Year: 2004
Type: review
Abstract: During the course of its lifecycle, a plant may experience extremes of temperature and moisture, excesses and deficiencies of minerals, and challenges from herbivores and pathogens, all of which provide specific signals for altered plant growth and development. Ca2+ has been widely implicated as an intracellular messenger of physiologically and environmentally induced signaling pathways in plants (Trewavas and Malho, 1998). Because cellular Ca2+ levels are tightly regulated, small changes in intracellular Ca2+ can provide information for the modification of enzyme activity and gene expression needed for subsequent responses. Signals may trigger changes in the cellular Ca2+ signature (for example, an oscillation in the cytosolic free Ca2+ concentration), which is then perceived by various intracellular Ca2+ sensors/binding proteins to regulate a series of signaling cascades (Snedden and Fromm, 1998; Zielinski, 1998). Ca2+ sensors can be classified into sensor responders and sensor relays (Sanders et al., 2002). Upon binding Ca2+, sensor responders change their conformation and modulate their own activity or function through intramolecular interactions. In plants, the best-characterized sensor responders are the Ca2+-dependent protein kinases (Harmon et al., 2001). Ca2+-dependent protein kinases have protein kinase and calmodulin-like Ca2+- binding domains in a single protein, which allows their direct activation by Ca2+ (for review, see Cheng et al., 2002; Hrabak et al., 2003). In contrast, sensor relays such as calmodulin communicate the changed conformation to interacting partner(s) such as protein kinases, resulting in a change in kinase activity. A number of mechanisms have been postulated to account for the wide-ranging role of Ca2+ in signaling in plants as well as for the specificity resulting from the Ca2+ signal. These include qualitative and quantitative information resident in the Ca2+ signal itself and in the Ca2+ sensors that perceive and translate the Ca2+ signal. The potential for different sensors to contribute to functional specificity is further magnified by the presence of families of interacting proteins such as protein kinases. This review focuses on recent progress in the characterization of the salt overly sensitive (SOS) 3 family of Ca2+ sensors and their associated SOS2 family of protein kinases. Recent genetic, molecular, and biochemical studies provide evidence that these two families of proteins interact differentially to form complexes that function in signaling pathways in Arabidopsis during growth and development and in response to abiotic stresses. In a genetic screen designed to identify components of the mechanisms controlling salt tolerance in Arabidopsis, several SOS genes were identified. One of these genes, SOS3, encodes a novel EF-hand Ca2+ sensor (Liu and Zhu, 1998). SOS3 shares significant sequence similarity with the regulatory calcineurin B subunit from yeast (Saccharomyces cerevisiae) and neuronal Ca2+ sensors from animals (Klee et al., 1988). In spite of this similarity with calcineurin B at the primary sequence level, it is now clear that Arabidopsis does not have calcineurin and that the SOS3 Ca2+- sensing protein activates a protein kinase and not a protein phosphatase. SOS3 is a small myristoylated protein that appears to have no enzymatic activity by itself; Ca2+ binding and myristoylation are required for SOS3 function in salt tolerance (Ishitani et al., 2000). The SOS2 gene encodes a novel Ser/Thr protein kinase that also functions in salt tolerance in Arabidopsis (Liu et al., 2000). SOS2 contains an N-terminal kinase catalytic domain similar to that found in the Suc nonfermenting1 (SNF1) and AMP-activated (AMPK) kinases (Hardie, 1999) and a novel C-terminal regulatory domain. SOS3 has been shown to interact physically with SOS2 in yeast two-hybrid assays as well as in vitro (Halfter et al., 2000). Moreover, SOS3 activates SOS2 kinase activity in a Ca2+- dependent manner and sos3/sos2 double-mutant analysis also indicates that SOS3 and SOS2 function in the same pathway (Halfter et al., 2000). The first target of the SOS3-SOS2 regulatory pathway to be identified is the plasma membrane Na+/H+ exchanger (antiporter) encoded by the SOS1 gene. SOS1 gene expression during salt stress is partially controlled by SOS3 and SOS2 (Shi et al., 2000), and activation of SOS1 Na+/H+ antiport activity requires SOS3 and SOS2 (Qiu et al., 2002). Recent studies using yeast have provided additional evidence for the interaction between SOS3 and SOS2 and their regulation of SOS1. Coexpression of SOS1, 2, and 3 dramatically enhanced the salt tolerance of a yeast mutant in which all endogenous Na+ transporters had been removed (Quintero et al., 2002). The SOS3-SOS2 kinase complex phosphorylates and activates SOS1 expressed in yeast, enhancing Na+ exclusion and increasing NaCl tolerance (Quintero