Minor participation of cAMP on the protein kinase phosphorylation of mitochondrial and cytosolic fractions from Ascaris suum: a comparative study with porcine heart muscle

In contrast to porcine heart muscle in which cAMP effectively activated the phosphorylation of cytosolic proteins, cAMP exerted a minor effect on the phosphorylation of proteins from the soluble fraction of Ascaris suum muscle. Similarly, cAMP did not enhance the kinase activity in the mitochondrial membranes from porcine heart and A. suum, although major differences in protein phosphorylation were observed between both fractions. However, cAMP-dependent protein kinases (PKA) were evidenced in the parasitic soluble mitochondrial fraction, since the phosphorylation of histone IIA and kemptide was augmented in this fraction, in the presence of cAMP. An increase in the phosphorylation of exogenously added A. suum phosphofructokinase was also obtained when cAMP was added to the parasite soluble mitochondrial fraction. The phosphorylation of phosphofructokinase by this fraction was inhibited when kemptide and cAMP were included in the reaction mixture, suggesting substrate competition for the same PKA. Although PKI (6-22), a reported inhibitor of the catalytic subunit of mammalian cAMP-dependent PKAs, did not affect the endogenous phosphorylation of proteins in the various A. suum fractions, an inhibition on the phosphorylation of exogenously added kemptide and phosphofructokinase was observed when PKI (6-22) was incubated with the parasite mitochondrial soluble fraction.     

Divalent cation hinder the solubilization of a tubulin kinase activity from Trypanosoma cruzi epimastigotes

Trypanosoma cruzi epimastigotes were extracted under various conditions in order to examine the role of divalent cations in the solubilization of microtubule proteins. When epimastigotes were homogenized in the presence of 5 mM Mg+2 and 5 mM Ca+2, a protein kinase responsible for phosphorylating tubulin, as well as the tubulin that became phosphorylated, remained tightly associated with the parasite particulate and detergent-resistant fractions. On the contrary, tubulin kinase and its substrate were predominantly released into the parasite cytosolic and detergent-soluble fractions, when epimastigotes were extracted in the presence of 5 mM EDTA and 5 mM EGTA. These evidences demonstrated a divalent cation-dependent solubilization of the enzyme responsible for the phosphorylation of tubulin in T. cruzi epimastigotes and suggested a tight association between tubulin and this kinase. Under all conditions tested, tubulin kinase activity in epimastigote extracts was lower than the addition of the corresponding value in the parasite cytosolic and membranous fractions, suggesting the presence of a kinase inhibitor or regulatory subunit which also seemed to be modulated by divalent cations. Additionally, inhibition experiments in the presence of heparin, 2,3-bisphosphoglycerate and GTP established that the parasite tubulin kinase corresponded to a protein kinase CK2.     

Protein phosphorylation in human peripheral blood lymphocytes. Subcellular distribution and partial characterization of adenosine 3'':5''-cyclic monophosphate-dependent protein kinase.

Cytoplasmic and membrane fractions prepared from human peripheral-blood lymphocytes both contained cyclic AMP-dependent protein kinase activity and endogenous protein kinase substrates. Protein kinase activity in the particulate fractions was not eluted with 0.25 M-NaCl, suggesting that it was not derived from non-specifically absorbed soluble cytoplasmic protein kinase. Nor was the particulate protein kinase activity eluted by treatment with cyclic AMP, suggesting that the catalytic subunit is membrane-bound and arguing against cyclic AMP-induced translocation of particulate activity. Cyclic AMP-dependent protein-phosphorylating activity in the cytoplasmic fraction was highly sensitive to inhibition by Mn2+, and was co-eluted from DEAE-cellulose primarily with type-I rabbit skeletal-muscle kinase. Cyclic AMP-dependent phosphorylating activity in the plasma-membrane fractions was stimulated at low [Mn2+] and inhibited only at high [Mn2+]. When solubilized with Nonidet P-40, plasma-membrane protein kinase was co-eluted from DEAE-cellulose with type-II rabbit muscle kinase. These differences, together with the strong association of the particulate kinases with the particulate fraction, suggest the possibility of compartmentalized protein phosphorylation in intact lymphocytes.     

