Molecular analyses of Toxoplasma gondii calmodulin-like domain protein kinase isoform 3.

Ca(2+) signaling is thought to play an important role in Toxoplasma gondii motility, including invasion of and egress from host cells. Recently, it has been reported that phosphorylation of the glideosome apparatus components of T. gondii occurs during invasion. To elucidate the role of T. gondii calmodulin-like domain protein kinase in the signaling pathway that bridges Ca(2+) stimulation and motility, we characterized T. gondii calmodulin-like domain protein kinase isoform 3 (TgCDPKif3). TgCDPKif3 is homologous to Plasmodium falciparum calcium-dependent protein kinase 1, which has been reported to phosphorylate P. falciparum glideosome components. TgCDPKif3 was purified as a fusion protein that was labeled with [gamma-(32)P]ATP, and the label was subsequently removed by phosphatase treatment. Phosphorylation was eliminated when the putative catalytic lysine residue of TgCDPKif3 was replaced with alanine. TgCDPKif3 phosphorylated Histone II(AS) as a representative substrate in a Ca(2+)-dependent manner at a high Ca(2+) concentration. TgCDPKif3 was localized to the apical ends of tachyzoites. TgCDPKif3 showed the translocation between intra- and extracellular tachyzoites. TgCDPKif3 could phosphorylate T. gondii aldolase 1 (TgALD1) in vitro. The interaction between TgCDPKif3 and TgALD1 was confirmed by the co-immunoprecipitation assay in mammal cells. We suggested that TgCDPKif3 could participate in the motility of T. gondii through the phosphorylation of glideosome complex member.

Novel Ca2+/calmodulin-dependent protein kinase expressed in actively growing mycelia of the basidiomycetous mushroom Coprinus cinereus.

We isolated cDNA clones for novel protein kinases by expression screening of a cDNA library from the basidiomycetous mushroom Coprinus cinereus. One of the isolated clones was found to encode a calmodulin (CaM)-binding protein consisting of 488 amino acid residues with a predicted molecular weight of 53,906, which we designated CoPK12. The amino acid sequence of the catalytic domain of CoPK12 showed 46% identity with those of rat Ca2+/CaM-dependent protein kinase (CaMK) I and CaMKIV. However, a striking difference between these kinases is that the critical Thr residue in the activating phosphorylation site of CaMKI/IV is replaced by a Glu residue at the identical position in CoPK12. As predicted from its primary sequence, CoPK12 was found to behave like an activated form of CaMKI phosphorylated by an upstream CaMK kinase, indicating that CoPK12 is a unique CaMK with different properties from those of the well-characterized CaMKI, II, and IV. CoPK12 was abundantly expressed in actively growing mycelia and phosphorylated various proteins, including endogenous substrates, in the presence of Ca2+/CaM. Treatment of mycelia of C. cinereus with KN-93, which was found to inhibit CoPK12, resulted in a significant reduction in growth rate of mycelia. These results suggest that CoPK12 is a new type of multifunctional CaMK expressed in C. cinereus, and that it may play an important role in the mycelial growth.

Functional, genetic and bioinformatic characterization of a calcium/calmodulin kinase gene in Sporothrix schenckii.

BACKGROUND: Sporothrix schenckii is a pathogenic, dimorphic fungus, the etiological agent of sporotrichosis, a subcutaneous lymphatic mycosis. Dimorphism in S. schenckii responds to second messengers such as cAMP and calcium, suggesting the possible involvement of a calcium/calmodulin kinase in its regulation. In this study we describe a novel calcium/calmodulin-dependent protein kinase gene in S. schenckii, sscmk1, and the effects of inhibitors of calmodulin and calcium/calmodulin kinases on the yeast to mycelium transition and the yeast cell cycle. RESULTS: Using the PCR homology approach a new member of the calcium/calmodulin kinase family, SSCMK1, was identified in this fungus. The cDNA sequence of sscmk1 revealed an open reading frame of 1,221 nucleotides encoding a 407 amino acid protein with a predicted molecular weight of 45.6 kDa. The genomic sequence of sscmk1 revealed the same ORF interrupted by five introns. Bioinformatic analyses of SSCMK1 showed that this protein had the distinctive features that characterize a calcium/calmodulin protein kinase: a serine/threonine protein kinase domain and a calmodulin-binding domain. When compared to homologues from seven species of filamentous fungi, SSCMK1 showed substantial similarities, except for a large and highly variable region that encompasses positions 330 - 380 of the multiple sequence alignment. Inhibition studies using calmodulin inhibitor W-7, and calcium/calmodulin kinase inhibitors, KN-62 and lavendustin C, were found to inhibit budding by cells induced to re-enter the yeast cell cycle and to favor the yeast to mycelium transition. CONCLUSION: This study constitutes the first evidence of the presence of a calcium/calmodulin kinase-encoding gene in S. schenckii and its possible involvement as an effector of dimorphism in this fungus. These results suggest that a calcium/calmodulin dependent signaling pathway could be involved in the regulation of dimorphism in this fungus. The results suggest that the calcium/calmodulin kinases of yeasts are evolutionarily distinct from those in filamentous fungi.

