Calmodulin-independent Protein Kinase Research Articles

Transcriptomic and proteomic analyses of embryogenic tissues in Picea balfouriana treated with 6-benzylaminopurine.

The cytokinin 6-benzylaminopurine (6-BAP) influences the embryogenic capacity of the tissues of Picea balfouriana during long subculture (after 3 months). Tissues that proliferate in 3.6 and 5 µM 6-BAP exhibit the highest and lowest embryogenic capacity, respectively, generating 113 ± 6 and 23 ± 3 mature embryos per 100 mg of tissue. In this study, a comparative transcriptomic and proteomic approach was applied to characterize the genes and proteins that are differentially expressed among tissues under the influence of different levels of 6-BAP. A total of 51 375 unigenes and 2617 proteins were obtained after quality filtering. There were 2770 transcripts for proteins found among these unigenes. Gene ontology (GO) analysis of the differentially expressed unigenes and proteins showed that they were involved in cell and binding activity and were enriched in ribosome and glutathione metabolism pathways. Ribosomal proteins, glutathione S-transferase proteins, germin-like proteins and calmodulin-independent protein kinases were up-regulated in the embryogenic tissues with the highest embryogenic ability (treated with 3.6 µM 6-BAP), which was validated via quantitative real-time polymerase chain reaction (qRT-PCR) analysis, and these proteins might serve as molecular markers of embryogenic ability. Data are available via Sequence Read Archive (SRA) and ProteomeXchange with identifier SRP042246 and PXD001022, respectively.

Membrane-associated protein kinase activities in the developing mesocarp of grape berry

Fruit development is a process involving various signals and gene expression. Protein phosphorylation catalyzed by protein kinases is known to play a key role in eukaryotic cell signalling and so may be involved in the regulation of fruit development. Using the method of exogenous substrate phosphorylation, we characterised the calcium-dependent and calmodulin-independent protein kinase (CDPK) activity and the myelin basic protein (MBP)-phosphoralating activity that could be due to a mitogen-activated protein kinase (MAPK)-like activity in the developing mesocarp of grape berry. The CDPK activity was shown to be predominantly localised in the plasma membrane, while the MAPK-like activity was predominantly associated with endomembranes. The assays of bivalent cation requirement showed that Mn2+ could to a certain extent replace Mg2+ in the incubation system for the protein kinase activities. Both CDPK and MAPK-like activities were resistant to heat treatment. The activities of the two enzymes were fruit developmental stage-specific with the highest activities of both enzymes in the lag growth phase before the ripening stage, suggesting strongly the important roles of the detected CDPK and MAPK-like activities in the fruit development.

Membrane-associated protein kinase activities in developing apple fruit.

Fruit development is a process involving various signals and gene expression. Protein phosphorylation catalysed by protein kinases is known to play a key role in eukaryotic cell signalling and so may be involved in the regulation of fruit development. Using the method of exogenous substrate phosphorylation, the activity of calcium-dependent and calmodulin-independent protein kinase (CDPK) that was stimulated by phosphatidylserine, and the myelin basic protein (MBP)-phosphorylating activity that could be due to a calcium-independent mitogen-activated protein kinase-like (MAPK-like) activity in the developing apple fruits were identified. The CDPK activity was shown to be predominantly localized in the plasma membrane, whereas in the presence of phosphatidylserine, the high activity of CDPK was detected in both plasma membrane and endomembranes. The MAPK-like activity was predominantly associated with endomembranes. The assays of bivalent cation requirement showed that Mn2+ could replace Mg2+ in the incubation system for the protein kinase activities and stimulate CDPK activity more than Mg2+. Heat treatment abolished CDPK but stimulated MAPK-like activity. The activities of the phosphatidylserine-stimulated CDPK and of the MAPK-like were fruit developmental stage-specific with higher activities of both enzymes in the early and middle developmental stages in comparison with the late developmental stage. These data suggest that the detected protein kinases may play an important role in the fruit development.

Two mRNA species encoding calcium-dependent protein kinases are differentially expressed in sexual organs of Marchantia polymorpha through alternative splicing.