et al., 2002). Expression of a constitutively activated SOS2 mutant also increased salt tolerance in yeast expressing SOS1, implying that SOS2 kinase activity is partially sufficient for SOS1 activation. These results provided functional evidence that the SOS proteins function in the same signaling pathway that mediates ion homeostasis and salt tolerance in Arabidopsis (Fig. 1; Zhu, 2002). In addition, recent studies demonstrating that overexpression of SOS1 or of constitutively active SOS2 mutant kinases improves the salt tolerance of Arabidopsis (Guo et al., 2003; Shi et al., 2003) suggest that co-overexpression of SOS1, SOS2, and SOS3 may dramatically increase salt tolerance in plants. Diagram showing that the SOS3-SOS2 signaling module functions in a salt stress-elicited Ca2+ signaling pathway, which mediates salt tolerance. Similarly, various SCaBP-PKS complexes have been implicated in Ca2+ signaling pathways in response to abscisic acid (ABA), sugar, high pH, or drought and cold stresses. Since the initial isolation and characterization of the SOS3 Ca2+ sensor and its interacting protein kinase SOS2, many genes encoding SCaBPs and SOS2-like protein kinases (PKS) have been identified in Arabidopsis through the Arabidopsis Genome Initiative (2000; Hrabak et al., 2003). Summarized here is recent progress in our understanding of the functional domains, biochemical properties, interaction specificities, gene expression patterns, and physiological functions of these Ca2+ sensors and their interacting protein kinases. Six SOS3-like Ca2+ sensor/binding proteins (designated SCaBP1-6) were identified in Arabidopsis from a search of the GenBank database, and the corresponding cDNAs were isolated by reverse transcriptase-PCR (Guo et al., 2001). SCaBP1, SCaBP5, and SCaBP6 had been previously referred to as the calcineurin B-like proteins calcineurin B-like (CBL)2, CBL1, and CBL3, respectively (Kudla et al., 1999). Three additional SCaBPs have recently been identified from Arabidopsis (Luan et al., 2002). Like SOS3, these SCaBPs have no apparent enzymatic activity by themselves and thus are sensor relays (Sanders et al., 2002). These SCaBP proteins are predicted to possess three typical EF-hand Ca2+-binding motifs with the Ca2+-binding loops flanked by E and F helices (Guo et al., 2001; Luan et al., 2002). The tripeptide LYD at the junction of the E helix and the Ca2+-binding loop in the second EF-hand is deleted in the sos3-1 mutant allele (Liu and Zhu, 1998), and this sequence is conserved in the SCaBPs. A recent study of the three-dimensional structure of SCaBP1/CBL2 has shown that this protein contains two pairs of EF-hand motifs; one pair (the first and fourth) binds two Ca2+ ions, whereas the other pair (the second and third) remains in an open conformation (Nagae et al., 2003a, 2003b). Although there is currently no experimental evidence for SOS3 binding to Na+, the possibility cannot be excluded that SOS3 might serve as a Na+ sensor based on the ability of Na+ to bind within the EF-hand motifs of other proteins (Henzl et al., 2000; Ward et al., 2003). Several SCaBPs, including SOS3 and SCaBP5, are associated with membrane fractions (Ishitani et al., 2000; Luan et al., 2002). This membrane localization is consistent with the idea that many Ca2+-signaling events are initiated by Ca2+ fluxes across membranes (Rudd and Franklin-Tong, 2001). At least three SCaBPs, SOS3, SCaBP4, and SCaBP5, contain a conserved N-myristoylation motif, MGxxxS/T[K] (Towler et al., 1988). This motif has been shown to be functional and is necessary for SOS3 function in salt tolerance (Ishitani et al., 2000) and for SOS3 to recruit SOS2 to the plasma membrane in yeast (Quintero et al., 2002; J.-K. Zhu and J.M. Pardo, unpublished data). It is possible that this cotranslational modification may help tether these Ca2+ sensors and their interacting protein kinases to specific membrane patches where Ca2+ flux takes place and/or where target proteins are localized or may contribute to pathway regulation by a Ca2+-myristoyl switch (Ishitani et al., 2000). Twenty-three novel Arabidopsis SOS2-like protein kinases (PKS2-24, also known as CBL-interacting protein kinases) have also been identified using yeast two-hybrid assays and database searches (Guo et al., 2001; Hrabak et al., 2003). All of these proteins contain a conserved FISL motif (also known as an NAF domain), a 21-amino acid sequence in the regulatory domain (Fig. 2; Halfter et al., 2000; Albrecht et al., 2001; Guo et al., 2001). cDNAs were isolated for all PKS proteins and their sequences, and domain structures were compared. As had been found for SOS2, the deduced amino acid sequences of all PKS kinases contain an N-terminal kinase catalytic domain and a C-terminal regulatory domain. Analysis of the N-terminal kinase domain indicated that the PKS kinases are related to the SNF1 kinase from yeast and the AMPK protein kinases