Casein Kinase II-Type Protein Kinase from Pea Cytoplasm and Its Inactivation by Alkaline Phosphatase in Vitro

A casein kinase II-type protein kinase has been purified from the cytosolic fraction of etiolated pea (Pisum sativum L.) plumules to about 90% purity as judged from Coomassie blue stained sodium dodecyl sulfate-polyacrylamide gels. This kinase has a tetrameric [alpha][alpha]'[beta]2 structure with a native molecular mass of 150 kD, and subunit molecular masses of 41 and 40 kD for the two catalytic subunits ([alpha] and [alpha]') and 35 kD for the putative regulatory subunit ([beta]).Casein and phosvitin can be used as artificial substrates for this kinase. Both serine and threonine residues were phosphorylated when mixed casein, [beta]-casein, or phosvitin were used as the substrate, whereas only serine was phosphorylated if [alpha]-casein or histone III-S was the substrate. The kinase activity was stimulated 130% by 0.5 mM spermine (the concentration required for 50% of maximal enzyme activity [A50] = 0.1 mM) and 80% by 2.5 mM spermidine (A50 = 0.4 mM), whereas putrescine and cadaverine had no effect. The kinase was very sensitive to inhibition by heparin (concentration for 50% inhibition [I50] = 0.025 [mu]g/mL). In contrast to most other casein kinase II-type protein kinases, this preparation was inhibited by K+ and Na+, with I50 values of 75 and 65 mM, respectively. Pretreatment of the purified kinase preparation in vitro with alkaline phosphatase caused a 5-fold decrease in its activity. Additionally, this kinase also lost its activity when its [beta] subunit was autophosphorylated in the absence of substrate. These results suggest that the activity of this casein kinase II protein kinase may be regulated by the phosphorylation state of two different sites in its multimeric structure.     

Phosphorylation of nucleolin by a nucleolar type NII protein kinase

Nucleolin [C23 or 100 kilodaltons (kDa)] is the major nucleolar phosphorylated protein in exponentially growing Chinese hamster ovary cells. A nucleolar cyclic nucleotide independent protein kinase copurified with nucleolin in a complex which could be dissociated by hydroxyapatite chromatography. The kinase was stimulated by spermine and inhibited by heparin and presented most of the properties of nuclear casein kinase NII. Kinetic analyses showed the apparent Km value for nucleolin (7 X 10(-4) mg/mL) to be lower than those for other casein kinase II substrates such as nuclear protein HMG 14 (0.15 mg/mL), topoisomerase I (0.025 mg/mL), or topoisomerase II (0.04 mg/mL). Similarly, Vmax values were higher for nucleolin than for other substrates. Nucleolin thus appears to be a natural preferential substrate of nucleolar casein kinase NII. The kinase phosphorylated nucleolin in vitro at serine residues in a 29-kDa CNBr fragment located near the amino terminus of the molecule. The enzyme labeled typical casein kinase II sites. These sites were found predominantly in two highly acidic tryptic fragments designated A (residues 21-49) and C (residues 180-221) which contained serines having at least two acidic residues on their carboxyl-terminal sides. These results demonstrate the existence in the nucleolus of a type of NII protein kinase that uses a protein involved in ribosome assembly as preferential substrate.     

Role for Tyrosine Phosphorylation of A-kinase Anchoring Protein 8 (AKAP8) in Its Dissociation from Chromatin and the Nuclear Matrix

