Inhibitory effect of calcium-binding protein regucalcin on protein kinase activity in the nuclei of regenerating rat liver

The effect of Ca²⁺-binding protein regucalcin on protein kinase activity in the nuclei of normal and regenerating rat livers was investigated. Protein kinase activity in the nuclei isolated from normal rat liver was significantly increased by addition of Ca²⁺ (500 μM) and calmodulin (10 μg/ml) in the enzyme reaction mixture. Nuclear protein kinase activity was significantly decreased in the presence of EGTA (1.0 mM), trifluoperazine (TFP; 20 μM), dibucaine (10⁻⁴ M), or staurosporine (10⁻⁷ M), indicating that Ca²⁺-dependent protein kinases are present in the nuclei. Protein kinase activity was significantly elevated in the liver nuclei obtained at 6 to 48 h after a partial hepatectomy. Hepatectomy-increased nuclear protein kinase activity was significantly decreased in the presence of EGTA (1.0 mM), TFP (20 μM), or staurosporine (10⁻⁷ M) in the enzyme reaction mixture. The presence of regucalcin (0.1–0.5 μM) caused a significant decrease in protein kinase activity in the nuclei obtained from normal and regenerating rat livers. Meanwhile, the nuclear protein kinase activity from normal and regenerating livers was significantly elevated in the presence of anti-regucalcin monoclonal antibody (50–200 ng/ml). The present study suggests that regucalcin plays a role in the regulation of protein kinase activity in the nuclei of proliferative liver cells. J. Cell. Biochem. 71:569–576, 1998. © 1998 Wiley-Liss, Inc.     

Determination of specific protein kinase activities using phosphorus-33

The immune complex kinase assay is the most widely applied method to assess the catalytic activity of protein tyrosine kinases. It offers the advantage that the activity of a single selected enzyme can be determined, and that the enzyme activity can be normalized for the amount of enzyme in a parallel immunoblotting experiment. Here, we describe the use of the recently introduced isotope phosphorus-33 for the protein kinase assay. The lower energy of ³³P, compared with the traditionally applied ³²P, allows the simultaneous examination of the amount of enzyme with ¹²⁵I-labeled antibodies. By analysing one and the same sample for both kinase activity and protein amount, the variation between parallel processed samples is avoided. Using this method, specific kinase activities can be calculated with high precision. The assay is particularly useful for the detection of cytokine and growth factor-induced activation of kinases, as changes in enzyme amounts by subcellular relocalization can be distinguished.     

Effect of triamcinolone treatment, starvation and diabetes on rat liver protein kinase activity

Triamcinolone administration for 3 days reduced protein kinase activity in normal and adrenalectomized rats by about one third. This was accompanied by a similar decrease in cyclic[³H] AMP-binding capacity. Alloxan diabetes reduced protein kinase activity as well as cyclic[³H] AMP-binding capacity by about 40%. Administration of insulin to diabetic rats produced an insignificant increase in protein kinase activity and did not improve cyclic[³H] AMP-binding capacity. In fasted rats, there was a gradual decrease in protein kinase activity to about one half of initial activity on the fourth day, whereas the decrease in cyclic[³H] AMP-binding capacity was less marked. The effect of starvation was observed in normal as well as adrenalectomized rats, indicating that the decrease due to fasting is not mediated by glucocorticoids. The findings suggest that the maintenance of liver protein kinase activity is under hormonal and nutritional control. This represents a long-term regulatory step at the stage of protein phosphorylation in addition to the rapid effect of hormones on cyclic AMP levels.     

