Insights into the Structural Regulation of Polo-Like Kinase Activity using AlphaFold

Abstract

The Polo Like Kinases including the major family member, PLK1 are key regulatory enzymes controlling the cell cycle and mitosis. PLK1 is associated with poor survival rates in cancer and has been extensively investigated as an oncology drug target. Each member of the Polo like kinase family (PLKs 1-5) have two subdomains with independent functions and include the well conserved N-terminal kinase domain (KD) and the C-terminal polobox domain (PBD). The PBD is involved in the recognition of substrates primed by other kinases and in the PLK1 context is responsible for subcellular localization to specific sites in the nucleus including centrosomes and kinetochores. While the phosphosubstrate recognition site in PLKs 1-3 is highly conserved, its role in PLKs 2 and 3 is not well characterized and phosphopeptides that inhibit PLK1 have dramatically lower affinity for PLKs 2 and 3. An additional role of the PBD is its ability through domain-domain interactions with the KD to regulate PLK1 activity by an autoinhibited state of PLK1, conceptually similar to that which occurs through other kinases. Other mechanisms regulating PLK activity include the interchange between monomeric and dimeric forms, which inhibit or activate PLK1 during the cell cycle. Furthermore, PLK1 may exist as heterodimers with PLK2 and/or PLK3 and thus play context dependent roles. Here, through the use of the AlphaFold (AF) algorithm, structural insights into regulation of activity of the PLK1 and other family members have been obtained. These include dramatically different tertiary arrangement of the individual domains in each individual PLK. Analysis of the domain-domain interactions, interdomain and intradomain loops in each PLK sheds light onto plausible mechanisms by which the activity of each PLK is regulated and provides insights into the selectivity of phosphopeptides. The results also suggest a mechanism for the heterodimerization of PLK1 and PLK2 which has been observed in the literature.