677.8 - Palmitoylation Targets the Calcineurin Phosphatase to the Phosphatidylinositol 4-kinase Complex at the Plasma Membrane

Martha Cyert (Stanford University), Idil Ulengin-Talkish (Stanford University), Matthew Parson (University of Victoria), Meredith Jenkins (University of Victoria), Jagoree Roy (Stanford University), Alexis Shih (University of British Columbia), Nicole St-Denis (University of Toronto), Gergo Gulyas (National Institute of Child Health and Human Development), Tamas Balla (National Institute of Child Health and Human Development), Anne-Claude Gingras (University of Toronto), Péter Várnai (Semmelweis University), Elizabeth Conibear (University of British Columbia), John Burke (University of Victoria)

Calcineurin (CN), the serine/threonine protein phosphatase and the target of immunosuppressants, is a critical regulator of Ca2+ signaling. To discover novel CN-regulated processes, we examined an understudied isoform, CNAb1, that is highly conserved across vertebrates and expressed in every human tissue. CN is a heterodimer of regulatory and catalytic subunits whose functions and regulation by Ca2+ and calmodulin are well understood for canonical CN isozymes. The few studies of CNAb1, demonstrate its unique physiological and biochemical properties, however, mechanisms that confer these properties and CNAb1- specific substrates remain unknown. Here, we show that the non-canonical C-terminus of CNAb1, generated by alternative 3’ pre-mRNA processing, confers unique intracellular localization, enzymatic regulation and substrate specificity for this isoform.  In vitro, CNAb1 displays distinct enzymatic properties, where instead of the auto-inhibitory domain that blocks the active site of canonical CN isoforms under non-signaling conditions, CNAβ1 is autoinhibited by a sequence motif at its C` tail that blocks substrate binding. In cells, unlike canonical cytosolic CN, CNAb1 localizes to the plasma membrane and Golgi due to palmitoylation of its divergent C-terminal tail. This palmitoylation, which is dynamically regulated by the ABHD17A depalmitoylase, targets CNAb1 to distinct set of membrane-associated interactors including the phosphatidylinositol 4-kinase (PI4KA) complex containing EFR3B, PI4KA, TTC7B and FAM126A. This complex recruits the cytosolic PI4KIIIA to the PM where it synthesizes phosphatidylinositol-4-phosphate (PI4P), a precursor of the critical signaling phospholipid, PI(4,5)P2, required for sustained Ca2+ signaling through GPCRs. Using hydrogen-deuterium exchange, we reveal multiple CN-PI4KA complex contacts, including a calcineurin-binding peptide motif in the disordered tail of FAM126A which we further establish as a novel calcineurin substrate. Using BRET-based detection of phosphoinositides in live cells, we show that CN inhibitors decrease PM PI4P production during Gq-coupled muscarinic receptor signaling, suggesting that CN dephosphorylates and promotes PI4KA complex activity. Together, this work not only discovers dynamic palmitoylation as a novel mechanism that confers unique localization, substrate specificity and regulation to CNAb1 but also uncovers a novel Ca2+/CN-mediated, feedback loop, that promotes PI4P replenishment at the PM during GPCR signaling.

M.S.C. and I.U.T.: National Institute of Health R01GM129236 and R35GM136243. J.E.B.: Discovery grant from the Natural Science and Engineering Research Council of Canada (NSERC-2020-04241, JEB) and Michael Smith Foundation for Health Research (JEB, Scholar Award 17686).
G.G. and T.B.: Intramural Research Program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development of the NIH.
A.C.G.: Canadian Institutes of Health Research (FDN 143301).
P.V.:Hungarian National Research, Development, and Innovation Fund (NKFIK134357).
E.C.: Canadian Institutes of Health Research (PJT-162184).