Regulation of the interaction of actin filaments with microtubule-associated protein 2 by calmodulin-dependent protein kinase II

Phosphorylation of microtubule-associated protein 2 (MAP 2) by Ca²⁺-, calmodulin-dependent protein kinase II (protein kinase II) inhibited the actin filament cross-linking activity of MAP 2. This inhibition required the presence of ATP, Mg²⁺, Ca²⁺ and calmodulin. The minimal concentration of MAP 2 required for gel formation of actin filaments was increased with increasing amounts of phosphate incorporated into MAP 2, and the phosphorylated MAP 2, into which 10.3 mol of phosphate/mol of protein had been incorporated, did not cause actin filaments to gel under the experimental conditions used. The phosphorylation of MAP 2 by Ca²⁺-, phospholipid-dependent protein kinase (protein kinase C) and cAMP-dependent protein kinase also inhibited the actin filament cross-linking activity of MAP 2. The extent and rate of phosphorylation of MAP 2 by protein kinase II were higher than those of the phosphorylation by protein kinase C and cAMP-dependent protein kinase. The interaction of actin filaments with MAP 2 was inhibited more by the actions of protein kinase II and protein kinase C than by cAMP-dependent protein kinase. The actin filament cross-linking activity of MAP 2 phosphorylated either by protein kinase II, cAMP-dependent protein kinase or protein kinase C was retrieved when phosphorylated MAP 2 was treated by protein phosphatase. These results indicate that the interaction of actin filaments with MAP 2 is regulated by the phosphorylation-dephosphorylation of MAP 2.     

A calcium-dependent but calmodulin-independent protein kinase from soybean

A calcium-dependent protein kinase activity from suspension-cultured soybean cells (Glycine max L. Wayne) was shown to be dependent on calcium but not calmodulin. The concentrations of free calcium required for half-maximal histone H1 phosphorylation and autophosphorylation were similar (≈2 micromolar). The protein kinase activity was stimulated 100-fold by ≥10 micromolar-free calcium. When exogenous soybean or bovine brain calmodulin was added in high concentration (1 micromolar) to the purified kinase, calcium-dependent and -independent activities were weakly stimulated (≤2-fold). Bovine serum albumin had a similar effect on both activities. The kinase was separated from a small amount of contaminating calmodulin by sodium dodecyl sulfate polyacrylamide gel electrophoresis. After renaturation the protein kinase autophosphorylated and phosphorylated histone H1 in a calcium-dependent manner. Following electroblotting onto nitrocellulose, the kinase bound ⁴⁵Ca²⁺ in the presence of KCl and MgCl₂, which indicates that the kinase itself is a high-affinity calcium-binding protein. Also, the mobility of one of two kinase bands in SDS gels was dependent on the presence of calcium. Autophosphorylation of the calmodulin-free kinase was inhibited by the calmodulin-binding compound N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W-7), showing that the inhibition of activity by W-7 is independent of calmodulin. These results show that soybean calcium-dependent protein kinase represents a new class of protein kinase which requires calcium but not calmodulin for activity.     

Side-effects of protein kinase inhibitors on ion channels

Protein kinases are one of the largest gene families and have regulatory roles in all aspects of eukaryotic cell function. Modulation of protein kinase activity is a desirable therapeutic approach for a number of human diseases associated with aberrant kinase activity, including cancers, arthritis and cardiovascular disorders. Several strategies have been used to develop specific and selective protein kinase modulators, primarily via inhibition of phosphorylation and down-regulation of kinase gene expression. These strategies are effective at regulating intracellular signalling pathways, but are unfortunately associated with several undesirable effects, particularly those that modulate ion channel function. In fact, the side-effects have precluded these inhibitors from being both useful experimental tools and therapeutically viable. This review focuses on the ion channel side-effects of several protein kinase inhibitors and specifically on those modulating K⁺, Na⁺ and Ca²⁺ ion channels. It is hoped that the information provided with a detailed summary in this review will assist the future development of novel specific and selective compounds targeting protein kinases both for experimental tools and for therapeutic approaches.     

cGMP-stimulated protein kinase phosphorylates pyruvate kinase in an anoxia-tolerant marine mollusc

