Project Summary/Abstract Active motility by apicomplexan parasites controls host cell invasion and egress, steps that are critical to the intracellular cycle of infection. These processes are governed by calcium-regulated secretion of adhesive proteins from micronemes, together with the concerted action of an actin-myosin motor that lies beneath the parasite plasma membrane. Prior studies in Toxoplasma gondii have established the importance of calcium- dependent protein kinases and protein kinase G (PKG) in controlling microneme secretion, motility, egress, and invasion. This cascade is tightly regulated and recent studies have also shown that the c1 isoform of protein kinase A (PKAc1) dampens calcium signaling to shut down microneme secretion and impair motility after invasion is complete. The balance between PKG vs. PKAc1 activities governs the decision to activate motility and promote egress vs. to remain intracellular. Although the role of PKAc1 in blocking premature egress is clear, neither its mechanism of regulation nor the targets that it phosphorylates in order to dampen calcium signaling and block egress have been elucidated. PKA activity is governed by production of cyclic AMP (cAMP) by adenylate cyclases (ACs) and its consumption by phosphodiesterases (PDEs). In preliminary studies, we have identified candidate ACs that control cAMP levels to regulate PKA. In the proposed studies, we will explore their functions to define how they temporally and spatially regulate PKA activity. We have also used a protein degradation approach to create conditional knockdowns of PKA isoforms and verify that PKAc1 controls egress in type II parasites. We will use the tightly regulated conditional knockdown approach to investigate downstream targets of PKAc1 that mediate the egress block using a combination of phosphoproteomic studies to identify substrates and downstream validation studies to test their roles in regulating egress. We will validate the role of specific phosphorylation sites in PKAc1 targets in suppressing calcium signaling, motility, and egress. Collectively, the proposed studies will elucidate the molecular pathway by which PKAc1 down regulates calcium levels, microneme secretion, and motility to prevent egress, thus counterbalancing the activating effects of PKG. Elucidating the molecular regulation of the PKG/PKA kinases in T. gondii will foster future studies to design inhibitors that block these pathways, thus providing new avenues for development of therapeutics.