Transgenic evaluation of activated mutant alleles of SOS2 reveals a critical requirement for its kinase activity and C-terminal regulatory domain for salt tolerance in Arabidopsis thaliana

In Arabidopsis thaliana, the calcium binding protein Salt Overly Sensitive3 (SOS3) interacts with and activates the protein kinase SOS2, which in turn activates the plasma membrane Na(+)/H(+) antiporter SOS1 to bring about sodium ion homeostasis and salt tolerance. Constitutively active alleles of SOS2 can be constructed in vitro by changing Thr(168) to Asp in the activation loop of the kinase catalytic domain and/or by removing the autoinhibitory FISL motif from the C-terminal regulatory domain. We expressed various activated forms of SOS2 in Saccharomyces cerevisiae (yeast) and in A. thaliana and evaluated the salt tolerance of the transgenic organisms. Experiments in which the activated SOS2 alleles were coexpressed with SOS1 in S. cerevisiae showed that the kinase activity of SOS2 is partially sufficient for SOS1 activation in vivo, and higher kinase activity leads to greater SOS1 activation. Coexpression of SOS3 with SOS2 forms that retained the FISL motif resulted in more dramatic increases in salt tolerance. In planta assays showed that the Thr(168)-to-Asp-activated mutant SOS2 partially rescued the salt hypersensitivity in sos2 and sos3 mutant plants. By contrast, SOS2 lacking only the FISL domain suppressed the sos2 but not the sos3 mutation, whereas truncated forms in which the C terminus had been removed could not restore the growth of either sos2 or sos3 plants. Expression of some of the activated SOS2 proteins in wild-type A. thaliana conferred increased salt tolerance. These studies demonstrate that the protein kinase activity of SOS2 is partially sufficient for activation of SOS1 and for salt tolerance in vivo and in planta and that the kinase activity of SOS2 is limiting for plant salt tolerance. The results also reveal an essential in planta role for the SOS2 C-terminal regulatory domain in salt tolerance.     

Biochemical characterization of the Arabidopsis protein kinase SOS2 that functions in salt tolerance

The Arabidopsis Salt Overly Sensitive 2 (SOS2) gene encodes a serine/threonine (Thr) protein kinase that has been shown to be a critical component of the salt stress signaling pathway. SOS2 contains a sucrose-non-fermenting protein kinase 1/AMP-activated protein kinase-like N-terminal catalytic domain with an activation loop and a unique C-terminal regulatory domain with an FISL motif that binds to the calcium sensor Salt Overly Sensitive 3. In this study, we examined some of the biochemical properties of the SOS2 in vitro. To determine its biochemical properties, we expressed and isolated a number of active and inactive SOS2 mutants as glutathione S-transferase fusion proteins in Escherichia coli. Three constitutively active mutants, SOS2T168D, SOS2T168D Delta F, and SOS2T168D Delta 308, were obtained previously, which contain either the Thr-168 to aspartic acid (Asp) mutation in the activation loop or combine the activation loop mutation with removal of the FISL motif or the entire regulatory domain. These active mutants exhibited a preference for Mn(2+) relative to Mg(2+) and could not use GTP as phosphate donor for either substrate phosphorylation or autophosphorylation. The three enzymes had similar peptide substrate specificity and catalytic efficiency. Salt overly sensitive 3 had little effect on the activity of the activation loop mutant SOS2T168D, either in the presence or absence of calcium. The active mutant SOS2T168D Delta 308 could not transphosphorylate an inactive protein (SOS2K40N), which indicates an intramolecular reaction mechanism of SOS2 autophosphorylation. Interestingly, SOS2 could be activated not only by the Thr-168 to Asp mutation but also by a serine-156 or tyrosine-175 to Asp mutation within the activation loop. Our results provide insights into the regulation and biochemical properties of SOS2 and the SOS2 subfamily of protein kinases.     