Summary A cystosolic protein kinase that phosphorylates pyruvate kinase (PK) in vitro has been identified in crude homogenates of heart, radular retractor, and foot muscle from the anoxia-tolerant marine whelk Busycon canaliculatum. Protein kinase action was measured by following changes in PK kinetic parameters: phosphorylated PK has a higher K _m value for phosphoenolpyruvate and a lower I₅₀ value for l-alanine. The crude protein kinase readily phosphorylated PK in a Mg²⁺-and ATP-dependent manner in the absence of any added effector. This activity was not affected by the addition of either cAMP (a stimulator of protein kinase A) or Ca²⁺ plus phorbol 12-myristate 13-acetate (stimulators of protein kinase C) to the incubation medium. Addition of cGMP to the homogenate, however, increased the rate of PK phosphorylation giving a 3–4-fold increase in the rate of change in PK kinetic parameters that was readily apparent after 5h. Complete time-courses of changes in PK kinetic parameters in the presence and absence of cGMP showed that cGMP increased the rate, but not the final extent, of PK phosphorylation. These results indicate that PK inactivation by enzyme phosphorylation in response to anoxia in whelk tissues may be mediated by a cyclic GMP stimulated protein kinase in response to changing levels of cGMP. This conclusion was further supported by data indicating that the total activity of protein kinase was the same in both anoxic and aerobic animals, and that the total PK phosphatase activity was also constant. Changes in PK phosphorylation during anoxia are not, therefore, the result of changes in the total amount of protein kinase or phosphatase.Abbreviations cAMP adenosine 3:5-monophosphate - cGMP guanosine 3:5-monophosphate - PK pyruvate kinase - PMA phorbol 12-myristate, 13-acetate - PEP phosphoenolpyruvate - K _m Michaelis constant - I ₅₀ inhibitor concentration that reduces enzyme activity by 50%     

Activation of protein kinase B (Akt/RAC-protein kinase) by cellular stress and its association with heat shock protein Hsp27

Protein kinase B (PKB, also named as Akt or RAC-protein kinase), that is activated by cellular stress such as heat shock and hyperosmotic treatment, was revealed to be activated by oxidative stress and by chemical stressors of CdCl₂ and NaAsO₂ by measuring the activity of the enzyme immunoprecipitated from the transfected COS-7 cells. Upon stress treatment, a 30-kDa phosphoprotein was co-immunoprecipitated with PKB from the cells metabolic labeled with [³²P]orthophosphate. The phosphoprotein was identified as Hsp27, a small heat shock protein, by immunoblot analysis and co-immunoprecipitation. The association of Hsp27 was specific to PKB as the heat shock protein was not co-immunoprecipitated with other protein kinases such as protein kinase C and PKN. When the cells were treated with H₂O₂, PKB was activated gradually and the association of Hsp27 with PKB increased concurrently with the enhancement of PKB activity. In heat-shocked cells, activation of PKB and the association of Hsp27 were detected immediately after the treatment, and the association of the heat shock protein decreased while PKB kept stimulated activity when the cells were further incubated at 37°C. These results suggest that Hsp27 is involved in the activation process of PKB in the signal transduction pathway of various forms of stress.     

A calcium-dependent protein kinase is present in tetrahymena

A Ca²⁺-dependent protein kinase of Tetrahymena thermophila has been partially purified and characterized. The molecular mass of the enzyme is less than that of similar enzymes (for example protein kinase C), being about 55 kDa. After purification and in the presence of Ca²⁺ the enzyme activity increased. The promoter of protein kinase C (PKC) activity, phorbol myristate acetate (PMA), increased the activity while the protein kinase inhibitor H-7 decreased the activity of the enzyme. The experiments demonstrate the presence, activity and similarity to vertebrate enzymes of a protein kinase at a low level of phylogeny.     

Properties and cellular distribution of cyclic AMP-dependent protein kinase from thymus

A protein kinase that catalyzes the phosphorylation of histone was partially purified from rat thymus, and the rate of histone phosphorylation was stimulated three- to fourfold by 1 × 10^?6 M adenosine 3′,5′-monophosphate (cyclic AMP). Thymic protein kinase was more active than the enzyme from spleen. Histone fractions f1, f2a, f2b, and f3 were all capable of serving as phosphate acceptors for the thymic protein kinase, and the rate of phosphorylation of each fraction was stimulated by cyclic AMP. The ability of various 3′,5′-mononucleotides to stimulate protein kinase activity was compared. Inosine 3′,5′-monophosphate (cyclic IMP) was the most effective substitute for cyclic AMP. The cellular distribution of cyclic AMP-dependent protein kinase and adenylate cyclase activities in the thymus was determined. Cyclic AMP-dependent protein kinase activity is present in both small thymocytes and residual thymic tissue. The specific activity of protein kinase from residual tissue, both for basal and cyclic AMP-stimulated enzyme, was greater than that of enzyme from small thymocytes. In contrast to this, adenylate cyclase activity is predominately localized in the thymocytes.     

