Uncovering The Unique Functions And Regulation Of The Palmitoylated Calcineurin Isoform, CNβ1

Calcineurin (CN), the serine/threonine protein phosphatase and the target of immunosuppressants FK506 and CysA, is a key regulator of Ca 2+ signaling with critical roles in the immune, cardiovascular and nervous systems. CN is a heterodimer of regulatory and catalytic subunits whose functions and regulation by Ca 2+ and calmodulin are well understood for canonical CN isozymes (CNalpha, CNβ2). In contrast, the CN β 1 isozyme, contains a catalytic subunit with a non‐canonical C‐terminus generated by alternative 3’ pre‐mRNA processing. The few studies of CNβ1, conserved in eukaryotes and broadly expressed in human tissues, demonstrate its unique physiological functions. For example, overexpression of the CNβ2 promotes cardiac hypertrophy through activation of NFAT, whereas CNβ1 is cardioprotective and does not dephosphorylate NFAT. However, CNβ1 specific substrates remain unknown. We determined that the unique C‐tail confers distinct regulatory properties , intracellular localization and function to CN β 1 . In vitro, CNβ1 displays distinct enzymatic properties. Instead of the auto‐inhibitory domain that blocks the active site of canonical CN isoforms under non‐signaling conditions, CNβ1 is autoinhibited by a sequence motif at its C` tail that blocks substrate binding. We show that CNβ1 localizes to the plasma membrane (PM), Golgi, and intracellular vesicles, in contrast to the cytosolic CNβ2, due to the palmitoylation of two conserved cysteine residues unique to its C‐tail. Palmitoylation allows CNβ1 to access substrates that are distinct from canonical CN isoforms. CNβ1 preferentially interacts with membrane proteins, and all members of a highly conserved PI4‐kinase complex, PI4KIIIA, TTC7B, FAM126A and EFR3B, were identified as CNβ1‐specific interactors by AP‐MS. 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)P 2 , required for sustained Ca 2+ signaling through GPCRs. We identify a CN binding motif in FAM126A, mutation of which results in FAM126A hyperphosphorylation, and prevents association of CNβ1 with the PI4KIIIA complex. Using BRET‐based detection of phosphoinositides in live cells, we show that CN promotes PI4P synthesis at the PM during signaling through the muscarinic receptor, and hypothesize that CNβ1 mediates this regulation by dephosphorylating FAM126A at the PM. Using pulse‐chase analysis in cells metabolically labelled with a palmitate analog, we demonstrate that palmitoylation of CNβ1 is dynamic and turns over rapidly. We are currently investigating whether dynamic palmitoylation regulates localization and activity CNβ1 activity in vivo during signaling. Together, these studies provide the first mechanistic understanding of CNβ1 and not only uncovers a novel Ca 2+ ‐dependent mechanism for activation of PI4P synthesis at the PM during signaling but also suggests palmitoylation as a novel mechanism that confers spatio/temporal regulation of calcineurin signaling in cells. Support or Funding Information NIGMS

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