Protein-tyrosine phosphorylation regulates a wide variety of cellular processes at the plasma membrane. Recently, we showed that nuclear tyrosine kinases induce global nuclear structure changes, which we called chromatin structural changes. However, the mechanisms are not fully understood. In this study we identify protein kinase A anchoring protein 8 (AKAP8/AKAP95), which associates with chromatin and the nuclear matrix, as a nuclear tyrosine-phosphorylated protein. Tyrosine phosphorylation of AKAP8 is induced by several tyrosine kinases, such as Src, Fyn, and c-Abl but not Syk. Nucleus-targeted Lyn and c-Src strongly dissociate AKAP8 from chromatin and the nuclear matrix in a kinase activity-dependent manner. The levels of tyrosine phosphorylation of AKAP8 are decreased by substitution of multiple tyrosine residues on AKAP8 into phenylalanine. Importantly, the phenylalanine mutations of AKAP8 inhibit its dissociation from nuclear structures, suggesting that the association/dissociation of AKAP8 with/from nuclear structures is regulated by its tyrosine phosphorylation. Furthermore, the phenylalanine mutations of AKAP8 suppress the levels of nuclear tyrosine kinase-induced chromatin structural changes. In contrast, AKAP8 knockdown increases the levels of chromatin structural changes. Intriguingly, stimulation with hydrogen peroxide induces chromatin structural changes accompanied by the dissociation of AKAP8 from nuclear structures. These results suggest that AKAP8 is involved in the regulation of chromatin structural changes through nuclear tyrosine phosphorylation.     

Role of PIP-Aquaporin Phosphorylation in Redox-Dependent Modulation of Osmotic Water Permeability in Plasmalemma from Roots of Pea Seedlings

Russian Journal of Plant Physiology - Investigations were directed to study the mechanism based on redox-dependent modulation of osmotic water permeability in plasmalemma from roots of pea (Pisum...     

Evidence for a UDP-Glucose Transporter in Golgi Apparatus-Derived Vesicles from Pea and Its Possible Role in Polysaccharide Biosynthesis

The Golgi apparatus in plant cells is involved in hemicellulose and pectin biosynthesis. While it is known that glucan synthase I is responsible for the formation of [beta]-l-4-linked glucose (Glc) polymers and uses UDP-Glc as a substrate, very little is known about the topography of reactions leading to the biosynthesis of polysaccharides in this organelle. We isolated from pea (Pisum sativum) stems a fraction highly enriched in Golgi apparatus-derived vesicles that are sealed and have the same topographical orientation that the membranes have in vivo. Using these vesicles and UDP-Glc, we reconstituted polysaccharide biosynthesis in vitro and found evidence for a luminal location of the active site of glucan synthase I. In addition, we identified a UDP-Glc transport activity, which is likely to be involved in supplying substrate for glucan synthase I. We found that UDP-Glc transport is protein mediated. Moreover, our results suggest that UDP-Glc transport is coupled to the exit of a luminal uridine-containing nucleotide via an antiporter mechanism. We suggest that UDP-Glc is transported into the lumen of Golgi and that Glc is transferred to a polysaccharide chain, whereas the nucleotide moiety leaves the vesicle by an antiporter mechanism.     

Phosphorylation of Mitogen-Activated Protein Kinase in Cultured Rat Cortical Glia by Stimulation of Metabotropic Glutamate Receptors

Abstract: Activation of metabotropic glutamate receptors (mGluRs) in glia results in significant physiological effects for both the glia and the neighboring neurons; but in many cases, the mGluR subtypes and signal transduction mechanisms mediating these effects have not been determined. In this study, we report that mGluR activation in primary cultures of rat cortical glia results in tyrosine phosphorylation of several proteins, including p44/p42 mitogen-activated protein kinases, also referred to as extracellular signal-regulated kinases (ERK1/2). Incubation of glial cultures with the general mGluR agonist 1-aminocyclopentane-1 S ,3 R -dicarboxylate and the mGluR group I-selective agonists ( RS )-3,5-dihydroxyphenylglycine (DHPG) and l -quisqualate resulted in increased tyrosine phosphorylation of ERK1/2. The group II-selective agonist (2 S ,2' R ,3' R )-2-(2',3'-dicarboxycyclopropyl)glycine and group III-selective agonist l (+)-2-amino-4-phosphonobutyric acid had no effect on tyrosine phosphorylation. DHPG-induced ERK1/2 phosphorylation could be inhibited by an antagonist that acts at group I or group II mGluRs but not by antagonists for group II and group III mGluRs. Protein kinase C (PKC) activators also induced ERK1/2 phosphorylation, but the PKC inhibitor bisindolylmaleimide I did not inhibit DHPG-induced ERK1/2 phosphorylation at a concentration that inhibited the response to phorbol 12,13-dibutyrate. These data suggest that mGluR activation of ERK1/2 in cultured glia is mediated by group I mGluRs and that this effect is independent of PKC activation. Furthermore, immunoblots with antibodies against various mGluR subtypes show expression of mGluR5, but no other mGluRs in our cultures. Taken together, these results suggest that mGluR5 stimulation results in tyrosine phosphorylation of ERK1/2 and other glial proteins.     