Independent modulation of hepatic protein kinase activities

The effects of hormonal status on protein kinase activity was examined in homogenates of rat liver. Protein kinase activity was evaluated from incorporation of ³²P from [γ-³²P]ATP into protamine or histone as receptor substrates. Protamine phosphorylation in the presence or absence of cyclic AMP exceeded histone phosphorylation by at least a factor or two. Hypophysectomy markedly increased protamine phosphorylation in the presence or absence of saturating amounts of cyclic AMP. In contrast, hypophysectomy only slightly increased cyclic AMP independent phosphorylation of histone. These results could not be accounted for by differences in ATPase or protein phosphase activities. Cortisone (2 mg/day × 3) decreased total protein kinase activity in livers of hypophysectomized rats when protamine was substrate, but had no effect on the total activity toward histone. Growth hormone (100 μg/day × 3) significantly increased histone, but not protamine phosphorylation in livers of hypophysectomized rats. Administration of 5 μg of triiodothyronine/day to hypophysectomized rats also markedly increased the phosphorylation of histone, but not protamine when saturating amounts of cyclic AMP were present. These results support the hypothesis that liver may contain more than one type of protein kinase activity and that the different protein kinase activities can be separately affected by hormones. Such control distal to cyclic AMP might allow selective modulation of cyclic AMP-dependent processes in cells which carry out more than one such process.     

Regulation of Ca2+/calmodulin-dependent protein kinase kinase β by cAMP signaling

BackgroundCa²⁺/calmodulin-dependent protein kinase kinase (CaMKK) is a pivotal activator of CaMKI, CaMKIV and 5’-AMP-activated protein kinase (AMPK), controlling Ca²⁺-dependent intracellular signaling including various neuronal, metabolic and pathophysiological responses. Recently, we demonstrated that CaMKKβ is feedback phosphorylated at Thr144 by the downstream AMPK, resulting in the conversion of CaMKKβ into Ca²⁺/CaM-dependent enzyme. However, the regulatory phosphorylation of CaMKKβ at Thr144 in intact cells and in vivo remains unclear.MethodsAnti-phosphoThr144 antibody was used to characterize the site-specific phosphorylation of CaMKKβ in immunoprecipitated samples from mouse cerebellum and in transfected mammalian cells that were treated with various agonists and protein kinase inhibitors. CaMKK activity assay and LC-MS/MS analysis were used for biochemical characterization of phosphorylated CaMKKβ.ResultsOur data suggest that the phosphorylation of Thr144 in CaMKKβ is rapidly induced by cAMP/cAMP-dependent protein kinase (PKA) signaling in CaMKKβ-transfected HeLa cells, that is physiologically relevant in mouse cerebellum. We confirmed that the catalytic subunit of PKA was capable of directly phosphorylating CaMKKβ at Thr144 in vitro and in transfected cells. In addition, the basal phosphorylation of CaMKKβ at Thr144 in transfected HeLa cells was suppressed by AMPK inhibitor (compound C). PKA-catalyzed phosphorylation reduced the autonomous activity of CaMKKβ in vitro without significant effect on the Ca²⁺/CaM-dependent activity, resulting in the conversion of CaMKKβ into Ca²⁺/CaM-dependent enzyme.ConclusioncAMP/PKA signaling may confer Ca²⁺-dependency to the CaMKKβ-mediated signaling pathway through direct phosphorylation of Thr144 in intact cells.General significanceOur results suggest a novel cross-talk between cAMP/PKA and Ca²⁺/CaM/CaMKKβ signaling through regulatory phosphorylation.     

