Regulatory protein phosphorylation of phosphoenolpyruvate carboxylase in the facultative crassulacean-acid-metabolism plant Mesembryanthemum crystallinum L.

Phosphoenolpyruvate PyrP carboxylase (PyrPC) and PyrPC kinase were copurified from dark-adapted leaves of the common ice plant Mesembryanthemum crystallinum L. with crassulacean-acid metabolism (CAM). Purification by (NH4)2SO4 fractionation, chromatography on Fractogel-DEAE and hydroxylapatite resulted in a PyrPC preparation with a specific activity of 23-25 U/mg protein and a protein kinase activity of 255 mumol Pi.mol-1 PyrPC.s-1. After in vitro phosphorylation, the most prominently phosphorylated polypeptide was identified as PyrPC by immunoblotting and sequencing. Phosphorylation of PyrPC in vitro by incubation with 400 microM MgATP decreased its sensitivity towards malate. When purified in the absence of the protease inhibitor chymostatin, PyrPC lost an N-terminal sequence of 128 amino acids. Although the carboxylation reaction was unaffected, the truncated PyrPC could neither be phosphorylated in vitro nor inhibited by malate. This result and data obtained by limited proteolysis concur with the hypothesis [Jiao, J.A. & Chollet, R. (1989) Arch. Biochem. Biophys. 283, 300-305] that Ser11 is the phosphorylation site of the CAM PyrPC of M. crystallinum. At pH 7.0, the Km for ATP of the protein kinase was 25 microM; phosphorylation of PyrPC was maximal after 30 min at pH 7.0. The kinase showed also activity with histone III-S but not with dephosphorylated casein. It was inhibited by malate. The results show, that reversible protein phosphorylation is an important factor in the regulation of PyrPC in the facultative CAM plant M. crystallinum, similar to C4 and constitutive CAM plants.     

Heterologous expression and biochemical characterization of two calcium-dependent protein kinase isoforms CaCPK1 and CaCPK2 from chickpea

In plants, calcium-dependent protein kinases (CPKs) constitute a unique family of enzymes consisting of a protein kinase catalytic domain fused to carboxy-terminal autoregulatory and calmodulin-like domains. We isolated two cDNAs encoding calcium-dependent protein kinase isoforms (CaCPK1 and CaCPK2) from chickpea. Both isoforms were expressed as fusion proteins in Escherichia coli. Biochemical analyses have identified CaCPK1 and CaCPK2 as Ca(2+)-dependent protein kinases since both enzymes phosphorylated themselves and histone III-S as substrate only in the presence of Ca(2+). The kinase activity of the recombinant enzymes was calmodulin independent and sensitive to CaM antagonists W7 [N-(6-aminohexyl)-5-chloro-1-naphthalene sulphonamide] and calmidazoilum. Phosphoamino acid analysis revealed that the isoforms transferred the gamma-phosphate of ATP only to serine residues of histone III-S and their autophosphorylation occurred on serine and threonine residues. These two isoforms showed considerable variations with respect to their biochemical and kinetic properties including Ca(2+) sensitivities. The recombinant CaCPK1 has a pH and temperature optimum of pH 6.8-8.6 and 35-42 degrees C, respectively, whereas CaCPK2 has a pH and temperature optimum of pH 7.2-9 and 35-42 degrees C, respectively. Taken together, our results suggest that CaCPK1 and CaCPK2 are functional serine/threonine kinases and may play different roles in Ca(2+)-mediated signaling in chickpea plants.     

Ca(2+)-calmodulin-dependent protein kinases Ia and Ib from rat brain I. Identification, purification, and structural comparisons.