Partial Purification and Characterization of a Ca2+-Dependent Protein Kinase from Pea Nuclei

Almost all the Ca²⁺-dependent protein kinase activity in nuclei purified from etiolated pea (Pisum sativum, L.) plumules is present in a single enzyme that can be extracted from chromatin by 0.3 molar NaCl. This protein kinase can be further purified 80,000-fold by salt fractionation and high performance liquid chromatography, after which it has a high specific activity of about 100 picomoles per minute per microgram in the presence of Ca²⁺ and reaches half-maximal activation at about 3 ×10⁻⁷ molar free Ca²⁺, without calmodulin. It is a monomer with a molecular weight near 90,000. It can efficiently use histone III-S, ribosomal S6 protein, and casein as artificial substrates, but it phosphorylates phosvitin only weakly. Its Ca²⁺-dependent kinase activity is half-maximally inhibited by 0.1 millimolar chlorpromazine, by 35 nanomolar K-252a and by 7 nanomolar staurosporine. It is insensitive to sphingosine, an inhibitor of protein kinase C, and to basic polypeptides that block other Ca²⁺-dependent protein kinases. It is not stimulated by exogenous phospholipids or fatty acids. In intact isolated pea nuclei it preferentially phosphorylates several chromatin-associated proteins, with the most phosphorylated protein band being near the same molecular weight (43,000) as a nuclear protein substrate whose phosphorylation has been reported to be stimulated by phytochrome in a calcium-dependent fashion.     

Phorbol Ester Binding Sites in the Fish Retina: Correlation with Stimulation of Endogenous Phosphorylation and Protein Kinase C Activation

Abstract: The injection of phorbol esters into the eyes of dark-adapted teleost fish can mimic light effects in the retina and induces corresponding synaptic plasticity of horizontal cells (HCs). It is therefore very likely that protein kinase C (PKC) mediates light-induced synaptic plasticity. In the present study, we investigated the distribution of PKC, the phorbol ester receptor, in isolated HCs and in the whole retina by using tritiated phorbol 12,13-dibutyrate ([³H]PDBu). The binding characteristics analyzed for HC homogenates and retinal homogenates revealed that [³H]PDBu binding is time dependent, specific, saturable, and reversible. Binding sites in HCs displayed a dissociation constant of 11.5 n M and a total number of 2.8 pmol/mg of protein. Autoradiography revealed that [³H]PDBu labeling is present in all retinal layers, including HCs, where it is associated with the somata. Furthermore, the treatment with PDBu strongly affected the endogenous phosphorylation of several membrane, cytosolic, and HC proteins and led to PKC activation as measured by H1 histone phosphorylation. In HCs, the treatment with PDBu in particular affected the amount of ³²P incorporated into a group of phosphoproteins (68, 56/58, 47, 28, and 15 kDa) that were recently shown to be affected by light adaptation. These proteins might therefore be considered as important components of the observed morphological and physiological synaptic plasticity of HCs in the course of light adaptation.     

Physiological Phosphorylation of Protein Kinase A at Thr-197 Is by a Protein Kinase A Kinase

