In brief

Using BioID, researchers found a close relationship between cancer-promoting protein PAK4 and a neighbouring protein, Afadin, which helps PAK4 stay anchored to cell membranes.

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Cancer cell stake-out reveals new associations

10 Aug 2022

A molecular tagging tool developed in Singapore identifies novel protein interactions on the surface of cancer cells.

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).

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Baskaran, Y., Tay, FP., Ng, EYW., Swa, CLF., Wee, S., et al. Proximity proteomics identifies PAK4 as a component of Afadin-Nectin junctions. Nature Communications 12, 5315 (2021) | article

About the Researchers

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Edward Manser

Senior Principal Investigator, Head, sGSK Laboratory

Institute of Molecular and Cell Biology (IMCB)
Edward Manser runs the multidisciplinary sGSK lab which investigates how cells regulate their shape. He obtained an undergraduate degree in Biochemistry at the University of Bristol, and PhD under the supervision of Peter M Bayley at the National Institute for Medical Research (NIMR), London. He has spent most of his career working in Singapore; he previously worked with several A*STAR initiatives, including the Neuroscience Research partnership (NRP). As a Research Fellow at IMCB, he developed an interest in the biochemical pathways downstream of the newly discovered Rho small GTPases and was promoted to principal investigator, working under the GSK-IMCB Trust. He also headed the IMCB Proteomic facility from 2003 to 2006. Currently, he holds an Associate Professor appointment and is a Senior Principal Investigator in IMCB.
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Yohendran Baskaran

Senior Research Fellow, sGSK lab

Institute of Molecular and Cell Biology (IMCB)
Yohendran Baskaran graduated from the National University of Singapore (NUS), Medical School and worked several years within the Ministry of Health, rotating through the various hospitals and in private practice before pursuing a Biochemistry degree at the University of Cambridge, UK. He then returned to Singapore and took up a PhD program at the Institute of Molecular and Cell Biology (IMCB)/NUS, graduated in 2009 and has since been part of the sGSK lab team that specialises in the study of the cell cytoskeleton. A major part of his work is to study PAK4, a member of the group II family of PAK proteins and he has published several papers on the regulation and activation of PAK4 and related proteins.
Felicia Pei-Ling Tay joined the Institute of Molecular and Cell Biology (IMCB) in 2003 right after she graduated from the National University of Singapore (NUS). She was part of Liu Ding Xiang’s lab and worked on virulence factors of several infectious viruses including SARS and IBV. In 2010, she continued her research journey with Edward Manser, working on cell adhesion and signalling pathways in tissue plasticity and remodelling. She was recruited in 2016 to work in Frederic Bard's lab on an IMCB-IPSEN industry collaborative project to uncover elusive host genes involved in botulinum neurotoxin intoxication in neurons.

This article was made for A*STAR Research by Wildtype Media Group