The Arabidopsis calcineurin B-like calcium sensor proteins (AtCBLs) interact with a group of serinethreonine protein kinases (AtCIPKs) in a calciumdependent manner. Here we identify a 24 amino acid domain (NAF domain) unique to these kinases as being required and suf®cient for interaction with all known AtCBLs. Mutation of conserved residues either abolished or signi®cantly diminished the af®nity of AtCIPK1 for AtCBL2. Comprehensive two-hybrid screens with various AtCBLs identi®ed 15 CIPKs as potential targets of CBL proteins. Database analyses revealed additional kinases from Arabidopsis and other plant species harbouring the NAF interaction module. Several of these kinases have been implicated in various signalling pathways mediating responses to stress, hormones and environmental cues. Full-length CIPKs show preferential interaction with distinct CBLs in yeast and in vitro assays. Our ®ndings suggest differential interaction af®nity as one of the mechanisms generating the temporal and spatial speci®city of calcium signals within plant cells and that different combinations of CBL±CIPK proteins contribute to the complex network that connects various extracellular signals to de®ned cellular responses.
Members of the Arabidopsis calcineurin B-like Ca 2 ؉ binding protein (AtCBL) family are differentially regulated by stress conditions. One AtCBL plays a role in salt stress; another is implicated in response to other stress signals, including drought, cold, and wounding. In this study, we identified a group of novel protein kinases specifically associated with AtCBL-type Ca 2 ؉ sensors. In addition to a typical protein kinase domain, they all contain a unique C-terminal region that is both required and sufficient for interaction with the AtCBL-type but not calmodulin-type Ca 2 ؉ binding proteins from plants. Interactions between the kinases and AtCBLs require micromolar concentrations of Ca 2 ؉ , suggesting that increases in cellular Ca 2 ؉ concentrations may trigger the formation of AtCBL-kinase complexes in vivo. Unlike most serine/threonine kinases, the AtCBL-interacting kinase efficiently uses Mn 2 ؉ to Mg 2 ؉ as a cofactor and may function as a Mn 2 ؉ binding protein in the cell. These findings link a new type of Ca 2 ؉ sensors to a group of novel protein kinases, providing the molecular basis for a unique Ca 2 ؉ signaling machinery in plant cells. INTRODUCTIONAmong the extracellular signals eliciting changes in Ca 2 ϩ concentration in the cytoplasm of plant cells are plant hormones, light, stress factors, and pathogenic or symbiotic elicitors (Knight et al., 1991(Knight et al., , 1996(Knight et al., , 1997Neuhaus et al., 1993; Trewavas and Knight, 1994; Ehrhardt et al., 1996;McAinsh et al., 1997; Wu et al., 1997). In addition, many intrinsic growth and developmental processes, such as elongation of the root hair and pollen tube, are accompanied by Ca 2 ϩ transients (Franklin-Tong et al., 1996; Felle and Hepler, 1997; Holdaway-Clarke et al., 1997; Wymer et al., 1997). Because different signals often elicit distinct and specific cellular responses, an interesting question is how do cells distinguish between the Ca 2 ϩ signals produced by different stimuli?Studies with both animal and plant cells suggest that a Ca 2 ϩ signal is represented not only by Ca 2 ϩ concentration but also by spatial and temporal information, including Ca 2 ϩ localization and oscillation (Franklin-Tong et al., 1996; Holdaway-Clarke et al., 1997; Dolmetsch et al., 1998;Li et al., 1998). Although such complexity in Ca 2 ϩ parameters may partially explain the specificity of cellular responses triggered by a particular stimulus, the signaling components that "sense" and "interpret" the Ca 2 ϩ signals hold the key to linking the changes in these parameters to specific cellular responses.If Ca 2 ϩ signaling pathways constitute "molecular relays," the first "runner" after Ca 2 ϩ should be a component that serves as the Ca 2 ϩ "sensor" to monitor changes in Ca 2 ϩ parameters. Such sensors often are proteins that bind Ca 2 ϩ and, in so doing, change conformation in a Ca 2 ϩ -dependent manner. Several families of Ca 2 ϩ sensors have been identified in higher plants. Perhaps the best known is the family of calmodulin (CaM) and CaM-related prot...
Members of the Arabidopsis calcineurin B-like Ca(2)+ binding protein (AtCBL) family are differentially regulated by stress conditions. One AtCBL plays a role in salt stress; another is implicated in response to other stress signals, including drought, cold, and wounding. In this study, we identified a group of novel protein kinases specifically associated with AtCBL-type Ca(2)+ sensors. In addition to a typical protein kinase domain, they all contain a unique C-terminal region that is both required and sufficient for interaction with the AtCBL-type but not calmodulin-type Ca(2)+ binding proteins from plants. Interactions between the kinases and AtCBLs require micromolar concentrations of Ca(2)+, suggesting that increases in cellular Ca(2)+ concentrations may trigger the formation of AtCBL-kinase complexes in vivo. Unlike most serine/threonine kinases, the AtCBL-interacting kinase efficiently uses Mn(2)+ to Mg(2)+ as a cofactor and may function as a Mn(2)+ binding protein in the cell. These findings link a new type of Ca(2)+ sensors to a group of novel protein kinases, providing the molecular basis for a unique Ca(2)+ signaling machinery in plant cells.
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