The structure of the C-terminal domain of the protein kinase AtSOS2 bound to the calcium sensor AtSOS3

The plant SOS2 family of protein kinases and their interacting activators, the SOS3 family of calcium-binding proteins, function together in decoding calcium signals elicited by different environmental stimuli. SOS2 is activated by Ca-SOS3 and subsequently phosphorylates the ion transporter SOS1 to bring about cellular ion homeostasis under salt stress. In addition to possessing the kinase activity, members of the SOS2 family of protein kinases can bind to protein phosphatase 2Cs. The crystal structure of the binary complex of Ca-SOS3 with the C-terminal regulatory moiety of SOS2 resolves central questions regarding the dual function of SOS2 as a kinase and a phosphatase-binding protein. A comparison with the structure of unbound SOS3 reveals the basis of the molecular function of this family of kinases and their interacting calcium sensors. Furthermore, our study suggests that the structure of the phosphatase-interaction domain of SOS2 defines a scaffold module conserved from yeast to human.     

Phosphorylation of SOS3-like calcium-binding proteins by their interacting SOS2-like protein kinases is a common regulatory mechanism in Arabidopsis

The Arabidopsis (Arabidopsis thaliana) genome encodes nine Salt Overly Sensitive3 (SOS3)-like calcium-binding proteins (SCaBPs; also named calcineurin B-like protein [CBL]) and 24 SOS2-like protein kinases (PKSs; also named as CBL-interacting protein kinases [CIPKs]). A general regulatory mechanism between these two families is that SCaBP calcium sensors activate PKS kinases by interacting with their FISL motif. In this study, we demonstrated that phosphorylation of SCaBPs by their functional interacting PKSs is another common regulatory mechanism. The phosphorylation site serine-216 at the C terminus of SCaBP1 by PKS24 was identified by liquid chromatography-quadrupole mass spectrometry analysis. This serine residue is conserved within the PFPF motif at the C terminus of SCaBP proteins. Phosphorylation of this site of SCaBP8 by SOS2 has been determined previously. We further showed that CIPK23/PKS17 phosphorylated CBL1/SCaBP5 and CBL9/SCaBP7 and PKS5 phosphorylated SCaBP1 at the same site in vitro and in vivo. Furthermore, the phosphorylation stabilized the interaction between SCaBP and PKS proteins. This tight interaction neutralized the inhibitory effect of PKS5 on plasma membrane H(+)-ATPase activity. These data indicate that SCaBP phosphorylation by their interacting PKS kinases is a critical component of the SCaBP-PKS regulatory pathway in Arabidopsis.     

Std1p (Msn3p) positively regulates the Snf1 kinase in Saccharomyces cerevisiae

The Snf1 protein kinase of the glucose signaling pathway in Saccharomyces cerevisiae is regulated by an autoinhibitory interaction between the regulatory and catalytic domains of Snf1p. Transitions between the autoinhibited and active states are controlled by an upstream kinase and the Reg1p-Glc7p protein phosphatase 1. Previous studies suggested that Snf1 kinase activity is also modulated by Std1p (Msn3p), which interacts physically with Snf1p and also interacts with glucose sensors. Here we address the relationship between Std1p and the Snf1 kinase. Two-hybrid assays showed that Std1p interacts with the catalytic domain of Snf1p, and analysis of mutant kinases suggested that this interaction is incompatible with the autoinhibitory interaction of the regulatory and catalytic domains. Overexpression of Std1p increased the two-hybrid interaction of Snf1p with its activating subunit Snf4p, which is diagnostic of an open, uninhibited conformation of the kinase complex. Overexpression of Std1p elevated Snf1 kinase activity in both in vitro and in vivo assays. These findings suggest that Std1p stimulates the Snf1 kinase by an interaction with the catalytic domain that antagonizes autoinhibition and promotes an active conformation of the kinase.     