Protein phosphatases in developing Dictyostelium discoideum cells

Protein phosphatase activities in developing Dictyostelium discoideum cells were investigated. Substrates were prepared by phosphorylation of histone H2b and kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly) using cAMP-dependent protein kinase. Two histone phosphatase activities (M_r 170 000 and 520 000) and one kemptide phosphatase activity (M_r 230 000) were found in the cytosolic cell fraction. Histone phosphatase was also present in the particulate fraction, kemptide phosphatase was not. All phosphatase activities were present throughout development. No differences in protein phosphatase activities were found in prespore and prestalk cells. A heat-stable factor which inhibits the particulate and both soluble histone phosphatase activities was isolated.     

Comparison of substrate recognition by protein kinase C (type III) between rat liver cytosolic and particulate fractions

1. Phosphorylation of rat liver endogenous substrates by protein kinase C (type III) was compared between cytosolic and particulate (mitochondria, microsomes and plasma membrane) fractions. 2. The rate and the maximum level of protein phosphorylation were several-fold higher in particulate fractions than in cytosolic fraction. 3. Protein phosphorylation in cytosolic fraction was dependent on both Ca2+ and phospholipid, but only Ca2+ was necessary in phosphorylation of particulate fractions. 4. These results suggest that protein kinase C (type III) has much more target proteins in particulate fractions rather than in cytosolic fraction and Ca2+ was important regulator in particulate protein phosphorylation.     

Cyclic AMP- and Ca2(+)-dependent protein kinases in Plasmodium falciparum

The cyclic AMP- and Ca2(+)-dependent protein kinase activities of Plasmodium falciparum were partially characterized after purification of parasites from host erythrocytes by N2 cavitation and Percoll gradient centrifugation. Proteins of molecular weights 80, 54, 51, and 31.5 kDa were phosphorylated in a cAMP-dependent manner in cytosolic extracts of isolated P. falciparum. Cytosolic extracts also contained cAMP-dependent histone II-A kinase activity with an average Vmax of 131.1 pmol/32P/min/mg protein and a Km for cAMP of 85nM. Upon photoaffinity labeling with [32P]-8-N3-cAMP, a 53-kDa protein was specifically labeled in parasite cytosol. A metabolically labeled protein of the same molecular weight was identified by cAMP-agarose affinity chromatography. The 53-kDa protein cochromatographed with cAMP-dependent histone II-A kinase activity on DEAE-cellulose, suggesting that it is the regulatory subunit of the kinase. Ca2(+)-dependent phosphorylation of proteins of molecular weights 195, 158, 51, 47.5, and 15 kDa was demonstrated in a membrane fraction from parasites free of the erythrocyte membrane. This activity was not stimulated by either calmodulin or phospholipid plus diacylglycerol and was absent from the membranes of uninfected erythrocytes. Of several exogenous substrates tested, none were found to be a substrate for this Ca2(+)-dependent kinase. Both cAMP- and Ca2(+)-dependent kinases phosphorylated serine and threonine residues.     

Arabidopsis casein kinase 1-like 6 contains a microtubule-binding domain and affects the organization of cortical microtubules,

Members of the casein kinase 1 (CK1) family are evolutionarily conserved eukaryotic protein kinases that are involved in various cellular, physiological, and developmental processes in yeast and metazoans, but the biological roles of CK1 members in plants are not well understood. Here, we report that an Arabidopsis (Arabidopsis thaliana) CK1 member named casein kinase 1-like 6 (CKL6) associates with cortical microtubules in vivo and phosphorylates tubulins in vitro. The unique C-terminal domain of CKL6 was shown to contain the signal that allows localization of CKL6 to the cortical microtubules. This domain on its own was sufficient to associate with microtubules in vivo and to bind tubulins in vitro. CKL6 was able to phosphorylate soluble tubulins as well as microtubule polymers, and its endogenous activity was found to associate with a tubulin-enriched subcellular fraction. Two major in vitro phosphorylation sites were mapped to serine-413 and serine-420 of tubulin beta. Ectopic expression of wild-type CKL6 or a kinase-inactive mutant form induced alterations in cortical microtubule organization and anisotropic cell expansion. Collectively, these results demonstrate that CKL6 is a protein kinase containing a novel tubulin-binding domain and plays a role in anisotropic cell growth and shape formation in Arabidopsis through the regulation of microtubule organization, possibly through the phosphorylation of tubulins.     