In plants, calcium-dependent calmodulin-independent protein kinases (CDPKs) are the predominant calcium-regulated protein kinases and their genes are encoded by a multigene family. A CDPK gene was cloned from a liverwort, Marchantia polymorpha, which showed a high level of sequence similarities to other higher plant CDPK genes. The liverwort CDPK gene consisted of 9 exons and 8 introns. The 6th and 7th exons (Exon 6A and Exon 6B) were almost identical except for 4-amino acid substitutions, both of which coded for EF-hands in the calcium-binding domain. RT-PCR analysis revealed that two species of mature mRNA containing either Exon 6A or Exon 6B were generated from a single CDPK gene by mutually exclusive alternative splicing. Both histidine-tagged fusion proteins derived from cDNAs containing either Exon 6A or Exon 6B exhibited calcium-dependent protein kinase activity in vitro. Preferential accumulation of the mature mRNA with Exon 6A detected in male sexual organ implies possible sexual control of the ratio between the two CDPK isozymes through alternative splicing. Functions and evolution of CDPKs are discussed based on the structure and expression of the liverwort CDPK gene.

Characterization of a maize Ca(2+)-dependent protein kinase phosphorylating phosphoenolpyruvate carboxylase.

Phosphoenolpyruvate carboxylase (PEPC) [EC 4.1.1.31] of plants undergoes regulatory phosphorylation in response to light or nutritional conditions. However, the nature of protein kinase(s) for this phosphorylation has not yet been fully elucidated. We separated a Ca(2+)-requiring protein kinase from Ca(2+)-independent one, both of which can phosphorylate maize leaf PEPC and characterized the former kinase after partial purification. Several lines of evidence indicated that the kinase is one of the characteristic Ca(2+)-dependent but calmodulin-independent protein kinase (CDPK). Although the M(r) of native CDPK was estimated to be about 100 kDa by gel permeation chromatography, in situ phosphorylation assay of CDPK in a SDS-polyacrylamide gel revealed that the subunit has an M(r) of about 50 kDa suggesting dimer formation or association with other protein(s). Several kinetic parameters were also obtained using PEPC as a substrate. Although the CDPK showed an ability of regulatory phosphorylation (Ser-15 in maize PEPC), no significant desensitization to feedback inhibitor, malate, could be observed presumably due to low extent of phosphorylation. The kinase was not specific to PEPC but phosphorylated a variety of synthetic peptides. The possible physiological role of this kinase was discussed.

Calcium modulates rapid protein phosphorylation/dephosphorylation in isolated eyespot apparatuses of the green alga Spermatozopsis similis

We present an initial characterization of protein kinase and phosphatase activities associated with isolated eyespot apparatuses, the organelle involved in blue/ green-light-mediated behavioural responses of flagellate green algae. In the presence of the phosphatase inhibitors okadaic acid and vanadate, rapid overall protein phosphorylation (t0.5 ≈ 10 s) was observed. The majority of protein kinase activities and their substrates were identified as integral or tightly-bound peripheral membrane proteins. While vanadate generally increased the phosphate incorporation into all phosphoproteins, okadaic acid specifically enhanced phosphorylation of proteins in the range of 39–43 kDa. In contrast to all other phosphoproteins in this subcellular fraction, two proteins with apparent molecular masses of 83 and 16 kDa shared remarkable similarities: (i) They exhibited a fast turnover of the 32P-label, even in the presence of phosphatase inhibitors, and (ii) their dephosphorylation was delayed at 10−8 M free Ca2+. In addition, the 16-kDa protein underwent thiophosphorylation. The general in-vitro phosphorylation pattern was strongly influenced by alterations of free Ca2+ in a concentration range known to affect responses related to phototactic and photophobic behaviour of this alga (10−8 M to 10−7 M). However, characteristics of Ca2+-calmodulin-dependent protein kinases were not observed, i.e. exogenous calmodulin and trifluoperazine had no significant effect on protein phosphorylation. Also exogenous lipids (phosphatidylserine, diacylglycerol), inhibitors of cGMP and cAMPdependent protein kinases and protein kinase C (H-7 and HA1004) as well as exogenously added cGMP and cAMP did not potentiate or inhibit protein phosphorylation. These characteristics of the kinase activity in our fraction most closely resemble those of the plant- and protist-specific group of Ca2+-dependent, calmodulin-independent protein kinases. In-situ phosphorylation experiments following electrophoretic separation revealed the presence of three putative Ca2+-dependent kinases or their catalytic subunits (77,48 and 47 kDa) in the eyespot preparation. In addition, a Ca2+-independent activity at 28 kDa was detected. Possible roles of reversible protein phosphorylation in eyespot apparatuses are discussed.