Autoinhibition of mixed lineage kinase 3 through its Src homology 3 domain

Mixed lineage kinase 3 (MLK3) is a serine/threonine protein kinase that functions as a mitogen-activated protein kinase kinase kinase to activate the c-Jun NH(2)-terminal kinase pathway. MLK3 has also been implicated as an I kappa B kinase kinase in the activation of NF-kappa B. Amino-terminal to its catalytic domain, MLK3 contains a Src homology 3 (SH3) domain. SH3 domains harbor three highly conserved aromatic amino acids that are important for ligand binding. In this study, we mutated one of these corresponding residues within MLK3 to deliberately disrupt the function of its SH3 domain. This SH3-defective mutant of MLK3 exhibited increased catalytic activity compared with wild type MLK3 suggesting that the SH3 domain negatively regulates MLK3 activity. We report herein that the SH3 domain of MLK3 interacts with full-length MLK3, and we have mapped the site of interaction to a region between the zipper and the Cdc42/Rac interactive binding motif. Interestingly, the SH3-binding region contains not a proline-rich sequence but, rather, a single proline residue. Mutation of this sole proline abrogates SH3 binding and increases MLK3 catalytic activity. Taken together, these data demonstrate that MLK3 is autoinhibited through its SH3 domain. The critical proline residue in the SH3-binding site of MLK3 is conserved in the closely related family members, MLK1 and MLK2, suggesting a common autoinhibitory mechanism among these kinases. Our study has revealed the first example of SH3 domain-mediated autoinhibition of a serine/threonine kinase and provides insight into the regulation of the mixed lineage family of protein kinases.     

The novel ankyrin-repeat containing kinase ARCK-1 acts as a suppressor of the Spalten signaling pathway during Dictyostelium development

Spalten (Spn), a member of the PP2C family of Ser/Thr protein phosphatases, is required for Dictyostelium cell-type differentiation and morphogenesis. We have identified a new protein kinase, ARCK-1, through a second site suppressor screen for mutants that allow spn null cells to proceed further through development. ARCK-1 has a C-terminal kinase domain most closely related to Ser/Thr protein kinases and an N-terminal putative regulatory domain with ankyrin repeats, a 14-3-3 binding domain, and a C1 domain, which is required for binding to RasB^GTP in a two-hybrid assay. Disruption of the gene encoding ARCK-1 results in weak, late developmental defects. However, overexpression of ARCK-1 phenocopies the spn null phenotype, consistent with Spn and ARCK-1 being on the same developmental pathway. Our previous analyses of Spn and the present analysis of ARCK-1 suggest a model in which Spn and ARCK-1 differentially control the phosphorylation state of a protein that regulates cell-type differentiation. Dephosphorylation of the substrate by Spn is required for cell-type differentiation. Control of ARCK-1 and Spn activities by upstream signals is proposed to be part of the developmental regulatory program mediating cell-fate decisions in Dictyostelium.     

Regulation of NDR protein kinase by hydrophobic motif phosphorylation mediated by the mammalian Ste20-like kinase MST3

NDR protein kinases are involved in the regulation of cell cycle progression and morphology. NDR1/NDR2 protein kinase is activated by phosphorylation on the activation loop phosphorylation site Ser281/Ser282 and the hydrophobic motif phosphorylation site Thr444/Thr442. Autophosphorylation of NDR is responsible for phosphorylation on Ser281/Ser282, whereas Thr444/Thr442 is targeted by an upstream kinase. Here we show that MST3, a mammalian Ste20-like protein kinase, is able to phosphorylate NDR protein kinase at Thr444/Thr442. In vitro, MST3 selectively phosphorylated Thr442 of NDR2, resulting in a 10-fold stimulation of NDR activity. MOB1A (Mps one binder 1A) protein further increased the activity, leading to a fully active kinase. In vivo, Thr442 phosphorylation after okadaic acid stimulation was potently inhibited by MST3KR, a kinase-dead mutant of MST3. Knockdown of MST3 using short hairpin constructs abolished Thr442 hydrophobic motif phosphorylation of NDR in HEK293F cells. We conclude that activation of NDR is a multistep process involving phosphorylation of the hydrophobic motif site Thr444/2 by MST3, autophosphorylation of Ser281/2, and binding of MOB1A.     