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.

Dual kinase-mediated regulation of PITK by CaMKII and GSK3.

Phosphatase Interactor Targeting K protein (PITK) was previously identified as a novel PP1 targeting subunit implicated in modulating the phosphorylation of the transcriptional regulator heterogeneous nuclear ribonucleoprotein K (hnRNP K) [Kwiek NC, Thacker DF, Datto MB, Megosh HB, Haystead TA. Cell Signal 18 (10) (2006) 1769.]. Through the phosphorylation of PITK at S1013 and S1017 (residues that flank or reside within a PP1C-binding motif), the binding of the PP1 catalytic subunit to PITK, and subsequently the activity of the holoenzyme, are discretely controlled. Herein, we demonstrate that PITK phosphorylation at S1013 and S1017 also dictates the subcellular localization of the holoenzyme. Whereas both wildtype-and an S1013,1017D-PITK mutant displayed a speckled nuclear localization, a constitutively dephosphorylated form of PITK (S1013,1017A-PITK) resulted in a diffuse localization throughout the cell including the cytoplasm. Additionally, through the use of unbiased proteomics techniques, we provide evidence for a dual kinase-mediated regulation of the PITK holoenzyme whereby PITK phosphorylation at S1017 is catalyzed by calcium/calmodulin-dependent kinase II-delta (CaMKIIdelta), promoting the subsequent phosphorylation of S1013 by glycogen synthase kinase-3 (GSK3) in vitro. Taken together, our findings provide further insight into the regulation of PITK, PP1, and hnRNP K by reversible phosphorylation.

CaMKIIbeta association with the actin cytoskeleton is regulated by alternative splicing.

The Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII)beta has morphogenic functions in neurons not shared by the alpha isoform. CaMKIIbeta contains three exons (v1, v3, and v4) not present in the CaMKIIalpha gene, and two of these exons (v1 and v4) are subject to differential alternative splicing. We show here that CaMKIIbeta, but not alpha, mediated bundling of F-actin filaments in vitro. Most importantly, inclusion of exon v1 was required for CaMKIIbeta association with the F-actin cytoskeleton within cells. CaMKIIbetae, which is the dominant variant around birth and lacks exon v1 sequences, failed to associate with F-actin. By contrast, CaMKIIbeta', which instead lacks exon v4, associated with F-actin as full-length CaMKIIbeta. Previous studies with CaMKIIbeta mutants have indicated a role of nonstimulated kinase activity in enhancing dendritic arborization. Here, we show that F-actin-targeted CaMKIIbeta, but not alpha, was able to phosphorylate actin in vitro even by nonstimulated basal activity in absence of Ca(2+)/CaM. In rat pancreatic islets and in skeletal muscle, the actin-associated CaMKIIbeta' and betaM were the predominant variants, respectively. Thus, cytoskeletal targeting may mediate functions of CaMKIIbeta variants also outside the nervous system.

Isolation of the Cdc45/Mcm2-7/GINS (CMG) complex, a candidate for the eukaryotic DNA replication fork helicase.

