Phosphorylation of the C-terminus of PI4KA inhibits lipid kinase activity

Phosphatidylinositol 4-kinase alpha (PI4KA) is a lipid kinase that generates phosphatidylinositol 4-phosphate (PI4P) from phosphatidylinositol (PI) at the plasma membrane (PM). PI4P generated by PI4KA is essential for both plasma membrane identity and for PIP2 and PIP3 signalling driven by the PLC and PI3K family of enzymes. While the structure of PI4KA is known, the regulatory mechanisms that control its activity are undefined. Here, we discovered PI4KA can be inhibited through phosphorylation of Y2090 in the kinase domain, which can be phosphorylated by multiple tyrosine kinases. Y2090 phosphorylation causes local conformational changes in the k12 C-terminal helix. PI4KA activity is predominantly controlled through membrane recruitment by EFR3, with Y2090 phosphorylation inhibiting EFR3 bound PI4KA. Phosphorylation of the k12 C-terminal helix is found in PI3Ks and PI4Ks, suggesting an evolutionarily conserved regulatory mechanism for diverse phosphoinositide kinases. Overall, our work reveals novel molecular insight into inhibitory post-translational regulation of PI4KA.