in animals (Hardie et al., 1998). Based on sequence similarity and domain structure, three subgroups of SNF1-related kinases (SnRK1-3) have been recognized in plants (Hardie, 2000; Hrabak et al., 2003); the PKS proteins are members of the plant-specific SnRK3 group (Halford et al., 2000; Hrabak et al., 2003). Mechanisms of activation of PKS kinases in vitro and in vivo. PKS proteins consist of an N-terminal kinase catalytic domain (PKS kinase) and a C-terminal regulatory domain. An activation loop (stippled bar) is shown in the kinase catalytic domain, whereas the regulatory domain contains a FISL motif (for interaction with SCaBPs), a PPI motif (for interaction with protein phosphatase 2Cs), and an unknown functional motif(s) at the C-terminal end (indicated by X). The kinase is proposed to be kept inactive by an intramolecular interaction between the catalytic and regulatory domains. In vitro, the kinase can be activated by a T to D mutation in amino acid 168 to mimic phosphorylation of the activation loop by a putative upstream kinase (A, top panel), by deletion of the autoinhibitory FISL motif (A, middle panel), or by a combination of the two mutations (A, bottom panel). In vivo, the kinase is proposed to be activated by the binding of Ca2+-SCaBP to the FISL motif (B, to1p panel), by phosphorylation of T168 in the activation loop (B, middle panel), or by a combination of both (B, bottom panel). An alignment of the deduced amino acid sequences of all 24 PKS kinases shows that the N-terminal kinase domains of these proteins are highly conserved and contain a putative “activation loop” (Guo et al., 2001; Gong et al., 2002b; Fig. 2A). The conserved FISL motif is found in the regulatory domain and is adjacent to the kinase catalytic domain (Guo et al., 2001; Fig. 2A). The FISL motif is responsible for binding to SCaBPs and has a consensus sequence XL (or M, I, F, Y) N (or T)AFD (or E, Q) I (or L, F, M)IS (or A, T, G, L, I) L (or M, F, Y, S, T, G)SXG (or F, D, E, S) F (or L, S) D (or N, S, G)LS (or A, E, G) G (or N, S, T, P) L (or F)FE (or D, G, A) (where X is any amino acid residue and residues in bold are absolutely conserved; Guo et al., 2001). Next to the FISL sequence is a protein phosphatase interaction (PPI) motif that mediates interaction with type 2C protein phosphatases (Ohta et al., 2003; Fig. 2A). The catalytic and regulatory domains of SOS2 have been shown to interact with each other, and this interaction requires the FISL motif (Guo et al., 2001; Fig. 2A). Intramolecular interaction in SOS2, and presumably other PKS proteins, is probably responsible for keeping the kinases inactive, as removal of the regulatory domain of SOS2 led to its constitutive activation (Guo et al., 2001; Fig. 2). It is likely that upon the binding of SCaBPs to the FISL motif, this intramolecular interaction is relieved, to the activation of the kinases (Fig. of kinase activity results from protein phosphorylation by and/or by regulatory domains or et al., 1998). As with other protein kinases, a for regulation of the PKS kinases may be phosphorylation of one or residues within the activation loop et al., 2000; and 2001). In the of specific SCaBP PKS kinases have kinase activity expressed and from as The activation loop in the kinase catalytic domain of the PKS proteins is between the conserved and sequences Hrabak et al., 2003). A residue in in this activation loop is conserved in all the PKS kinases. constitutively active of the kinases were through a of the T residue with D to partially mimic phosphorylation by an upstream (Guo et al., 2001; Gong et al., Fig. 2A). These results suggest that activation loop by an upstream may an role in the regulation of PKS kinase activity in (Fig. Although the upstream kinase is it is possible that the protein phosphatase that bind to PKS may function to the activation loop (Ohta et al., 2003). the putative activation loop of these PKS kinases, two additional and are also conserved et al., J.-K. Zhu, unpublished data). any of these residues to D in SOS2 activates the kinase in vitro et al., These results suggest that may be for activation of SOS2 (or kinase activity. studies these three residues are phosphorylation in SOS2 or other PKS kinases in vivo. analysis has shown that the FISL motif in the regulatory domain of the SOS2 protein is autoinhibitory to the phosphorylation of the PKS kinases. FISL motif deletion in SOS2 and several other PKS proteins that were also constitutively active (Guo et al., 2001; Gong et al., 2002b; Fig. 2A). the T168 to D mutation with the FISL SOS2 and PKS kinases were (Guo et al., 2001; Gong et al., 2002b; et al., 2002; Fig. 2A). It is PKS activation by Ca2+-SCaBP and by activation loop phosphorylation place in vivo. both activation mechanisms may function and (Fig. The biochemical of the PKS kinases are not well in to the in and PKS kinases from plants. PKS proteins in are active in not in the of