Two Ca(2+)-calmodulin (CaM)-dependent protein kinases were purified from rat brain using as substrate a synthetic peptide based on site 1 (site 1 peptide) of the synaptic vesicle-associated protein, synapsin I. One of the purified enzymes was an approximately 89% pure protein of M(r) = 43,000 which bound CaM in a Ca(2+)-dependent fashion. The other purified enzyme was an apparently homogenous protein of M(r) = 39,000 accompanied by a small amount of a M(r) = 37,000 form which may represent a proteolytic product of the 39-kDa enzyme. The 39-kDa protein bound CaM in a Ca(2+)-dependent fashion. Gel filtration analysis indicated that both enzymes are monomers. The 43- and 39-kDa enzymes are named Ca(2+)-CaM-dependent protein kinases Ia and Ib (CaM kinases Ia, Ib), respectively. The specific activities of CaM kinases Ia and Ib were similar (5-8 mumol/min/mg protein). CaM kinase Ia (but not CaM kinase Ib) activity was enhanced by addition of a CaM-Sepharose column wash (non-binding) fraction suggesting the existence of an "activator" of CaM kinase Ia. Both kinases phosphorylated exogenous substrates (site 1 peptide and synapsin I) in a Ca(2+)-CaM-dependent fashion and both kinases underwent autophosphorylation. CaM kinase Ia autophosphorylation was Ca(2+)-CaM-dependent and occurred exclusively on threonine while CaM kinase Ib autophosphorylation showed Ca(2+)-CaM independence and occurred on both serine and threonine. Proteolytic digestion of autophosphorylated CaM kinases Ia and Ib yielded phosphopeptides of differing M(r). These characteristics, as well as enzymatic and regulatory properties (DeRemer, M. F., Saeli, R. J. Brautigen, D. L., and Edelman, A. M. (1992) J. Biol. Chem. 267, 13466-13471), indicate that CaM kinases Ia and Ib are distinct and possibly previously unrecognized enzymes.     

Competing carboxylases: circadian and metabolic regulation of Rubisco in C3 and CAM Mesembryanthemum crystallinum L

The temporal co-ordination of ribulose 1·5-bisphosphate carboxylase/oxygenase (Rubisco) and phosphoenolpyruvate carboxylase (PEPc) activities by Mesembryanthemum crystallinum L. in C(3) and crassulacean acid metabolism (CAM) modes was investigated under conventional light-dark (LD) and continuous light (LL) conditions. When C(3) , net CO(2) assimilation rate increased during each subjective night under LL with maximum carboxylation unrelated to Rubisco activation state. The CAM circadian rhythm of CO(2) uptake was more pronounced, with CO(2) assimilation rate maximal towards the end of each subjective night. In vivo and in vitro techniques were integrated to map carboxylase enzyme regulation to the framework provided by CAM LL gas exchange activity. Rubisco was activated in vitro throughout each subjective dark period and consistently deactivated at each subjective dawn, similar to that observed at true dawn in constitutive CAM species. Instantaneous carbon isotope discrimination showed in vivo carboxylase co-dominance during the CAM subjective night, initially by Rubisco and latterly C(4) (PEPc), despite both enzymes seemingly activated in vitro. The circadian rhythm in titratable acidity accumulation was progressively damped over successive subjective nights, but maintenance of PEPc carboxylation capacity ensures that CAM plants do not become progressively more 'C(3) -like' with time under LL.     

Sucrose induces rapid activation of CfSAPK, a mitogen-activated protein kinase, in Cephalostachyum fuchsianum Gamble cells

Sucrose was recently demonstrated to function as a molecular signal. However, sucrose-specific sensing and signalling pathways remain largely undefined. Here, we show that Cephalostachyum fuchsianum sucrose-activated protein kinase (CfSAPK) is transiently and specifically activated by sucrose in C. fuchsianum Gamble suspension cells. The result suggested that CfSAPK participates in a sucrose-signalling pathway. CfSAPK was partially purified from sucrose-treated cells and further analysed. Kinase activity assays revealed that CfSAPK preferentially used myelin basic protein (MBP) as substrate in vitro and strongly phosphorylate MBP threonine residue(s) and weakly phosphorylated MBP serine residue(s). Of the divalent cations tested, Mg(2+) was required for CfSAPK activation. Phosphatase treatment of CfSAPK abolished its kinase activity, indicating that phosphorylation is required for CfSAPK activation. Seven internal tryptic peptides identified from CfSAPK matched mitogen-activated protein kinases (MAPKs) in plants. CfSAPK cDNA was cloned using RT-PCR and rapid amplification of cDNA ends (RACE). CfSAPK cDNA encodes a 382-amino acid protein with a calculated molecular mass of 43,466.9 Da. The CfSAPK protein contains all 11 conserved kinase subdomains found in other Ser/Thr kinases. The amino acids sequence of CfSAPK is highly homologous to group A MAPKs in monocotyledon plants.     