Cloning and characterization of a maize pollen-specific calcium-dependent calmodulin-independent protein kinase.

A calcium-dependent calmodulin-independent protein kinase (CDPK) has been cloned from maize (Zea mays). The sequence predicts a 550-amino acid (predicted molecular mass is 60 kDa) protein with two major functional domains: an N-terminal catalytic domain highly homologous to protein kinases and a C-terminal domain resembling calmodulins. Northern analysis shows that the expression of the maize CDPK gene is pollen specific and that its transcription is restricted to late stages of pollen development. Western blots reveal a major abundance of CDPK protein at the stage of pollen germination. In vitro germination and pollen tube growth are impaired upon addition of a calmodulin antagonist (calmidazolium), CDPK inhibitors (W-7), and antisense oligonucleotides directed against CDPK mRNA. These observations indicate that the function of the pollen-specific maize CDPK protein is required for germination and pollen tube growth.

Glucose activates the multifunctional Ca2+/calmodulin-dependent protein kinase II in isolated rat pancreatic islets.

The influence of the insulin secretagogues, glucose and K+, to activate the multifunctional, Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) in isolated rat pancreatic islets has been examined. Glucose (28 mM) and K+ (40 mM) were demonstrated to induce a 1.89 +/- 0.19- and 1.75 +/- 0.15-fold increase, respectively, in phosphorylation of a subunit of CaM kinase II immunoprecipitated by an anti-CaM kinase II alpha antibody. In intact islets, glucose and K+ also induced the generation of an autonomous, Ca2+/calmodulin-independent protein kinase II activity characteristic of autophosphorylated enzyme. Maximal activation, 2.9 +/- 0.2- and 3.0 +/- 0.5-fold for glucose and K+, respectively, relative to basal glucose control, was achieved at 2.5-5 min followed by a decline to near basal levels by 20 min. Glucose induced the production of autonomous CaM kinase II activity that, in terms of -fold stimulation, correlated closely with the extent of insulin release over a glucose concentration range of 3-28 mM. This stimulated activity was completely prevented by an inhibitor of glucose metabolism, mannoheptulose. These data demonstrate that the exposure of islets to stimulatory glucose concentrations activates CaM kinase II. The close correlation of enzyme activation with insulin secretion is consistent with the hypothesis that CaM kinase II plays an important role in the regulation of insulin secretion or related beta-cell processes.

Topology and phosphorylation of soybean nodulin-26, an intrinsic protein of the peribacteroid membrane.

Soybean nodulin-26, a homologue of bovine eye lens major intrinsic protein (MIP-26), is an integral protein of the peribacteroid membrane in symbiotic root nodules. It comprises 271 amino acids with six potential transmembrane domains and lacks an amino-terminal signal sequence. A full-length nodulin-26 cDNA and its various deletion derivatives were transcribed in vitro after linking them to bacteriophage T3 promoter. In vitro translation of these transcripts in a rabbit reticulocyte lysate, in the presence or absence of canine pancreatic microsomal membranes, suggested that nodulin-26 is cotranslationally inserted into the microsomes without a cleavable signal peptide. The first two transmembrane domains (103 amino acids) of the protein are sufficient for microsomal membrane insertion. Membrane-translocated nodulin-26 binds to Con-A and is sensitive to endoglycosidase-H treatment, suggesting that it is glycosylated. Native nodulin-26 from root nodules retains its sugar moiety as it, too, binds to Con-A. Chemical cleavage mapping at cysteine residues, a trypsin protection assay, and the Con-A binding affinity of nodulin-26 suggested that both the NH2 and COOH termini of this protein are on the cytoplasmic surface of the peribacteroid membrane, while the glycosidic residue is on the surface of the membrane facing the bacteroids. In vitro phosphorylation experiments showed that nodulin-26 is a major phosphorylated protein in the peribacteroid membrane. This phosphorylation is mediated by a Ca(2+)-dependent, calmodulin-independent protein kinase located in the peribacteriod membrane. Externally supplied acid phosphatase dephosphorylates this protein, but alkaline phosphatase does not. Based on its homology with several eukaryotic and prokaryotic channel-type membrane proteins, nodulin-26 may form a channel translocating specific molecules to the bacteroids during endosymbiosis in legume plants.

