The unique C terminus of the calcineurin isoform CNAß1 confers non-canonical regulation of enzyme activity by Ca2+ and calmodulin.

Calcineurin, the conserved Ca2+/calmodulin-regulated phosphatase and target of immunosuppressants, plays important roles in the circulatory, nervous, and immune systems. Calcineurin activity strictly depends on Ca2+ and Ca2+-bound calmodulin (Ca2+/CaM) to relieve autoinhibition of the catalytic subunit (CNA) by its C terminus. The C terminus contains two regulatory domains, the autoinhibitory domain (AID) and calmodulin-binding domain (CBD), which block the catalytic center and a conserved substrate-binding groove, respectively. However, this mechanism cannot apply to CNAß1, an atypical CNA isoform generated by alternative 3'-end processing, whose divergent C terminus shares the CBD common to all isoforms, but lacks the AID. We present the first biochemical characterization of CNAß1, which is ubiquitously expressed and conserved in vertebrates. We identify a distinct C-terminal autoinhibitory four-residue sequence in CNAß1, 462LAVP465, which competitively inhibits substrate dephosphorylation. In vitro and cell-based assays revealed that the CNAß1-containing holoenzyme, CNß1, is autoinhibited at a single site by either of two inhibitory regions, CBD and LAVP, which block substrate access to the substrate-binding groove. We found that the autoinhibitory segment (AIS), located within the CBD, is progressively removed by Ca2+ and Ca2+/CaM, whereas LAVP remains engaged. This regulatory strategy conferred higher basal and Ca2+-dependent activity to CNß1, decreasing its dependence on CaM, but also limited maximal enzyme activity through persistence of LAVP-mediated autoinhibiton during Ca2+/CaM stimulation. These regulatory properties may underlie observed differences between the biological activities of CNß1 and canonical CNß2. Our insights lay the groundwork for further studies of CNß1, whose physiological substrates are currently unknown.

Calcineurin, the Ca2+-dependent phosphatase, regulates Rga2, a Cdc42 GTPase-activating protein, to modulate pheromone signaling.

Calcineurin, the conserved Ca2+/calmodulin-activated phosphatase, is required for viability during prolonged exposure to pheromone and acts through multiple substrates to down-regulate yeast pheromone signaling. Calcineurin regulates Dig2 and Rod1/Art4 to inhibit mating-induced gene expression and activate receptor internalization, respectively. Recent systematic approaches identified Rga2, a GTPase-activating protein (GAP) for the Cdc42 Rho-type GTPase, as a calcineurin substrate. Here we establish a physiological context for this regulation and show that calcineurin dephosphorylates and positively regulates Rga2 during pheromone signaling. Mating factor activates the Fus3/MAPK kinase, whose substrates induce gene expression, cell cycle arrest, and formation of the mating projection. Our studies demonstrate that Fus3 also phosphorylates Rga2 at inhibitory S/TP sites, which are targeted by Cdks during the cell cycle, and that calcineurin opposes Fus3 to activate Rga2 and decrease Cdc42 signaling. Yeast expressing an Rga2 mutant that is defective for regulation by calcineurin display increased gene expression in response to pheromone. This work is the first to identify cross-talk between Ca2+/calcineurin and Cdc42 signaling and to demonstrate modulation of Cdc42 activity through a GAP during mating.

The molecular mechanism of substrate engagement and immunosuppressant inhibition of calcineurin.

Ser/thr phosphatases dephosphorylate their targets with high specificity, yet the structural and sequence determinants of phosphosite recognition are poorly understood. Calcineurin (CN) is a conserved Ca(2+)/calmodulin-dependent ser/thr phosphatase and the target of immunosuppressants, FK506 and cyclosporin A (CSA). To investigate CN substrate recognition we used X-ray crystallography, biochemistry, modeling, and in vivo experiments to study A238L, a viral protein inhibitor of CN. We show that A238L competitively inhibits CN by occupying a critical substrate recognition site, while leaving the catalytic center fully accessible. Critically, the 1.7 Å structure of the A238L-CN complex reveals how CN recognizes residues in A238L that are analogous to a substrate motif, "LxVP." The structure enabled modeling of a peptide substrate bound to CN, which predicts substrate interactions beyond the catalytic center. Finally, this study establishes that "LxVP" sequences and immunosuppressants bind to the identical site on CN. Thus, FK506, CSA, and A238L all prevent "LxVP"-mediated substrate recognition by CN, highlighting the importance of this interaction for substrate dephosphorylation. Collectively, this work presents the first integrated structural model for substrate selection and dephosphorylation by CN and lays the groundwork for structure-based development of new CN inhibitors.