Ser1928 is a common site for Cav1.2 phosphorylation by protein kinase C isoforms

Voltage-dependent Ca(2+) channel (Ca(v)1.2, L-type Ca(2+) channel) function is highly regulated by hormones and neurotransmitters in large part through the activation of kinases and phosphatases. Regulation of Ca(v)1.2 by protein kinase C (PKC) is of significant physiologic importance, mediating, in part, the cardiac response to hormonal regulation. Although PKC has been reported to mediate activation and/or inhibition of Ca(v)1.2 function, the molecular mechanisms mediating the response have not been definitively elucidated. We show that PKC forms a macromolecular complex with the alpha(1c) subunit of Ca(v)1.2 through direct interaction with the C terminus. This interaction leads to phosphorylation of the channel in response to activators of PKC. We identify Ser(1928) as the residue that is phosphorylated by PKC in vitro and in vivo. Ser(1928) has been identified previously as the site mediating, in part, the protein kinase A up-regulation of channel activity. Thus, the protein kinase A and PKC signaling pathways converge on the Ca(v)1.2 complex at Ser(1928) to increase channel activity. Our results identify two mechanisms leading to regulation of Ca(v)1.2 activity by PKC: pre-association of the channel with PKC isoforms and phosphorylation of specific sites within the alpha(1c) subunit.     

Characterization of a Hank’s Type Serine/Threonine Kinase and Serine/Threonine Phosphoprotein Phosphatase in Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen that causes infections in eye, urinary tract, burn, and immunocompromised patients. We have cloned and characterized a serine/threonine (Ser/Thr) kinase and its cognate phosphoprotein phosphatase. By using oligonucleotides from the conserved regions of Ser/Thr kinases of mycobacteria, an 800-bp probe was used to screen P. aeruginosa PAO1 genomic library. A 20-kb cosmid clone was isolated, from which a 4.5-kb DNA with two open reading frames (ORFs) were subcloned. ORF1 was shown to encode Ser/Thr phosphatase (Stp1), which belongs to the PP2C family of phosphatases. Overlapping with the stp1 ORF, an ORF encoding Hank's type Ser/Thr kinase was identified. Both ORFs were cloned in pGEX-4T1 and expressed in Escherichia coli. The overexpressed proteins were purified by glutathione-Sepharose 4B affinity chromatography and were biochemically characterized. The Stk1 kinase is 39 kDa and undergoes autophosphorylation and can phosphorylate eukaryotic histone H1. A site-directed Stk1 (K86A) mutant was shown to be incapable of autophosphorylation. A two-dimensional phosphoamino acid analysis of Stk1 revealed strong phosphorylation at a threonine residue and weak phosphorylation at a serine residue. The Stp1 phosphatase is 27 kDa and is an Mn(2+)-, but not a Ca(2+)- or a Mg(2+)-, dependent Ser/Thr phosphatase. Its activity is inhibited by EDTA and NaF, but not by okadaic acid, and is similar to that of PP2C phosphatase.     

Phosphoenolpyruvate carboxylase kinase is controlled by a similar signaling cascade in CAM and C(4) plants

In Crassulacean acid metabolism (CAM) plants, phosphoenolpyruvate carboxylase (PEPC) is subject to day-night regulatory phosphorylation of a conserved serine residue in the plant enzyme's N-terminal domain. The dark increase in PEPC-kinase (PEPC-k) activity is under control of a circadian oscillator, via the enhanced expression of the corresponding gene (1). The signaling cascade leading to PEPC-k up-regulation was investigated in leaves and mesophyll cell protoplasts of the facultative, salt-inducible CAM species, Mesembryanthemum crystallinum. Mesophyll cell protoplasts had the same PEPC-k activity as leaves from which they were prepared (i.e., high at night, low during the day). However, unlike C(4) protoplasts (2), CAM protoplasts did not show marked PEPC-k up-regulation when isolated during the day and treated with a weak base such as NH(4)Cl. Investigations using various pharmacological reagents established the operation, in the darkened CAM leaf, of a PEPC-k cascade including the following components: a phosphoinositide-dependent phospholipase C (PI-PLC), inositol 1,4,5 P (IP(3))-gated tonoplast calcium channels, and a putative Ca(2+)/calmodulin protein kinase. These results suggest that a similar signaling machinery is involved in both C(4) (2, 3) and CAM plants to regulate PEPC-k activity, the phosphorylation state of PEPC, and, thus, carbon flux through this enzyme during CAM photosynthesis.     

