” This complex in turn can reorganise the cell-cell junctions in cancer cells, allowing them to thrive.
The team continues to follow the trail of breadcrumbs that emerged from this study: for example, PAK4 phosphorylation sites on neighbouring proteins. “The most important goal is to generate biomarkers of PAK4 activity, allowing us to investigate for the first time which types of cancer are driven by PAK4's action,” explained Manser.
The A*STAR-affiliated researchers contributing to this research are from the Institute of Molecular and Cell Biology (IMCB).