Altered properties of cAMP-dependent protein kinase in H-ras-transformed NIH3T3 cells

cAMP-dependent protein kinase activity in the soluble fraction was decreased in both v-H-ras-transformed and activated-c-H-ras-transformed NIH3T3 cells as compared with that in NIH3T3 cells. Both of the elution profile of type II cAMP-dependent protein kinase from DEAE-cellulose and the electrophoretic behavior of its regulatory subunits in the particulate fraction of H-ras-transformed cells are different from those of control NIH3T3 cells. These results suggest that ras protein causes the alterations of some properties of cAMP-dependent protein kinases.     

Parietal cell protein kinases. Selective activation of type I cAMP-dependent protein kinase by histamine

cAMP-dependent protein kinases have been characterized in parietal cells isolated from rabbit gastric mucosa. Both Type I and Type II cAMP-dependent protein kinase isozymes are present in these cells. Type II isozymes were detected in 900, 14,000, and 100,000 X g particulate fractions as well as 100,000 X g cytosolic fractions; Type I isozymes were found predominately in the cytosolic fraction. When parietal cells were stimulated with histamine, an agent that elevates intracellular cAMP content and initiates parietal cell HCl secretion, cAMP-dependent protein kinase activity was increased in homogenates of these cells as measured by an increase in the cAMP-dependent protein kinase activity ratio. Histamine activation of cAMP-dependent protein kinase was correlated with parietal cell acid secretory responses which were measured indirectly as increased cellular uptake of the weak base, [14C]aminopyrine. These results suggest that cAMP-dependent protein kinase(s) is involved in the control of parietal cell HCl secretion. The parietal cell response to histamine may be compartmentalized because histamine appears to activate only a cytosolic Type I cAMP-dependent protein kinase isozyme, as determined by three different techniques including 1) ion exchange chromatography; 2) Sephadex G-25 to remove cAMP and allow rapid reassociation of the Type II but not the Type I isozyme; and 3) 8-azido-[32P]cAMP photoaffinity labeling. Forskolin, an agent that directly stimulates adenylate cyclases, was found to activate both the Type I and Type II isozymes. Several cAMP-dependent protein kinases were also detected in parietal cell homogenates, including a Ca2+-phospholipid-sensitive or C kinase and two casein kinases which were tentatively identified as casein kinase I and II. At least two additional protein kinases with a preference for serine or lysine-rich histones, respectively, were also detected. The function of these enzymes in parietal cells remains to be shown.     

Early effects of TPA on protein kinase activity in murine thymocytes. Reduction of protein kinase C activity in the cytosol and increase of Ca2+ and phospholipid-independent kinase activity in the particulate fractions

Brief treatment of intact thymocytes with TPA and other tumor promoters causes a reduction in protein kinase C activity from the cytosol and an increase in kinase activity in the particulate fraction. In contrast to the activity in the cytosol, which is absolutely dependent on the addition of Ca2+, phosphatidylserine and diolein, the activity in the particulate fraction is independent of these agents. Analysis of target specificity of the particulate kinase activity using exogenous and endogenous substrates suggests that the increased phosphorylation in the particulate fraction is catalysed by protein kinase C with altered catalytic properties. Although interleukin-1 and TPA are both co-mitogens for murine thymocytes, interleukin-1 does not share with TPA its property to alter protein kinase activity in the cytosolic and particulate fractions.     

Characterization of protein-tyrosine kinase activity in the canine prostate.

Following the measurement of the phosphorylation of the substrate poly(Glu80Na,Tyr20) and the analysis of the alkali-resistant phosphorylation of endogenous proteins, the protein-tyrosine kinase of the canine prostate was partially characterized with regard to its subcellular localization, as well as certain kinetic and molecular properties. This kinase was mainly found in the cytosolic fraction (75%); however, its specific activity was similar to that of the residual enzyme present in the particulate fraction. Conditions for optimal activity of both fractions were determined. Under these conditions, several endogenous phosphoproteins (44-63 kilodaltons upon electrophoresis) were alkali resistant and phosphotyrosine was present in all of the major ones (pp63, pp57, pp52, and pp44). The particulate protein-tyrosine kinase activity was partially solubilized (58%) with 0.5% Triton X-100; this percentage was increased to 85% in the presence of 0.25 M KCl. Upon gel filtration, both cytosolic and particulate kinases showed an apparent molecular mass of 44 kilodaltons; these enzymes also phosphorylated similar major alkali-resistant phosphoproteins. The soluble protein-tyrosine kinase, with a sedimentation coefficient of 4.0S and an isoelectric point of 5.5, could be separated from arginine esterase and prostatic acid phosphatase.     

