Research - Schumaker Lab

Mechanisms controlling calcium-mediated development

Calcium has emerged as a critical component of many pathways in plants, underlying growth and development by linking perception of endogenous and environmental cues to cellular responses. Because cellular calcium levels are tightly regulated, small changes in cytosolic calcium concentration provides information for the modification of enzyme activity and gene expression needed for the subsequent responses. Calcium signals are perceived, modified and propagated through the action of proteins that bind calcium -- the appropriate response to a specific calcium signal requires activation of specific calcium-binding proteins. In plants, a number of calcium-binding proteins have been identified including calmodulin (CaM), CaM-like proteins, Salt-Overly-Sensitive (SOS3)-like/Calcineurin B-like (CBL) proteins and calcium-dependent protein kinases (2, 4, 15). When calcium binds to one of these sensors, the structural and/or enzymatic properties of the sensor change and its subsequent interactions with target proteins can alter solute transport, enzymatic activities, cytoskeletal orientation, protein phosphorylation cascades and gene expression (2, 9). The specific response is determined by the diverse tissue expression patterns of the calcium-binding protein, by changes in expression of the calcium-binding protein in response to developmental and environmental stimuli, by the affinity of the calcium-binding protein for calcium, by the range of target proteins with which the calcium-binding proteins interact as well as by the abundance and activity of these target proteins.

In a genetic screen designed to identify components of the mechanisms controlling salt tolerance in Arabidopsis, several SOSgenes wereidentified (Fig. 1 – SOS model). One of these genes, SOS3, encodes a novel EF-hand calcium sensor (14). SOS3 is a small myristoylated proteinthat appears to have no enzymatic activity on its own; calcium binding and myristoylation are required for SOS3 function in salt tolerance (10). SOS3 has been shown to interact physically with the serine/threonine protein kinase SOS2 in yeast two-hybrid assays as well as in vitro (13). SOS3 activates SOS2 kinase activity in a calcium-dependent manner (8). The SOS3/SOS2 complex activates the expression and activity of SOS1, a plasma membrane Na+/H+ exchanger involved in theregulation of cellular Na+ ion homeostasis during salt stress (17, 18, 21).

Nine SOS3-like calcium sensor/binding proteins (also identified as AtCBL calcium-binding proteins) have been identified in Arabidopsis (12). Like SOS3, these calcium-binding proteins have no apparent enzymatic activity by themselves and thus are sensor relays (20). The CBL proteins are predicted to possess three to four typical EF-hand calcium-binding motifs flanked by E and F helices (16). Several calcium-binding proteins, including SOS3/CBL4 and CBL1, are associated with membrane fractions (10, 16); this membrane localization is consistent with the idea that many calcium signaling events are initiated by calcium fluxes across membranes (19). At least three calcium-binding proteins, SOS3/CBL4, CBL5 and CBL1, contain a conserved N-myristoylation motif (10). It is possible that this co-translational modification may help tether these calcium sensors to specific membrane patches where calcium flux takes place and/or where target proteins are localized or may contribute to pathway regulation by a calcium-myristoyl switch (10).

Members of the CBL family have been implicated in calcium-dependent responses to environmental signals when the plant experiences abiotic stress (3, 7, 14). With funding from the Energy Biosciences Program at the Department of Energy, we are working to add to our understanding of the functions of this important family of calcium-binding proteins. To do this, we are determining: (i) the extent and nature of CBL interactions with other proteins (in vitro and in vivo interaction assays), (ii) the developmental processes regulated by the CBL proteins (phenotypic analysis of loss-of-function mutants, localization of CBL gene activities and protein accumulation) and (iii) if CBL interactions result in additional functions during calcium signaling.

The CBL proteins share sequence similarity to the regulatory B subunit of calcineurin. Calcineurin has been identified in organisms from yeast to mammals as a heterodimer with a calcium-binding regulatory subunit (Calcineurin B) and a catalytic calcium/calmodulin-regulated protein phosphatase subunit (Calcineurin A). In Arabidopsis, a Calcineurin A-like 2B-type protein phosphatase has not been identified; instead, a family of SNF1-like serine/threonine protein kinases (SOS2-like or Calcineurin B-like Interacting Protein Kinases, CIPKs) has been identified as calcium-dependent targets for the CBLs (5, 8, 11, 21), interacting through a FISL or NAF domain in the CIPK protein (1, 6, 7). Each CBL protein may interact with several CIPK proteins and certain CIPK proteins can interact with several CBLs. Common interactions between CBLs and CIPKs may allow cross-talk between signaling cascades while preferential associations between CBLs and CIPKs may give rise to specific signaling cascades.

The Arabidopsis genome contains twenty-five CIPKs (12) which can be divided into two sub-groups based on the presence or absence of introns. To add to our understanding of the functions of this novel family of protein kinases, we are determining: (i) the developmental processes mediated by the CIPK proteins (phenotypic analysis of single and higher order mutants, localization of CIPK gene activities and protein accumulation), (ii) which CBLs regulate CIPK activity (in vitro and in vivo interaction assays), (iii) the substrate targets for CIPK phosphorylation, and (iv) the biochemical mechanisms that regulate CIPK substrate phosphorylation.

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