Phosphoinositide-dependent kinase 1 (PDK1) is at the hub of many signalling pathways, activating PKB and PKC isoenzymes, as well as p70 S6 kinase and perhaps PKA. PDK1 action is determined by colocalization with substrate and by target site availability, features that may enable it to operate in both resting and stimulated cells.

ORAI1 constitutes the store-operated Ca 2+ release-activated Ca 2+ (CRAC) channel crucial for life. Whereas ORAI1 activation by Ca 2+ -sensing STIM proteins is known, still obscure is how ORAI1 is turned off through Ca 2+ -dependent inactivation (CDI), protecting against Ca 2+ toxicity. Here we identify a spatially-restricted Ca 2+ /cAMP signaling crosstalk critical for mediating CDI. Binding of Ca 2+ -activated adenylyl cyclase 8 (AC8) to the N-terminus of ORAI1 positions AC8 near the mouth of ORAI1 for sensing Ca 2+ . Ca 2+ permeating ORAI1 activates AC8 to generate cAMP and activate PKA. PKA, positioned by AKAP79 near ORAI1, phosphorylates serine-34 in ORAI1 pore extension to induce CDI whereas recruitment of the phosphatase calcineurin antagonizes the effect of PKA. Notably, CDI shapes ORAI1 cytosolic Ca 2+ signature to determine the isoform and degree of NFAT activation. Thus, we uncover a mechanism of ORAI1 inactivation, and reveal a hitherto unappreciated role for inactivation in shaping cellular Ca 2+ signals and NFAT activation.

In mammals exclusively, the pore-forming Ca2+ release-activating Ca2+ (CRAC) channel subunit, Orai1, occurs in two forms due to alternative translation initiation. The longer, mammal-specific Orai1α contains an additional 63 amino acids upstream of the conserved start site for Orai1β, which occurs at methionine 64 in Orai1α. Orai1 participates in the generation of three distinct Ca2+ currents, including two store-operated currents, Icrac, which involves the Ca2+-sensing protein STIM1 activation of Orai1 channels, and Isoc, which involves a tertiary interaction among Orai1, the transient receptor potential (TRP) family member TRPC1, and STIM1. Orai1 is also a pore-forming subunit of an arachidonic acid (leukotriene C4)-regulated current Iarc that involves interactions of Orai1, Orai3, and STIM1. Here, we evaluated the roles of the two Orai1 forms in the Ca2+ currents Icrac, Isoc, and Iarc. We found that Orai1α and Orai1β are largely interchangeable for Icrac and Isoc, although Orai1α exhibits stronger inhibition by Ca2+. Only the mammalian-specific Orai1α functions in the arachidonic acid-regulated current, Iarc. Thus, alternative translation initiation of Orai1 message produces at least three types of Ca2+ channels with distinct signaling and regulatory properties.