Identification of a novel phosphorylation motif for CDPKs: phosphorylation of synthetic peptides lacking basic residues at P-3/P-4

The Ca(2+)-dependent protein kinases (CDPKs) are members of a large subfamily of protein kinases in plants that have been implicated in the control of numerous aspects of plant growth and development. One known substrate of the CDPKs is the ER-located ACA2 calcium pump, which is regulated by phosphorylation of Ser(45). In the present study, a synthetic peptide based on the known regulatory phosphorylation site (RRFRFTANLS(45)KRYEA) was efficiently phosphorylated in vitro by CDPKs but not a plant SNF1-related protein kinase. Phosphorylation of the Ser(45)-ACA2 peptide was surprising because the sequence lacks basic residues at P-3/P-4 (relative to the phosphorylated Ser at position P) that are considered to be essential recognition elements for CDPKs. We demonstrate that phosphorylation of the Ser(45)-ACA2 peptide is dependent on the cluster of basic residues found N-terminal (P-6 to P-9) as well as C-terminal (P + 1/P + 2) to the phosphorylated Ser. The results establish a new general phosphorylation motif for CDPKs: [Basic-Basic-X-Basic]-phi-X(4)-S/T-X-Basic (where phi is a hydrophobic residue). The motif predicts a number of new phosphorylation sites in plant proteins. Evidence is presented that the novel motif may explain the phosphorylation by CDPKs of Ser271 in the aquaporin PM28A.     

Cloning and functional characterization of NtCPK4, a new tobacco calcium-dependent protein kinase

A cDNA clone, encoding calcium (Ca2+)-dependent protein kinase (CDPK or CPK), was isolated from tobacco (Nicotiana tabacum). The full-length cDNA of 2360 bp contains an open reading frame for NtCPK4 consisting of 572 amino acid residues. Sequence alignment indicated that NtCPK4 shared high similarities with other CPKs and some CPK-related protein kinases (CRKs). Biochemical analyses showed that NtCPK4 phosphorylated itself and calf thymus histones fraction III-S (histone III-S) in a calcium-dependent manner, and the K0.5 of calcium activation was 0.29 microM or 0.25 microM with histone III-S or syntide-2 as substrates, respectively. The Vmax and Km were 588 nmol min-1 mg-1 and 176 microg ml-1, respectively, when histone III-S was used as substrate, while they were 2415 nmol min-1 mg-1 and 58 microM, respectively, with syntide-2 as substrate. In addition, the phosphorylation of NtCPK4 occurred on threonine residue, as shown by capillary electrophoresis analyses. All of these data demonstrated that NtCPK4 was a serine/threonine protein kinase. NtCPK4 as a low copy gene was expressed in all tested organs including the root, leaf, stem, and flower of tobacco, while its expression was temporally and spatially modulated in both productive and vegetative tissues during tobacco growth and development. NtCPK4 expression was also increased in response to the treatment of gibberellin or NaCl. Our study suggested that NtCPK4 might play vital roles in plant development and responses to environmental stimuli.     

Isolation and characterization of a novel calcium/calmodulin-dependent protein kinase, AtCK, from arabidopsis