Phosphorylation of the catalytic subunit of cyclic AMP-dependent protein kinase, or protein kinase A, on Thr-197 is required for optimal enzyme activity, and enzyme isolated from either animal sources or bacterial expression strains is found phosphorylated at this site. Autophosphorylation of Thr-197 occurs in Escherichia coli and in vitro but is an inefficient intermolecular reaction catalyzed primarily by active, previously phosphorylated molecules. In contrast, the Thr-197 phosphorylation of newly synthesized protein kinase A in intact S49 mouse lymphoma cells is both efficient and insensitive to activators or inhibitors of intracellular protein kinase A. Using [³⁵S]methionine-labeled, nonphosphorylated, recombinant catalytic subunit as the substrate in a gel mobility shift assay, we have identified an activity in extracts of protein kinase A-deficient S49 cells that phosphorylates catalytic subunit on Thr-197. The protein kinase A kinase activity partially purified by anion-exchange and hydroxylapatite chromatography is an efficient catalyst of protein kinase A phosphorylation in terms of both a low K_m for ATP and a rapid time course. Phosphorylation of wild-type catalytic subunit by the kinase kinase activates the subunit for binding to a pseudosubstrate peptide inhibitor of protein kinase A. By both the gel shift assay and a [γ-³²P]ATP incorporation assay, the enzyme is active on wild-type catalytic subunit and on an inactive mutant with Met substituted for Lys-72 but inactive on a mutant with Ala substituted for Thr-197. Combined with the results from mutant subunits, phosphoamino acid analysis suggests that the enzyme is specific for phosphorylation of Thr-197.     

Phosphorylation of Nuclear Proteins in Myelinating Oligodendrocytes and Its Control by Cyclic AMP

Oligodendroglial nuclei isolated from rat brains at different stages of myelinogenesis (10, 18, and 30 days of age) were incubated with [gamma-32P]ATP and extracted with 0.75 M perchloric acid to yield a fraction of nonacidic chromatin proteins. The protein extracts were then analyzed by polyacrylamide gel electrophoresis. The phosphorylation pattern of these proteins was found to be different for different age groups. In 10-day-old rat oligodendrocytes the most extensive phosphorylation occurred in low molecular mass species (less than 30 kDa), in contrast to fractions obtained from 18- and 30-day-old rat oligodendrocytes which showed a significantly higher labeling of the proteins with molecular masses greater than 30 kDa. The phosphorylation of the latter species was greatly stimulated by the presence of cyclic AMP in the incubation media. The results suggest that the phosphorylation of specific nuclear proteins, which may play a regulatory role at different stages of oligodendroglial maturation and myelinogenesis, may be at least partially modulated by intracellular cyclic AMP.     

Direct Phosphorylation and Activation of a Mitogen-Activated Protein Kinase by a Calcium-Dependent Protein Kinase in Rice

The mitogen-activated protein kinase (MAPK) is a pivotal point of convergence for many signaling pathways in eukaryotes. In the classical MAPK cascade, a signal is transmitted via sequential phosphorylation and activation of MAPK kinase kinase, MAPK kinase (MKK), and MAPK. The activation of MAPK is dependent on dual phosphorylation of a TXY motif by an MKK, which is considered the sole kinase to phosphorylate and activate MAPK. Here, we report a novel regulatory mechanism of MAPK phosphorylation and activation besides the canonical MAPK cascade. A rice (Oryza sativa) calcium-dependent protein kinase (CDPK), CPK18, was identified as an upstream kinase of MAPK (MPK5) in vitro and in vivo. Curiously, CPK18 was shown to phosphorylate and activate MPK5 without affecting the phosphorylation of its TXY motif. Instead, CPK18 was found to predominantly phosphorylate two Thr residues (Thr-14 and Thr-32) that are widely conserved in MAPKs from land plants. Further analyses reveal that the newly identified CPK18-MPK5 pathway represses defense gene expression and negatively regulates rice blast resistance. Our results suggest that land plants have evolved an MKK-independent phosphorylation pathway that directly connects calcium signaling to the MAPK machinery.     

Cyclic GMP-dependent Stimulation of Serotonin Transport Does Not Involve Direct Transporter Phosphorylation by cGMP-dependent Protein Kinase