The protein Cdc45 plays a critical but poorly understood role in the initiation and elongation stages of eukaryotic DNA replication. To study Cdc45's function in DNA replication, we purified Cdc45 protein from Drosophila embryo extracts by a combination of traditional and immunoaffinity chromatography steps and found that the protein exists in a stable, high-molecular-weight complex with the Mcm2-7 hexamer and the GINS tetramer. The purified Cdc45/Mcm2-7/GINS complex is associated with an active ATP-dependent DNA helicase function. RNA interference knock-down experiments targeting the GINS and Cdc45 components establish that the proteins are required for the S phase transition in Drosophila cells. The data suggest that this complex forms the core helicase machinery for eukaryotic DNA replication.

Mass of the postsynaptic density and enumeration of three key molecules.

The total molecular mass of individual postsynaptic densities (PSDs) isolated from rat forebrain was measured by scanning transmission EM. PSDs had a mean diameter of 360 nm and molecular mass of 1.10 +/- 0.36 GDa. Because the mass represents the sum of the molecular masses of all of the molecules comprising a PSD, it becomes possible to derive the number of copies of each protein, once its relative mass contribution is known. Mass contributions of PSD-95, synapse-associated protein (SAP)97, and alpha-Ca2+/calmodulin-dependent protein kinase II (CaMKII) were determined by quantitative gel electrophoresis of PSD fractions. The number of PSD-95 molecules per average PSD, contributing 2.3% of the mass of the PSD, was calculated to be 300, whereas the number of SAP97 molecules, contributing 0.9% of the mass of the PSD, was 90. The alpha-CaMKII holoenzymes, which contribute 6% of the mass when brains are homogenized within 2 min of interrupting blood flow, have 80 holoenzymes associated with a typical PSD. When blood flow is interrupted 15 min before homogenization, the average mass of PSDs increases by approximately 40%. The additional alpha-CaMKII associated with PSDs accounts for up to 20% of this mass increase, representing the addition of 100-200 alpha-CaMKII holoenzymes.

Neuronal Ca2+/calmodulin-dependent protein kinase II--discovery, progress in a quarter of a century, and perspective: implication for learning and memory.

Much has been learned about the activity-dependent synaptic modifications that are thought to underlie memory storage, but the mechanism by which these modifications are stored remains unclear. A good candidate for the storage mechanism is Ca2+/calmodulin-dependent protein kinase II (CaM kinase II). CaM kinase II is one of the most prominent protein kinases, present in essentially every tissue but most concentrated in brain. Although it has been about a quarter of a century since the finding, CaM kinase II has been of the major interest in the region of brain science. It plays a multifunctional role in many intracellular events, and the expression of the enzyme is carefully regulated in brain regions and during brain development. Neuronal CaM kinase II regulates important neuronal functions, including neurotransmitter synthesis, neurotransmitter release, modulation of ion channel activity, cellular transport, cell morphology and neurite extension, synaptic plasticity, learning and memory, and gene expression. Studies concerning this kinase have provided insight into the molecular basis of nerve functions, especially learning and memory, and indicate one direction for studies in the field of neuroscience. This review presents the molecular structure, properties and functions of CaM kinase II, as a major component of neurons, based mainly developed on findings made in our laboratory.

Assay and purification of calmodulin-dependent protein kinase.

Posttranslational modification has long been recognized as a way in which the properties of proteins may be subtly altered after synthesis of the polypeptide chain is complete. Amongst the moieties most commonly encountered covalently attached to proteins are oligosaccharides, phosphate, acetyl, formyl, and nucleosides. Posttranslational covalent modification of protein by phosphorylation is one of the most prevalent and best understood mechanisms employed in cellular regulation (1-9). Protein kinases catalyze the transfer of the γ-phosphoryl group of adenosine triphosphate (ATP) to an acceptor protein substrate. The activity of the enzyme is determined by the transfer of (32)P (labeled γ-phosphate) from [γ-(32)P] ATP to protein substrate.

Biochemical characterization of Ca2+/calmodulin dependent protein kinase from Candida albicans.