Protein phosphorylation is one of the major mechanisms by which eukaryotic cells transduce extracellular signals into intracellular responses. Calcium/calmodulin (Ca(2+)/CaM)-dependent protein phosphorylation has been implicated in various cellular processes, yet little is known about Ca(2+)/CaM-dependent protein kinases (CaMKs) in plants. From an Arabidopsis expression library screen using a horseradish peroxidase-conjugated soybean calmodulin isoform (SCaM-1) as a probe, we isolated a full-length cDNA clone that encodes AtCK (Arabidopsis thaliana calcium/calmodulin-dependent protein kinase). The predicted structure of AtCK contains a serine/threonine protein kinase catalytic domain followed by a putative calmodulin-binding domain and a putative Ca(2+)-binding domain. Recombinant AtCK was expressed in E. coli and bound to calmodulin in a Ca(2+)-dependent manner. The ability of CaM to bind to AtCK was confirmed by gel mobility shift and competition assays. AtCK exhibited its highest levels of autophosphorylation in the presence of 3 mM Mn(2+). The phosphorylation of myelin basic protein (MBP) by AtCK was enhanced when AtCK was under the control of calcium-bound CaM, as previously observed for other Ca(2+)/CaM-dependent protein kinases. In contrast to maize and tobacco CCaMKs (calcium and Ca(2+)/CaM-dependent protein kinase), increasing the concentration of calmodulin to more than 3 microgram suppressed the phosphorylation activity of AtCK. Taken together our results indicate that AtCK is a novel Arabidopsis Ca(2+)/CaM-dependent protein kinase which is presumably involved in CaM-mediated signaling.     

Autophosphorylation and subcellular localization dynamics of a salt- and water deficit-induced calcium-dependent protein kinase from ice plant

A salinity and dehydration stress-responsive calcium-dependent protein kinase (CDPK) was isolated from the common ice plant (Mesembryanthemum crystallinum; McCPK1). McCPK1 undergoes myristoylation, but not palmitoylation in vitro. Removal of the N-terminal myristate acceptor site partially reduced McCPK1 plasma membrane (PM) localization as determined by transient expression of green fluorescent protein fusions in microprojectile-bombarded cells. Removal of the N-terminal domain (amino acids 1-70) completely abolished PM localization, suggesting that myristoylation and possibly the N-terminal domain contribute to membrane association of the kinase. The recombinant, Escherichia coli-expressed, full-length McCPK1 protein was catalytically active in a calcium-dependent manner (K0.5 = 0.15 microm). Autophosphorylation of recombinant McCPK1 was observed in vitro on at least two different Ser residues, with the location of two sites being mapped to Ser-62 and Ser-420. An Ala substitution at the Ser-62 or Ser-420 autophosphorylation site resulted in a slight increase in kinase activity relative to wild-type McCPK1 against a histone H1 substrate. In contrast, Ala substitutions at both sites resulted in a dramatic decrease in kinase activity relative to wild-type McCPK1 using histone H1 as substrate. McCPK1 undergoes a reversible change in subcellular localization from the PM to the nucleus, endoplasmic reticulum, and actin microfilaments of the cytoskeleton in response to reductions in humidity, as determined by transient expression of McCPK1-green fluorescent protein fusions in microprojectile-bombarded cells and confirmed by subcellular fractionation and western-blot analysis of 6x His-tagged McCPK1.     

Calyculin A-induced endothelial cell shape changes are independent of [Ca(2+)](i) elevation and may involve actin polymerization

Changes in endothelial cell (EC) shape result in inter-EC gap formation and subsequently regulate transendothelial passage. In this work, we investigated the effects of protein phosphorylation (induced by inhibition of protein phosphatases) on EC shape changes. Treatment of bovine pulmonary artery endothelial cells (BPAEC) with calyculin A (100 nM, an inhibitor of protein Ser/Thr phosphatases 1 and 2A) resulted in cell retraction, surface bleb formation and cell rounding. Trypan blue and electrophysiological experiments suggested that the plasma membrane of these rounded cells maintained functional integrity. Calyculin A-induced morphological changes were strongly inhibited by staurosporine, but not affected by specific inhibitors of the myosin light chain (MLC) kinase, protein kinases A, C and G, and tyrosine kinases. The calyculin A effects were not mimicked by phorbol myristate acetate, dibutyryl cAMP, 8-bromo-cGMP or ionomycin. Cytochalasin B (an inhibitor of actin polymerization) almost completely abolished such shape changes while colchicine (an inhibitor of microtubule polymerization) had no inhibitory effect at all. Ca(2+) imaging experiments showed that the morphological changes were not associated with any global or local cytosolic Ca(2+) concentration ([Ca(2+)](i)) elevation. The results suggest that calyculin A unmasked the basal activities of some protein Ser/Thr kinases other than MLC kinase and protein kinases A, C and G; these unknown kinases might cause BPAEC shape changes by a mechanism involving actin polymerization but not [Ca(2+)](i) elevation.     