The crystal structure of plant-specific calcium-binding protein AtCBL2 in complex with the regulatory domain of AtCIPK14

Calcium signals mediate a multitude of plant responses to external stimuli. Calcineurin B-like (CBL) proteins and their target kinases, CBL-interacting protein kinases (CIPKs), represent important relays in plant calcium signaling. CBL interacts with CIPK through a conserved motif (NAF/FISL motif) within the C-terminal regulatory domain. To better understand the functional role of the CBL-CIPK system, we determined the crystal structure of AtCBL2 in complex with the regulatory domain of AtCIPK14 at 1.2 Å resolution. The NAF/FISL motif is inserted into a hydrophobic crevice within AtCBL2, accompanied by a large displacement of the helices and loop on the opposite side of the NAF/FISL motif from the C-terminal region, which shields the hydrophobic crevice in free form. Ca²⁺ are coordinated within four EF hands in AtCBL2 in bound form. This calcium coordination pattern differs from that in the structure of the SOS3-SOS2 complex previously reported. Structural comparison of the two structures shows that the recognition of CBL by CIPK is performed in a similar manner, but inherent interactions confer binding affinity and specificity.     

A novel transmembrane Ser/Thr kinase complexes with protein phosphatase-1 and inhibitor-2

Protein kinases and protein phosphatases exert coordinated control over many essential cellular processes. Here, we describe the cloning and characterization of a novel human transmembrane protein KPI-2 (Kinase/Phosphatase/Inhibitor-2) that was identified by yeast two-hybrid using protein phosphatase inhibitor-2 (Inh2) as bait. KPI-2 mRNA was predominantly expressed in skeletal muscle. KPI-2 is a 1503-residue protein with two predicted transmembrane helices at the N terminus, a kinase domain, followed by a C-terminal domain. The transmembrane helices were sufficient for targeting proteins to the membrane. KPI-2 kinase domain has about 60% identity with its closest relative, a tyrosine kinase. However, it only exhibited serine/threonine kinase activity in autophosphorylation reactions or with added substrates. KPI-2 kinase domain phosphorylated protein phosphatase-1 (PP1C) at Thr(320), which attenuated PP1C activity. KPI-2 C-terminal domain directly associated with PP1C, and this required a VTF motif. Inh2 associated with KPI-2 C-terminal domain with and without PP1C. Thus, KPI-2 is a kinase with sites to associate with PP1C and Inh2 to form a regulatory complex that is localized to membranes.     

Sucrose regulated expression of a Ca2+-dependent protein kinase (TaCDPK1) gene in excised leaves of wheat.

Sucrose (Suc) can influence the expression of a large number of genes and thereby regulates many metabolic and developmental processes. However, the Suc sensing and the components of the ensuing signaling transduction pathway leading to the regulation of gene expression are not fully understood. We have shown that protein kinases and phosphatases are involved in the Suc induced expression of fructosyltransferase (FT) genes and fructan accumulation by an hexokinase independent pathway in wheat (Triticum aestivum). In the present study, using an RT-PCR based strategy, we have cloned a calcium-dependent protein kinase (TaCDPK1) cDNA that is upregulated during Suc treatment of excised wheat leaves. The deduced amino-acid sequence of CDPK1 has high sequence similarity (>70%) to known CDPKs from both monocots and dicots. Based on sequence homology, TaCDPK1 sequence shows a variable domain preceding a catalytic domain, an autoinhibitory function domain, and a C-terminal calmodulin-domain containing 4 EF-hand calcium-binding motifs, along with a N-myristoylation motif in the N-terminal variable domain. The recombinant Escherichia coli expressed TaCDPK1 was able to phosphorylate histone III-S in a calcium dependent manner in in vitro assays. The TaCDPK1 gene expression, as determined by quantitative RT-PCR, is induced by Suc and this effect is repressed by the inhibitors of the putative components of the Suc signal transduction pathway (calcium, Ser/Thr protein kinases and protein phosphatases). We propose that TaCDPK1 is involved in the Suc induced signaling pathway in wheat leaves.     