A multifunctional Ca2+/calmodulin dependent protein kinase was purified approximately 650 fold from cytosolic extract of Candida albicans. The purified preparation gave a single band of 69 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis with its native molecular mass of 71 kDa suggesting that the enzyme is monomeric. Its activity was dependent on calcium, calmodulin and ATP when measured at saturating histone IIs concentration. The purified Ca2+/CaMPK was found to be autophosphorylated at serine residue(s) in the presence of Ca2+/calmodulin and enzyme stimulation was strongly inhibited by W-7 (CaM antagonist) and KN-62 (Ca2+/CaM dependent PK inhibitor). These results confirm that the purified enzyme is Ca2+/CaM dependent protein kinase of Candida albicans. The enzyme phosphorylated a number of exogenous and endogenous substrates in a Ca2+/calmodulin dependent manner suggesting that the enzyme is a multifunctional Ca2+/calmodulin-dependent protein kinase of Candida albicans.

Purification and characterization of a Ca(2+)-dependent/calmodulin-stimulated protein kinase from moss chloronema cells.

We have demonstrated the presence of a Ca2+-dependent/calmodulin-stimulated protein kinase (PK) in chloronema cells of the moss Funaria hygrometrica. The kinase, with a molecular mass of 70,000 daltons (PK70), was purified to homogeneity using ammonium sulphate fractionation, DEAE-cellulose chromatography, and calmodulin (CaM)-agarose affinity chromatography. The kinase activity was stimulated at a concentration of 50 mM free Ca2+, and was further enhanced 3-5-fold with exogenously added 3-1000 nm moss calmodulin (CaM). Autophosphorylation was also stimulated with Ca2+ and CaM. Under in vitro conditions, PK70 phosphorylated preferentially lysine-rich substrates such as HIIIS and HVS. This PK shares epitopes with the maize Ca2+-dependent/calmodulin-stimulated PK (CCaMK) and also exhibits biochemical properties similar to the maize, lily, and tobacco CCaMK. We have characterized it as a moss CCaMK.

Calcium/calmodulin-dependent protein kinase I in Xenopus laevis.

Calcium/calmodulin (CaM) dependent protein kinase I (CaM-KI) is a member of a well-defined multi-functional CaM-K family, but its physiological and developmental functions have yet to be determined. Here, we have cloned two cDNAs encoding CaM-KI from a Xenopus laevis (X. laevis) oocyte cDNA library. One is a novel isoform of CaM-KI, named CaM-KI LiKbeta (XCaM-KI LiKbeta). The other is an alpha isoform of CaM-KI (XCaM-KIalpha), which is a highly related to previously cloned mammalian isoform. XCaM-KIalpha was constantly expressed through embryogenesis, whereas XCaM-KI LiKbeta expression dramatically increased in the neurula stage. Both XCaM-KI isoforms exhibited kinase activity in a Ca(2+)/CaM-dependent manner. Overexpression of a constitutively active mutant of CaM-KI isoforms inhibited cell cleavage in X. laevis embryos and caused a marked change of cell morphology in Hela cells. Taken together, these results suggest that CaM-KI plays a role in cell-structure regulation during early embryonic development.

Purification and crystallization of the N-terminal domain from the human doublecortin-like kinase.

The unique doublecortin-like tandem of two homologous domains is found in certain microtubule-associated proteins such as doublecortin (DCX) and doublecortin-like kinase (DCLK). It is responsible for interactions with tubulin/microtubules and regulates microtubule dynamics. Here, the expression and purification of the tandem from human DCLK (residues 49-280) and of the isolated domains (residues 49-154 and 176-280) and the successful crystallization of the N-terminal domain (N-DCLK) are reported. High-quality wild-type crystals were obtained and a complete native data set was collected to 1.5 A resolution. The crystals belong to space group C2, with unit-cell parameters a = 85.98, b = 29.62, c = 40.33 A, beta = 101.3 degrees. Crystals of SeMet-substituted N-DCLK (Leu120Met) were also obtained, but they exhibit the symmetry of space group P2(1), with unit-cell parameters a = 38.81, b = 29.43, c = 40.1 A, beta = 115.7 degrees.

Differential functional properties of calmodulin-dependent protein kinase IIgamma variants isolated from smooth muscle.