A type-1 casein kinase from yeast phosphorylates both serine and threonine residues of casein. Identification of the phosphorylation sites

A protein kinase (casein kinase 1A) active on casein and phosvitin but not on histones has been purified to near homogeneity from yeast cytosol and meets most criteria for being considered a type-1 casein kinase: it is a monomeric enzyme exhibiting an Mr of about 27 kDa by sucrose gradient centrifugation: it is not affected by inhibitors of type-2 casein kinases, such as heparin and polyglutamate, and shows negligible affinity for GTP. It also readily phosphorylates the residue Ser-22 of beta-casein located within the sequence -Ser(P)-Ser(P)-Ser(P)-Glu-Glu-Ser22-Ile-Thr-Arg- which is typically affected by casein kinases of the first class. On the other hand, casein kinase 1A displays the unusual property of phosphorylating threonine residue(s) in both whole casein and alpha s1-casein. The threonine residue phosphorylated in alpha s1-casein and accounting for most of the 32P incorporated into this protein by casein kinase 1A has been identified as Thr-49, which occurs in the sequence -Ser(P)-Glu-Ser(P)-Thr(P*)49-Glu-Asp-Gln-, whose two Ser(P) residues are already phosphorylated in the native protein. It is concluded that some type-1 casein kinases can also phosphorylate threonine residues provided they fulfil definite structural requirements, probably an acidic cluster near their N-terminal side.     

Calcium-independent activation of salicylic acid-induced protein kinase and a 40-kilodalton protein kinase by hyperosmotic stress

Reversible protein phosphorylation/dephosphorylation plays important roles in signaling the plant adaptive responses to salinity/drought stresses. Two protein kinases with molecular masses of 48 and 40 kD are activated in tobacco cells exposed to NaCl. The 48-kD protein kinase was identified as SIPK (salicylic acid-induced protein kinase), a member of the tobacco MAPK (mitogen-activated protein kinase) family that is activated by various other stress stimuli. The activation of the 40-kD protein kinase is rapid and dose-dependent. Other osmolytes such as Pro and sorbitol activate these two kinases with similar kinetics. The activation of 40-kD protein kinase is specific for hyperosmotic stress, as hypotonic stress does not activate it. Therefore, this 40-kD kinase was named HOSAK (high osmotic stress-activated kinase). HOSAK is a Ca(2+)-independent kinase and uses myelin basic protein (MBP) and histone equally well as substrates. The kinase inhibitor K252a rapidly activates HOSAK in tobacco cells, implicating a dephosphorylation mechanism for HOSAK activation. Activation of both SIPK and HOSAK by high osmotic stress is Ca(2+) and abscisic acid (ABA) independent. Furthermore, mutation in SOS3 locus does not affect the activation of either kinase in Arabidopsis seedlings. These results suggest that SIPK and 40-kD HOSAK are two new components in a Ca(2+)- and ABA-independent pathway that may lead to plant adaptation to hyperosmotic stress.     

Rho-associated kinase phosphorylates MARCKS in human neuronal cells

Myristoylated alanine-rich C kinase substrate (MARCKS) is a filamentous actin bundling protein and has multiple sites for phosphorylation, by which the biochemical function is negatively regulated. However, the role of such phosphorylation in physiological functions, particularly in neuronal functions, is not well understood. Using a phosphorylation-site specific antibody, we detected the phosphorylation of MARCKS at Ser159 by various protein kinases. Rho-kinase, protein kinase A, and protein kinase C, could introduce (32)P into human recombinant MARCKS in vitro and the phosphorylation site was confirmed to be the Ser159 residue. In human neuronal teratoma (NT-2) cells, lysophosphatidic acid (LPA) induced MARCKS phosphorylation dose- and time-dependently. This phosphorylation was sensitive to Rho-kinase inhibitor HA1077. However, the phosphorylation induced by PDBu was lesser sensitive. In a skinned NTera-2 cell system, Ca(2+)-independent and GTP gamma S/ATP-stimulated phosphorylation at Ser159 was also sensitive to pre-treatment C3 toxin and HA1077. These findings suggest that the Ser159 residue of MARCKS is a target of LPA-stimulated Rho-kinase in neuronal cells.