Calcium-activated protein kinase from soluble and membrane fractions of maize coleoptiles

This paper describes the results of experiments in which phenyl Sepharose was used to partially purify Ca2+-activated protein kinase (CPK) from maize soluble and membrane-solubilized proteins. It is shown that CPK has very similar properties to Ca2+-activated, calmodulin independent protein kinase from other plant tissues, and that chromatography on phenyl Sepharose resolves two closely related forms of CPK from both soluble and membrane-solubilized proteins. The amount of each of these forms differs in the two fractions, and it is suggested that the kinase requiring EGTA for elution from phenyl Sepharose at high pH may be either a non-proteolitically digested form or an acylated form of CPK.

Okadaic acid, a phosphatase inhibitor, produces a Ca2+ and calmodulin-independent contraction of smooth muscle

The effects of okadaic acid, a phosphoprotein phosphatase inhibitor, on the contractile response and on myosin light chain phosphorylation were studied in intact lamb tracheal smooth muscle. The effects of okadaic acid were compared to the response of the same fibers stimulated with 1 microM methacholine, a concentration that induces 90% of maximal force. Okadaic acid (50 microM) produced a slow but maximal contraction that was accompanied by an increase in phosphorylation of the 20 kDa light chain of myosin. The myosin light chain phosphorylation pattern induced by okadaic acid, however, differed from that induced by methacholine. Ca2+ depletion, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a calmodulin antagonist and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), a protein kinase C inhibitor, blocked or attenuated methacholine-induced contractions but had no significant effect on force development or myosin light chain phosphorylation induced by okadaic acid. These results suggest that phosphorylation of the 20 kDa light chain of myosin is essential for smooth muscle contraction; they also suggest that okadaic acid either uncovers or activates an apparently Ca2+ and calmodulin-independent protein kinase activity that phosphorylates the 20 kDa light chain of myosin at multiple sites.

Activation of the multifunctional Ca2+/calmodulin-dependent protein kinase by autophosphorylation: ATP modulates production of an autonomous enzyme.

The multifunctional Ca2+/calmodulin-dependent protein kinase purified from rat brain cytosol undergoes an intramolecular self-phosphorylation or autophosphorylation. Autophosphorylation produces two strikingly different effects on kinase activity that are dependent on the level of ATP used in the reaction. At low but saturating levels of ATP (5 microM), autophosphorylation causes a 75% reduction in kinase activity, with the residual activity still retaining a dependence on Ca2+ and calmodulin. By contrast, at high but physiological levels of ATP (500 microM), the kinase is converted by autophosphorylation to a form that is autonomous of Ca2+ and calmodulin, with no accompanying reduction in activity. The extent of phosphate incorporation does not determine whether the kinase becomes inhibited or autonomous. Autophosphorylated kinase shows the functional change characteristic of the ATP concentration used during the reaction--inhibited at low ATP and autonomous at high ATP--even when compared at the same level of incorporated phosphate. ATP appears to regulate the site(s) phosphorylated during activation of the kinase and thereby modulates the dual effects of autophosphorylation. Events triggered by transient elevations of cellular Ca2+ may be potentiated and retained by generation of the Ca2+/calmodulin-independent protein kinase activity.

Properties of protein kinases in brain coated vesicles.

Coated vesicles prepared from bovine brain contained cyclic nucleotides- and Ca2+-calmodulin-independent protein kinases which in the presence of Mg2+ catalyzed the phosphorylation of an endogenous 48,000 Mr protein of coated vesicles (C-48), phosvitin and troponin T. Phosvitin was phosphorylated either in the presence of ATP or GTP. The phosphorylation of C-48, on the other hand, was specific for ATP. Heparin inhibited the phosphorylation of phosvitin but not that of C-48. Mn2+ inhibited the phosphorylation of phosvitin, while Mn2+ substituted for Mg2+ in the phosphorylation of C-48. When the coated vesicles were prepared in the presence of NaF, C-48 contained 2.5-2.8 mol of phosphate/mol. On incubation with Mg2+ and ATP, C-48 incorporated 1.2-1.6 mol of phosphate/mol. With C-48 as a substrate, the value of its apparent Km for ATP was 6 microM. With phosvitin as a substrate, the value of its apparent Km was 20 microM. The phosphorylated amino acid residues in the phosvitin were identified as serine and threonine. Phosphothreonine was detected in C-48. These results suggest that brain coated vesicles possess two different classes of protein kinase, a casein kinase II and C-48 kinase.