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 PIP 2 and PIP 3 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 that PI4KA lipid kinase activity can be inhibited through tyrosine phosphorylation of the kα12 helix of the kinase domain (Y2090). This site can be phosphorylated by multiple tyrosine kinases. Structural studies using cryo-EM and HDX-MS defined the mechanism of how Y2090 phosphorylation inhibits activity, with this being driven through local conformational changes in the kα12 C-terminal helix of the PI4KA kinase domain. PI4KA activity is predominantly controlled through membrane recruitment by EFR3, with phosphorylation inhibiting the EFR3 tethered PI4KA complex. Phosphorylation of the kα12 C-terminal helix is found in multiple PI3Ks and PI4Ks, suggesting this may be an evolutionarily conserved regulatory mechanism for this family of phosphoinositide kinases. Overall, our work reveals novel molecular insight into inhibitory post-translational regulation of PI4KA.