The serotonin transporter (SERT) is responsible for reuptake of serotonin (5-hydroxytryptamine) after its exocytotic release from neurons. It is the primary target for antidepressants and stimulants, including “ecstasy” (3,4-methylenedioxymethamphetamine). SERT is regulated by several processes, including a cyclic GMP signaling pathway involving nitric oxide synthase, guanylyl cyclase, and cGMP-dependent protein kinase (PKG). Here, we show that SERT was phosphorylated in a PKG Iα-dependent manner in vitro, but that SERT was not a direct substrate of PKG. We generated an analog-sensitive gatekeeper residue mutant of PKG Iα (M438G) that efficiently used the ATP analog N⁶-benzyl-ATP. This mutant, but not the wild type (WT) kinase, used the ATP analog to phosphorylate both a model peptide substrate as well as an established protein substrate of PKG (vasodilator-stimulated phosphoprotein). PKG Iα M438G effectively substituted for the WT kinase in stimulating SERT-mediated 5-hydroxytryptamine transport in cultured cells. Addition of either WT or mutant PKG Iα M438G to membranes containing SERT in vitro led to radiolabel incorporation from [γ-³³P]ATP but not from similarly labeled N⁶-benzyl-ATP, indicating that SERT was phosphorylated by another kinase that could not utilize the ATP analog. These results are consistent with the proposed SERT phosphorylation site, Thr-276, being highly divergent from the consensus PKG phosphorylation site sequence, which we verified through peptide library screening. Another proposed SERT kinase, the p38 mitogen-activated protein kinase, could not substitute for PKG in this assay, and p38 inhibitors did not block PKG-dependent phosphorylation of SERT. The results suggest that PKG initiates a kinase cascade that leads to phosphorylation of SERT by an as yet unidentified protein kinase.     

Autophosphorylation of Polo-like Kinase 4 and Its Role in Centriole Duplication

Centrosome duplication occurs once every cell cycle in a strictly controlled manner. Polo-like kinase 4 (PLK4) is a key regulator of this process whose kinase activity is essential for centriole duplication. Here, we show that PLK4 autophosphorylation of serine S305 is a consequence of kinase activation and enables the active fraction to be identified in the cell. Active PLK4 is detectable on the replicating mother centriole in G1/S, with the proportion of active kinase increasing through interphase to reach a maximum in mitosis. Activation of PLK4 at the replicating daughter centriole is delayed until G2, but a level equivalent to the replicating mother centriole is achieved in M phase. Active PLK4 is regulated by the proteasome, because either proteasome inhibition or mutation of the degron motif of PLK4 results in the accumulation of S305-phosphorylated PLK4. Autophosphorylation probably plays a role in the process of centriole duplication, because mimicking S305 phosphorylation enhances the ability of overexpressed PLK4 to induce centriole amplification. Importantly, we show that S305-phosphorylated PLK4 is specifically sequestered at the centrosome contrary to the nonphosphorylated form. These data suggest that PLK4 activity is restricted to the centrosome to prevent aberrant centriole assembly and sustained kinase activity is required for centriole duplication.     

Protein Kinase A-dependent Phosphorylation of Rap1 Regulates Its Membrane Localization and Cell Migration

The small G protein Rap1 can mediate “inside-out signaling” by recruiting effectors to the plasma membrane that signal to pathways involved in cell adhesion and cell migration. This action relies on the membrane association of Rap1, which is dictated by post-translational prenylation as well as by a stretch of basic residues within its carboxyl terminus. One feature of this stretch of acidic residues is that it lies adjacent to a functional phosphorylation site for the cAMP-dependent protein kinase PKA. This phosphorylation has two effects on Rap1 action. One, it decreases the level of Rap1 activity as measured by GTP loading and the coupling of Rap1 to RapL, a Rap1 effector that couples Rap1 GTP loading to integrin activation. Two, it destabilizes the membrane localization of Rap1, promoting its translocation into the cytoplasm. These two actions, decreased GTP loading and decreased membrane localization, are related, as the translocation of Rap1-GTP into the cytoplasm is associated with its increased GTP hydrolysis and inactivation. The consequences of this phosphorylation in Rap1-dependent cell adhesion and cell migration were also examined. Active Rap1 mutants that lack this phosphorylation site had a minimal effect on cell adhesion but strongly reduced cell migration, when compared with an active Rap1 mutant that retained the phosphorylation site. This suggests that optimal cell migration is associated with cycles of Rap1 activation, membrane egress, and inactivation, and requires the regulated phosphorylation of Rap1 by PKA.