Changes in protein kinase and protein phosphatase properties during the cycle of asparaginase activity in Leptosphaeria michotti

Regulation of the cyclic activity of asparaginase (obtained as a purified protein complex) by a reversible auto-phosphorylation process has been previously reported in the fungus Leptosphaeria michotii (West) Sacc. In the present study, the protein complex was purified in the presence of either a mixture of 3 protein phosphatase inhibitors (fluoride, vanadate and molybdate) or EGTA, during the cycle of asparaginase activity, and the protein kinase and protein phosphatase activities characterized. (I) At the phase of increasing asparaginase activity, a Ca²⁺/calmodulin-dependent kinase activity was identified by (a) its inhibition by calmidazolium, reversed by calmodulin, and its inhibition by EGTA, but not by poly(Glu/Tyr 4:1)n. dichloro-(ribofuranosyl)-benzimidazole or polylysine (b) an increasing level of calmodulin bound to the complex, as estimated by enzyme-linked immunosorbent assay (ELISA). (2) At the phase of decreasing asparaginase activity, the Ca²⁺-calmodulin-dependent kinase activity disappeared and a little calmodulin remained associated with the complex: phosphorylation of the complex was increased several-fold by 1 nM okadaic acid and 25 nM inhibitor-2, and was not affected by EGTA, indicating a protein phosphatase-2A-like activity. (3) When asparaginase activity was low, a little calmodulin was bound to the complex. The kinase could phosphorylate casein and phosvitin. was inhibited by poly(Glu/Tyr 4:1)n. dichloro-(ribofuranosyl)-benzimidazole and heparin, stimulated by polylysine and not affected by calmidazolium or EGTA, just as a casein kinase 2. A Ca²⁺-dependent but calmodulin-independent protein phosphatase activity, not affected by okadaic acid and inhibitor-2. was then identified. We postulate the presence in the complex, of (a) only one protein kinase and one protein phosphatase, whose properties could change during the cycle of asparaginase activity: (b) two Ca²⁺/-binding proteins: first calmodulin, which could bind to Ca²⁺ and the casein kinase-2 form to give a Ca²⁺/calmodulin-dependent kinase, which could become Ca²⁺/calmodulin-independent following an auto-phosphorylation process: second a protein homologous to calmodulin, able to bind to the protein phosphatase-2A catalytic subunit to give a protein phosphatase-2B catalytic subunit.     

Ca2+-dependent protein kinase from alfalfa (Medicago varia): Partial purification and autophosphorylation

A calcium-dependent protein kinase (CDPK) was purified to 1400-fold from the soluble fraction of alfalfa (Medicago varia) cells by ammonium sulfate fractionation, Sephacryl-300, DEAE-Sephacel, Phenyl-Sepharose and Hydroxylapatite column chromatography. The enzyme is mainly monomeric. During the course of the purification steps a 50 kDa phosphoprotein doublet and a 56 kDa phosphoprotein copurified with the CDPK activity. Mobility shift of these proteins have been shown by SDS PAGE in Ca²⁺ free conditions. Tests on enzyme activity after separation by native gel electrophoresis revealed two protein kinase activities in our enzyme preparation and the phosphorylation of the 50 kDa and 56 kDa proteins. We suggest that these proteins are the autophosphorylated forms of calcium dependent protein kinases. Preincubation of the CDPK in ATP resulted in a marked increase in enzyme activity, but did not alter the Ca²⁺ sensitivity of the protein kinase.     

Mitogen-activated protein kinase (MAP kinase) activation in Xenopus oocytes: Roles of MPF and protein synthesis

Mitogen-activated protein kinase (MAP kinase) is a serine/threonine kinase whose enzymatic activity is thought to play a crucial role in mitogenic signal transduction and also in the progesterone-induced meiotic maturation of Xenopus oocytes. We have purified MAP kinase from Xenopus oocytes and have shown that the protein is present in metaphase ll oocytes under two different forms: an inactive 41-kD protein able to autoactivate and to autophosphorylate in vitro, and an active 42-kD kinase resolved into two tyrosine phosphorylated isoforms on 2D gels. During meiotic maturation, MAP kinase becomes tyrosine phosphorylated and activated following the activation of the M-phase promoting factor (MPF), a complex between the p34^cdc2 kinase and cyclin B. In vivo, MAP kinase activity displays a different stability in metaphase l and in metaphase II: protein synthesis is required to maintain MAP kinase activity in metaphase I but not in metaphase II oocytes. Injection of either MPF or cyclin B into prophase oocytes promotes tyrosine phosphorylation of MAP kinase, indicating that its activation is a downstream event of MPF activation. In contrast, injection of okadaic acid, which induces in vivo MPF activation, promotes only a very weak tyrosine phosphorylation of MAP kinase, suggesting that effectors other than MPF are required for the MAP kinase activation. Moreover, in the absence of protein synthesis, cyclin B and MPF are unable to promote in vivo activation of MAP kinase, indicating that this activation requires the synthesis of new protein(s). © 1993 Wiley-Liss, Inc.     