Cloning and characterization of MST4, a novel Ste20-like kinase

MST4, a novel member of the germinal center kinase subfamily of human Ste20-like kinases, was cloned and characterized. Composed of a C-terminal regulatory domain and an N-terminal kinase domain, MST4 is most closely related to mammalian Ste20 kinase family member MST3. Both the kinase and C-terminal regulatory domains of MST4 are required for full activation of the kinase. Northern blot analysis indicates that MST4 is ubiquitously distributed, and the MST4 gene is localized to chromosome Xq26, a disease-rich region, by fluorescence in situ hybridization. Although some members of the MST4 family function as upstream regulators of mitogen-activated protein kinase cascades, expression of MST4 in 293 cells was not sufficient to activate or potentiate extracellular signal-regulated kinase, c-Jun N-terminal kinase, or p38 kinase. An alternatively spliced isoform of MST4 (MST4a) was isolated by yeast two-hybrid interaction with the catalytic domain of Raf from a human fetal brain cDNA library and also found in a variety of human fetal and adult tissues. MST4a lacks an exon encoding kinase subdomains IX-XI that stabilizes substrate binding. The existence of both MST4 isoforms suggests that the MST4 kinase activity is highly regulated, and MST4a may function as a dominant-negative regulator of the MST4 kinase.     

Structure of Mycobacterium tuberculosis PknB supports a universal activation mechanism for Ser/Thr protein kinases

A family of eukaryotic-like Ser/Thr protein kinases occurs in bacteria, but little is known about the structures and functions of these proteins. Here we characterize PknB, a transmembrane signaling kinase from Mycobacterium tuberculosis. The intracellular PknB kinase domain is active autonomously, and the active enzyme is phosphorylated on residues homologous to regulatory phospho-acceptors in eukaryotic Ser/Thr kinases. The crystal structure of the PknB kinase domain in complex with an ATP analog reveals the active conformation. The predicted fold of the PknB extracellular domain matches the proposed targeting domain of penicillin-binding protein 2x. The structural and chemical similarities of PknB to metazoan homologs support a universal activation mechanism of Ser/Thr protein kinases in prokaryotes and eukaryotes.     

A 3-phosphoinositide-dependent protein kinase-1 (PDK1) docking site is required for the phosphorylation of protein kinase Czeta (PKCzeta ) and PKC-related kinase 2 by PDK1

Members of the AGC subfamily of protein kinases including protein kinase B, p70 S6 kinase, and protein kinase C (PKC) isoforms are activated and/or stabilized by phosphorylation of two residues, one that resides in the T-loop of the kinase domain and the other that is located C-terminal to the kinase domain in a region known as the hydrophobic motif. Atypical PKC isoforms, such as PKCzeta, and the PKC-related kinases, like PRK2, are also activated by phosphorylation of their T-loop site but, instead of possessing a phosphorylatable Ser/Thr in their hydrophobic motif, contain an acidic residue. The 3-phosphoinositide-dependent protein kinase (PDK1) activates many members of the AGC subfamily of kinases in vitro, including PKCzeta and PRK2 by phosphorylating the T-loop residue. In the present study we demonstrate that the hydrophobic motifs of PKCzeta and PKCiota, as well as PRK1 and PRK2, interact with the kinase domain of PDK1. Mutation of the conserved residues of the hydrophobic motif of full-length PKCzeta, full-length PRK2, or PRK2 lacking its N-terminal regulatory domain abolishes or significantly reduces the ability of these kinases to interact with PDK1 and to become phosphorylated at their T-loop sites in vivo. Furthermore, overexpression of the hydrophobic motif of PRK2 in cells prevents the T-loop phosphorylation and thus inhibits the activation of PRK2 and PKCzeta. These findings indicate that the hydrophobic motif of PRK2 and PKCzeta acts as a "docking site" enabling the recruitment of PDK1 to these substrates. This is essential for their phosphorylation by PDK1 in cells.     

Studies on the regulatory domain of Ca2+/calmodulin-dependent protein kinase II. Functional analyses of arginine 283 using synthetic inhibitory peptides and site-directed mutagenesis of the alpha subunit