Multiple mechanisms actively target the SUN protein UNC-84 to the inner nuclear membrane.

Approximately 100 proteins are targeted to the inner nuclear membrane (INM), where they regulate chromatin and nuclear dynamics. The mechanisms underlying trafficking to the INM are poorly understood. The Caenorhabditis elegans SUN protein UNC-84 is an excellent model to investigate such mechanisms. UNC-84 recruits KASH proteins to the outer nuclear membrane to bridge the nuclear envelope (NE), mediating nuclear positioning. UNC-84 has four targeting sequences: two classical nuclear localization signals, an INM sorting motif, and a signal conserved in mammalian Sun1, the SUN--nuclear envelope localization signal. Mutations in some signals disrupt the timing of UNC-84 nuclear envelope localization, showing that diffusion is not sufficient to move all UNC-84 to the NE. Thus targeting UNC-84 requires an initial step that actively transports UNC-84 from the peripheral endoplasmic reticulum to the NE. Only when all four signals are simultaneously disrupted does UNC-84 completely fail to localize and to function in nuclear migration, meaning that at least three signals function, in part, redundantly to ensure proper targeting of UNC-84. Multiple mechanisms might also be used to target other proteins to the INM, thereby ensuring their proper and timely localization for essential cellular and developmental functions.

UNC-83 coordinates kinesin-1 and dynein activities at the nuclear envelope during nuclear migration.

Nuclei migrate during many events, including fertilization, establishment of polarity, differentiation, and cell division. The Caenorhabditis elegans KASH protein UNC-83 localizes to the outer nuclear membrane where it recruits kinesin-1 to provide the major motor activity required for nuclear migration in embryonic hyp7 cells. Here we show that UNC-83 also recruits two dynein-regulating complexes to the cytoplasmic face of the nucleus that play a regulatory role. One consists of the NudE homolog NUD-2 and the NudF/Lis1/Pac1 homolog LIS-1, and the other includes dynein light chain DLC-1, the BicaudalD homolog BICD-1, and the Egalitarian homologue EGAL-1. Genetic disruption of any member of these two complexes caused nuclear migration defects that were enhanced in some double mutant animals, suggesting that BICD-1 and EGAL-1 function in parallel to NUD-2. Dynein heavy chain mutant animals also had a nuclear migration defect, suggesting these complexes function through dynein. Deletion analysis indicated that independent domains of UNC-83 interact with kinesin and dynein. These data suggest a model where UNC-83 acts as the cargo-specific adaptor between the outer nuclear membrane and the microtubule motors kinesin-1 and dynein. Kinesin-1 functions as the major force generator during nuclear migration, while dynein is involved in regulation of bidirectional transport of the nucleus.

UNC-83 is a nuclear-specific cargo adaptor for kinesin-1-mediated nuclear migration.

Intracellular nuclear migration is essential for many cellular events including fertilization, establishment of polarity, division and differentiation. How nuclei migrate is not understood at the molecular level. The C. elegans KASH protein UNC-83 is required for nuclear migration and localizes to the outer nuclear membrane. UNC-83 interacts with the inner nuclear membrane SUN protein UNC-84 and is proposed to connect the cytoskeleton to the nuclear lamina. Here, we show that UNC-83 also interacts with the kinesin-1 light chain KLC-2, as identified in a yeast two-hybrid screen and confirmed by in vitro assays. UNC-83 interacts with and recruits KLC-2 to the nuclear envelope in a heterologous tissue culture system. Additionally, analysis of mutant phenotypes demonstrated that both KLC-2 and the kinesin-1 heavy chain UNC-116 are required for nuclear migration. Finally, the requirement for UNC-83 in nuclear migration could be partially bypassed by expressing a synthetic outer nuclear membrane KLC-2::KASH fusion protein. Our data support a model in which UNC-83 plays a central role in nuclear migration by acting to bridge the nuclear envelope and as a kinesin-1 cargo-specific adaptor so that motor-generated forces specifically move the nucleus as a single unit.