In situ regulation of cell-cell communication by the cAMP-dependent protein kinase and protein kinase C

The effects of cAMP-dependent protein kinase A and protein kinase C on cell-cell communication have been examined in primary ovarian granulosa cells microinjected with purified components of these two regulatory cascades. These cells possess connexin43 ( 1)-type gap junctions, and are well-coupled electrotonically and as judged by the cell-to-cell transfer of fluorescent dye. Within 2–3 min after injection of the protein kinase A inhibitor (PKI) communication was sharply reduced or ceased, but resumed in about 3 min with the injection of the protein kinase A catalytic subunit. A similar resumption also occurred in PKI-injected cells after exposure to follicle stimulating hormone. Microinjection of the protein kinase C inhibitor protein caused a transient cessation of communication that spontaneously returned within 15–20 min. Treatment of cells with activators of protein kinase C, TPA or OAG for 60 min caused a significant reduction in communication that could be restored within 2–5 min by the subsequent injection of either the protein kinase C inhibitor or the protein kinase A catalytic subunit. With a longer exposure to either protein kinase C activator communication could not be restored and this appeared to be related to the absence of aggregates of connexin43 in membrane as detected immunologically. In cells injected with alkaline phosphatase communication stopped but returned either spontaneously within 20 min or within 2–3 min of injecting the cell with either the protein kinase A catalytic subunit or with protein kinase C. When untreated cells were injected with protein kinase C communication diminished or ceased within 5 min. Collectively these results demonstrate that cell-cell communication is regulated by both protein kinase A and C, but in a complex interrelated manner, quite likely by multiple phosphorylation of proteins within or regulating connexin-43 containing gap junctions.Abbreviations C catalytic subunit of protein kinase A - CKI protein kinase C inhibitor protein - Cx connexin protein - dbcAMP N⁶,2-O-dibutyryladenosine 3:5-cyclic monophosphate - OAG 1-oleoyl-2-acetyl-sn-glycerol - protein kinase A cAMP-dependent protein kinase - protein kinase C Ca²⁺-sensitive phospholipid-dependent protein kinase - PKI protein kinase A inhibitor protein - R regulatory subunit of protein kinase A - TRA 12-O-tetradecanoylphorbol-13-acetate - 8Br-cAMP 8-bromoadenosine 3:5 cyclic monophosphate     

Reversible inhibition of cyclic AMP-dependent protein kinase by insulin

Summary Extracts of fasted rat diaphragms, previously treated with or without insulin were assayed for glycogen synthase, protein kinase and cyclic [³H]-AMP binding. Treatment with insulin produced an elevation in the % of glycogen synthase I and a concurrent decrease in cyclic AMP-dependent protein kinase activity and cyclic [³H]-AMP binding. Analysis of extracts by disc gel electrophoresis demonstrated the inhibition of cyclic [3H]-AMP binding to involve the Type I protein kinase holoenzyme. Inhibition of protein kinase activity was most apparent in the presence of 0.2 µM cyclic AMP, with enzymatic activity of the insulin-treated extracts typically 60–65% of control. Higher assay concentrations diminished the difference between control and insulin-treated extracts and concentrations greater than 20 µm abolished it.The inhibition of cyclic AMP-dependent protein kinase activity after insulin was a transient and labile phenomenon. The effect was independent of ATP concentration in the assay, but was sensitive to the pH of tissue extraction, requiring a pH of 7.0 to 8.4 to be observed.Insulin-mediated inhibition of protein kinase activity was reversed upon preincubation of extracts at 0–2°. Relatively concentrated homogenates (<4 µl buffer/mg tissue) yielded extracts which exhibited little or no inhibition of protein kinase activity compared to extracts prepared from more dilute (6–10 µl/mg) homogenates. A model for the inhibition of the cyclic-AMP dependent protein kinase by an insulin-generated inhibitor which becomes directly associated with the Type 1 holoenzyme is proposed.Abbreviations cyclic AMP (cAMP) Adenosine 3,5-monophosphate - Tricine N-Tris (Hydroxy-methyl) methyl glycine - G-6-P glucose-6-phosphate - MES 2-[N-morpholino]ethane sulfonic acid A preliminary report was communicated to the 61st meeting of the F.A.S.E.B., April, 1977.     