constitutively active PKS has characterization of of the biochemical of these These PKS kinases a for and not protein including protein, and three from the sequences of protein kinase or (Halfter et al., 2000; Gong et al., and for with or have been and the PKS kinases, including SOS2, and to for phosphorylation and et al., This may the of these kinases in a complex during activation et al., The apparent of of the constitutively activated PKS kinases for and has also been The of kinase activity on or typical The apparent from to and from to for and and inactive SOS2 that have different structures have been to the PKS kinase The protein which the FISL domain and C-terminal amino have been to the mutant N which a conserved amino acid in the catalytic required for changed to Gong et al., This that other kinases and et al., 2000; et al., SOS2 takes place an intramolecular other PKS kinases using a similar remains to be In the physiological of the PKS kinases are several of evidence that SOS1, a Na+/H+ is one physiological of SOS2 in the C-terminal of the SOS1 protein contains several putative consensus motifs found in the of the SOS2 a SOS1 protein from yeast membranes be in vitro by the mutant (Quintero et al., 2002). SOS3-SOS2 activates the Na+/H+ antiport activity of SOS1 (Qiu et al., 2002). Recent evidence indicates that on the membrane may also be or for activity from proteins in isolated from and of Arabidopsis (Qiu et al., 2003). activity in and this activity be activated SOS2 protein in Coexpression of SOS2 and the in yeast growth on high levels of Ca2+ and activated activity et al., 2003). SOS2 regulation of the activity of these transporters not on the presence or activity of SOS3 and appears to mechanisms that not changes in phosphorylation of the transporters (Qiu et al., 2003; Cheng et al., J.-K. Zhu, unpublished data). SOS2 regulation of Na+/H+ on the and plasma membrane additional that this kinase an role in the of cellular Na+ homeostasis and is a of the salt tolerance in In to for SOS2, these studies suggest between the regulation of Na+ and Ca2+ homeostasis in In and in vitro assays have been to the interaction specificity of members of the SCaBP and PKS families (Halfter et al., 2000; et al., 2000; Guo et al., 2002). SCaBPs can interact with PKS kinases and PKS kinases interact with several SCaBP proteins can interact with one protein, and several SCaBP members not interact with any of the PKS kinases et al., 2001; Guo et al., that these members interact with other PKS proteins or have protein As of these specificities, SOS3 with SOS2 (Halfter et al., 2000), whereas with SOS2 and shows interaction with (Guo et al., 2001). An in interaction between and has also been (Guo et al., 2002). These are the highly conserved FISL motif in the kinase regulatory domain (Guo et al., 2001). within the FISL motif and/or in structures likely which PKS with which and the interaction specificity between the SCaBP and PKS proteins may provide the for of the functional specificity of these proteins in plant SOS2, as well as several other PKS proteins, has been shown to interact with the type 2C protein and (Guo et al., 2002; et al., 2003). A novel protein domain of amino acid residues in the SOS2 kinase C-terminal regulatory as the PPI motif, is necessary and sufficient for interaction with the protein The PPI motif is not conserved in the PKS protein kinases, also in the and from various including (Ohta et al., 2003). in conserved amino acid residues in the PPI motif the interaction of SOS2 with In contrast, a mutation in the FISL motif the interaction between SOS2 and SOS3, has no on the interaction between SOS2 and A protein domain in the protein phosphatase (Ohta et al., 2003). The interaction between SOS2 and is by the mutation in the domain, and the mutation increased tolerance to salt as well as in plants (Ohta et al., 2003). the interaction specificity of several PKS and proteins PKS members were found to interact with whereas with (Ohta et al., 2003). in the PPI motif of PKS may a PKS with or other related protein example, residues and of SOS2 are conserved in and which all interact with not In contrast, these two residues are changed to and T in which likely with with in the domain of the protein phosphatase may its interaction with a specific Although the protein with SOS2, of and within the domain with corresponding residues and in led to an interaction with SOS2 as as that of (Ohta et al., 2003). The functional specificity of the SCaBPs and PKS kinases is not by their biochemical properties, and their interacting proteins, also by the and expression of their Expression of a number of the SCaBPs and PKS kinases have been SCaBP genes are expressed differentially in various and in response to (Kudla et al., Guo et al., 2002). example, is expressed in whereas SCaBP6 is constitutively expressed in and Expression of or SCaBP6 is not induced by or