Six variants of calmodulin-dependent protein kinase IIgamma were isolated from a ferret-aorta smooth-muscle cDNA library. Variant G-2 is generated by a novel alternative polyadenylation, utilizing a site contained in an intron. The last 77 residues of the association domain are replaced with 99 residues of a unique sequence containing Src homology 3-domain-binding motifs, which alter catalytic activity. Variant C-2 has an eight-residue deletion in an ATP-binding motif and does not autophosphorylate Thr(286), but does phosphorylate exogenous substrate. Two variants, B and J, autodephosphorylate. Four variants differing only in the variable domain have differing catalytic activities, despite identical sequences in the catalytic domains. Thus structural features determined by variable and association domains are important for the catalytic activity of calmodulin-dependent protein kinase II.

Effect of (auto)phosphorylation on the kinetic behavior of the Ca2+/calmodulin-dependent protein kinase II from horse brain.

After extraction and purification, the kinetic behavior of the Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II) from horse brain was investigated as a function of ATP and a synthetic substrate, syntide-2. Both phospho- and dephospho- forms of the enzyme obey a bi-bi random mechanism. The K(M)s for ATP (K(M,ATP)) and syntide-2 (K(M,syntide-2)) were determined as equal to 80 and 30 microM, respectively. However, the maximum reaction yield is decreased by 50% when the enzyme is (auto)phosphorylated. In addition, this phosphorylated form of the enzyme leads to the formation of a totally Ca(2+)-independent state of activity.

A Ca2+/CaM-dependent kinase from pea is stress regulated and in vitro phosphorylates a protein that binds to AtCaM5 promoter.

An immuno-homologue of maize Ca2+/calmodulin (CaM)-dependent protein kinase with a molecular mass of 72 kDa was identified in pea. The pea kinase (PsCCaMK) was upregulated in roots in response to low temperature and increased salinity. Exogenous Ca2+ application increased the kinase level and the response was faster than that obtained following stress application. Low temperature-mediated, but not salinity-mediated stress kinase increase was inhibited by the application of EGTA and W7, a CaM inhibitor. The purification of PsCCaMK using immuno-affinity chromatography resulted in coelution of the kinase with another polypeptide of molecular mass 40 kDa (p40). Western blot revealed the presence of PsCCaMK in nuclear protein extracts and was found to phosphorylate p40 in vitro. Gel mobility shift and South-Western analysis showed that p40 is a DNA-binding protein and it interacted specifically with one of the cis acting elements of the Arabidopsis CaM5 gene (AtCaM5) promoter. The binding of p40 to the specific elements in the AtCaM5 promoter was dependent of its dephosphorylated state. Our results suggest that p40 could be an upstream signal component of the stress responses.

Characterization, physical location and expression of the genes encoding calcium/calmodulin-dependent protein kinases in maize (Zea mays L.).

The maize genomic sequence and cDNA encoding a calcium/calmodulin-dependent protein kinase homolog were isolated and identified. The deduced peptide (MCK2) from this cDNA shared high amino acid identity (91.2%) with maize MCK1. These two genes were physically mapped onto chromosomes by fluorescence in situ hybridization using the first introns of the genes as gene-specific probes. While the MCK1 gene was assigned to a locus on the long arm of chromosome 9, the MCK2 gene was localized to a locus on the long arm of chromosome 1. Both of these genes were expressed in roots, leaves, stems and flowers, and the expression patterns of MCK were verified by RNA in situ hybridization. These results indicated that MCK expression is temporally and spatially regulated during maize growth and development.

Cloning and differential expression of new calcium, calmodulin-dependent protein kinase II isoforms in Xenopus laevis oocytes and several adult tissues.

The different oligomers composing the high molecular weight calcium/calmodulin-dependent protein kinase II (CaMKII) holoenzyme, previously shown to be transiently activated during Xenopus oocyte maturation, migrate on SDS-PAGE as proteins of 83, 72, 62, 56, and 52 kDa and have all been cloned. The holoenzyme consists of the CaMKII isoforms gammaB, gammaC, and delta12, already described in other species, while gammaJ, gammaK, gammaL, gammaM, and gammaN are now described for the first time. The gamma-isoforms are splice variants of the gamma-gene, containing in their variable region different combinations of known exons and one, two or three novel exons. Semi-quantitative RT-PCR revealed that all isoforms identified in prophase oocytes are also expressed in adult tissues with a tissue-specific expression pattern. At least thirty different CaMKII isoforms could be identified in different Xenopus adult tissues, most of which are described here for the first time.