A gibberellin-regulated protein phosphorylated by a putative Ca2+-dependent protein kinase is G-protein mediated in rice root

The GA-signal transduction pathways downstream to the Gα protein in rice seedling root were investigated using in-gel kinase assay and in vitro protein phosphorylation techniques with a Gα protein defective mutant, d1. A 50-kDa protein kinase was detected downstream to Gα protein in the membrane fraction of rice seedling roots using an in-gel kinase assay with histone III-S as a substrate. The activity of a 50-kDa protein kinase increased in the wild-type rice by gibberellin (GA₃) treatment, but did not change in the d1 mutant. This protein kinase activity was inhibited by the Ca²⁺ chelator ethyleneglycol-bis-(beta-aminoethylether)-N,N,N ¹,N ¹-tetraacetic acid (EGTA), protein kinase inhibitors, staurosporine and H7, and calmodulin antagonist, trifluoperazine, suggesting that the 50-kDa protein kinase is a putative plant Ca²⁺-dependent protein kinase (CDPK). The activity of the 50-kDa putative CDPK reached its highest level at 3 h after GA₃ treatment and then gradually declined with time. In order to identify the endogenous substrate for 50-kDa putative CDPK, two-dimensional polyacrylamide gel electrophoresis followed by in vitro protein phosphorylation was carried out. The phosphorylation activity of an endogenous protein PP30, identified as an unknown protein having molecular weight 30 kDa and isoelectric point 5.8 was increased in the wild-type rice by GA₃ treatment, compared with the d1 mutant. The addition of GA₃ treated membrane fraction, which predominantly represent a 50-kDa putative CDPK further increased the phosphorylation of PP30. Almost similar to GA₃ treatment, phosphorylation activity of PP30 was also increased by the treatment with cholera toxin in the wild-type rice but not in d1 mutant. These results suggest that the 50-kDa putative CDPK and an unknown protein, PP30 promoted by GA₃ treatment are G-protein mediated in rice seedling roots.     

Purification and properties of a nuclear protein kinase from rat mammary gland

A nuclear protein kinase that shows a high degree of substrate specificity for the phosphorylation of the acidic proteins casein, phosvitin and non-histone chromatin proteins, rather than the basic proteins histones and protamine, was partially purified from lactatingrat mammary gland. The enzyme is associated with the acidic protein fraction of chromatin. Nuclear kinase requires Co²⁺ for activity, and other bivalent cations such as Mg²⁺ and Mn²⁺ can substitute partially for Co²⁺. The kinase is further activates (2–3-fold) by various salts, their concentration for maximum stimulation being: NaCl, 150mm; KCl, 200mm; sodium acetate, 300mm. The sedimentation coefficient of the nuclear kinase is 8.9S and its mol.wt. is approx. 300000 by gel-exclusion chromatography. The enzyme is not activated by cyclic AMP or cyclic GMP and is inhibited neither by the regulatory subunit of mammary cyclic AMP-dependent protein kinase nor by the heat-stable protein kinase inhibitor from ox heart. Analysis of ³²P-labelled protein products reveals that the kinase transfers the terminal phosphate of ATP to serine and threonine residues of proteins. The enzyme, however, has specificity for the phosphorylation of threonine in casein and serine in phosvitin. Molecular size and enzymic characteristics of the nuclear protein kinase are clearly different from those of the cytosol enzyme previously characterized.