The regulatory role of Arg283 in the autoinhibitory domain of Ca2+/calmodulin-dependent protein kinase II was investigated using substituted inhibitory synthetic peptides and site-directed mutation of the expressed kinase. In the synthetic peptide corresponding to the autoinhibitory domain (residues 281-309) of Ca2+/calmodulin-dependent protein kinase II, substitution of Arg283 by other residues increased the IC50 values of the peptides in the following order: Arg much less than Lys much less than Gln much less than Glu. Site-directed mutations of Arg283 to glutamic acid and glutamine in the kinase alpha subunit cDNA were transcribed and translated in vitro. The expressed enzymes had the same total kinase activities, determined in the presence of Ca2+/CaM, but the Glu283 mutant had a slightly higher Ca2(+)-independent kinase activity (5.46 +/- 0.88%) compared to the wild-type Arg283 (1.86 +/- 0.71%) and the Gln283 mutant (2.15 +/- 0.60%). When the expressed kinases were subjected to limited autophosphorylation on ice to monitor generation of the Ca2(+)-independent activity, the Arg283 kinase attained maximal Ca2(+)-independent activity (about 20%) within 30 s, whereas the Gln283 and Glu283 mutants attained maximal Ca2(+)-independence only after about 40 min of autophosphorylation. The results indicate that Arg283 is a very important determinant for the regulatory autophosphorylation of Thr286 that generates the Ca2(+)-independent activity but is not essential for the other multiple autophosphorylations within Ca2+/calmodulin-dependent protein kinase II, and that Arg283 is only one of several important residues for the inhibitory potency of the autoinhibitory domain.     

Generation of autonomous activity of Ca(2+)/calmodulin-dependent protein kinase kinase β by autophosphorylation

Ca(2+)/calmodulin-dependent protein kinase kinases (CaMKKs) phosphorylate and activate specific downstream protein kinases, including CaMKI, CaMKIV, and 5'-AMP-activated protein kinase, which mediates a variety of Ca(2+) signaling cascades. CaMKKs have been shown to undergo autophosphorylation, although their role in enzymatic regulation remains unclear. Here, we found that CaMKKα and β isoforms expressed in nonstimulated transfected COS-7 cells, as well as recombinant CaMKKs expressed in and purified from Escherichia coli, were phosphorylated at Thr residues. Introduction of a kinase-dead mutation completely impaired the Thr phosphorylation of these recombinant CaMKK isoforms. In addition, wild-type recombinant CaMKKs were unable to transphosphorylate the kinase-dead mutants, suggesting that CaMKK isoforms undergo Ca(2+)/CaM-independent autophosphorylation in an intramolecular manner. Liquid chromatography-tandem mass spectrometry analysis identified Thr(482) in the autoinhibitory domain as one of the autophosphorylation sites in CaMKKβ, but phosphorylation of the equivalent Thr residue (Thr(446)) in the α isoform was not observed. Unlike CaMKKα that has high Ca(2+)/CaM-dependent activity, wild-type CaMKKβ displays enhanced autonomous activity (Ca(2+)/CaM-independent activity, 71% of total activity). This activity was significantly reduced (to 37%) by substitution of Thr(482) with a nonphosphorylatable Ala, without significant changes in Ca(2+)/CaM binding. In addition, a CaMKKα mutant containing the CaMKKβ regulatory domain was shown to be partially phosphorylated at Thr(446), resulting in a modest elevation of its autonomous activity. The combined results indicate that, in contrast to the α isoform, CaMKKβ exhibited increased autonomous activity, which was caused, at least in part, by autophosphorylation at Thr(482), resulting in partial disruption of the autoinhibitory mechanism.     

Unconventional myosin VIIA is a novel A-kinase-anchoring protein

To gain an insight into the cellular function of the unconventional myosin VIIA, we sought proteins interacting with its tail region, using the yeast two-hybrid system. Here we report on one of the five candidate interactors we identified, namely the type I alpha regulatory subunit (RI alpha) of protein kinase A. The interaction of RI alpha with myosin VIIA tail was demonstrated by coimmunoprecipitation from transfected HEK293 cells. Analysis of deleted constructs in the yeast two-hybrid system showed that the interaction of myosin VIIA with RI alpha involves the dimerization domain of RI alpha. In vitro binding assays identified the C-terminal "4.1, ezrin, radixin, moesin" (FERM)-like domain of myosin VIIA as the interacting domain. In humans and mice, mutations in the myosin VIIA gene underlie hereditary hearing loss, which may or may not be associated with visual deficiency. Immunohistofluorescence revealed that myosin VIIA and RI alpha are coexpressed in the outer hair cells of the cochlea and rod photoreceptor cells of the retina. Our results strongly suggest that myosin VIIA is a novel protein kinase A-anchoring protein that targets protein kinase A to definite subcellular sites of these sensory cells.