whereas expression is by these and by the (Luan et al., a potential of the Ca2+ sensor in and abiotic stress responses. have been to the of SCaBP gene expression in Arabidopsis (Guo et al., 2002). in and of and also in the and of plants (Guo et al., 2002). These results suggest that different members of the SCaBP family may function in at specific and in response to PKS members also specific expression example, is expressed in and is not in of plants et al., In contrast, expression in is that in or et al., is also in the in other is the These PKS genes also expression in response to a of stresses. example, expression is by and is not by cold et al., no significant or of expression is any of these The expression of the SCaBPs and PKS kinases functions for their gene in plant growth and as well as in the response of the plant to The expression of SCaBP and PKS genes and the of these proteins that may have physiological functions in the The in functions of several SCaBP and PKS proteins have been recently using and reverse genetic changes in protein as well as regulation of gene these proteins have been implicated in the response to and signaling and in cellular regulation (Fig. an role in plant growth and as well as in the response of the plant to is evidence that changes in protein phosphorylation may be an of Because is known to Ca2+ it likely that PKS kinases and SCaBPs also a role in the response of the plant to Arabidopsis plants with altered levels of the were for their response to plants a constitutively active form of were to during and whereas the kinase gene by et al., Arabidopsis in which or its interacting had been were found to be to during and In addition, of a number of genes and 1998), including and enhanced in the and (Guo et al., 2002). These results that and related Ca2+ sensors and interacting protein kinases may be of signaling in plants (Guo et al., 2002). Because has been shown to the of Ca2+ second messenger in have that may intracellular Ca2+ levels in response to and regulate Ca2+ (Guo et al., 2002). with may be for the of Ca2+ It is possible that the complex Ca2+ oscillation Ca2+- and et that overexpression of enhanced drought cold of stress the mutant in Arabidopsis increased cold and drought of stress a mutant of CBL-interacting protein kinase et that this kinase is in The mutant plants were sensitive type to during and and expression of a number of and genes altered in the In a manner similar to plant can as signaling that gene expression and in plants and 2002). Recent evidence indicates that the PKS proteins may also be in a constitutively active expressed in Arabidopsis plants, the plants were to high levels of Based on with between the and plants in their to the or Gong et al., it is likely that functions in Ca2+ signaling in response to a and that its function may be of the pathway et al., 1999). of and proteins help to the role of this kinase in responses. The SOS2-like protein, has been shown to be in the regulation of intracellular homeostasis through its on the plasma membrane Guo et al., unpublished data). with SCaBP1, not with by or have been to the role of this kinase in the response to various with the growth of were to growth at and to at a assays using isolated plasma membrane that the activity of the plasma membrane in the mutant with activity in isolated from plants. activated protein kinase in vitro, activity in the mutant to phosphorylates the of the plasma membrane and this phosphorylation in the presence of the interacting Ca2+-binding protein, Guo et al., unpublished data). The novel families of SCaBP Ca2+ sensors and PKS protein kinases to Ca2+- that and Ca2+ signals during plant growth and development and in response to abiotic (Fig. Although significant progress has been in our understanding of the biochemical and physiological of of these proteins, many to be mechanisms Ca2+ sensing and kinase activity this the structures of the SCaBP-PKS complexes to be are the upstream that the or residues in the putative activation loop of the PKS is the between the regulation of PKS activity in by Ca2+-SCaBP and by the upstream mechanisms may in vivo, and may function and are the in functions of the SCaBP and PKS This information be using and reverse genetic the physiological and of each SCaBP-PKS pair be required to their In studies of and mutant plants, it be to at the of as well as in response to and abiotic stresses. are the in of the PKS progress has been to SCaBP-PKS kinase signaling pathways in Arabidopsis, as is known these signaling in other plant These signaling are likely conserved different plant the number and combination of the SCaBPs and may studies that genetic, and to the of SCaBP and PKS proteins in Ca2+ signaling in plants.
Source: PLANT PHYSIOLOGY
Institution: University of Arizona
Language: English
Subfield: Plant Science
Domain: Life Sciences
Open Access status: bronze
OpenAlex ID: W2016618351