Possible implication of the Golgi apparatus casein kinase in the phosphorylation of vesicle docking protein p115 Ser-940: a study with peptide substrates

Phosphorylation of human vescicle docking protein p115 at Ser-942 (homologous to Ser-940 in rat p115) promotes its dissociation from the Golgi membrane. Here we show that a peptide encompassing the 934--950 sequence of p115 is unaffected or poorly phosphorylated by a variety of Ser/Thr protein kinases with the notable exception of the Golgi apparatus casein kinase (G-CK) which phosphorylates it with an efficiency comparable to that of its optimal peptide substrates. In contrast phosphorylation of the p115 peptide by protein kinase CK2 is negligible compared to that of the specific peptide substrates of this kinase. Phosphorylation by G-CK is abolished if a conserved cluster of acidic residues at position between n + 4 and n + 9 (EDDDDE) is replaced by a neutral stretch (GAGAGA). These data strongly support the view that G-CK but not the other two classes of ubiquitous "casein kinases" (CK1 and CK2) is the natural phosphorylating agent of p115.     

Early effects of TPA on protein kinase activity in murine thymocytes : Reduction of protein kinase C activity in the cytosol and increase of Ca and phospholipid-independent kinase activity in the particulate fractions

Brief treatment of intact thymocytes with TPA and other tumor promoters causes a reduction in protein kinase C activity from the cytosol and an increase in kinase activity in the pariculate fraction. In contrast to the activity in the cytosol, which is absolutely dependent on the addition of Ca²⁺, phosphatidylserine and diolein, the activity in the particulate fraction is independent of these agents. Analysis of target specificity of the particulate kinase activity using exogenous and endogenous substrates suggests that the increased phosphorylation in the particulate fraction is catalysed by protein kinase C with altered catalytic properties. Although interleukin-1 and TPA are both co-mitogens for murine thymocytes, interleukin-1 does not share with TPA its property to alter protein kinase activity in the cytosolic and particulate fractions.     

Phosphatidylinositol kinase,phosphatidylinositol-4-phosphate kinase and diacylglycerol kinase activities in rat brain subcellular fractions

Subcellular fractions isolated and purified from rat brain cerebral cortices were assayed for phosphatidylinositol (PI-), phosphatidylinositol-4-phosphate (PIP-), and diacylglycerol (DG-) kinase activities in the presence of endogenous or exogenously added lipid substrates and [γ-³²P]ATP. Measurable amounts of all three kinase activities were observed in each subcellular fraction, including the cytosol. However, their subcellular profiles were uniquely distinct. In the absence of exogenous lipid substrates, PI-kinase specific activity was greatest in the microsomal and non-synaptic plasma membrane fractions (150–200 pmol/min per mg protein), whereas PIP-kinase was predominantly active in the synaptosomal fraction (136 pmol/min per mg protein). Based on percentage of total protein, total recovered PI-kinase activity was most abundant in the cytosolic, synaptosomal, microsomal and mitochondrial fractions (4–11 nmol/min). With the exception of the microsomal fraction, a similar profile was observed for PIP-kinase activity when assayed in the presence of exogenous PIP (4 nmol/20 mg protein in a final assay volume of 0.1 ml). Exogenous PIP (4 nmol/20 mg protein) inhibited PI-kinase activity in most fractions by 40–70%, while enhancing PIP-kinase activity. PI- and PIP-kinase activities were observed in the cytosolic fraction when assayed in the presence of exogenously added PI or PIP, respectively, but not in heat-inactivated membranes containing these substrates. When subcellular fractions were assayed for DG-kinase activity using heat-inactivated DG-enriched membranes as substrate, DG-kinase specific activity was predominantly present in the cytosol. However, incubation of subcellular fractions in the presence of deoxycholate resulted in a striking enhancement of DG-kinase activities in all membrane fractions. These findings demonstrate a bimodal distribution between particulate and soluble fractions of all three lipid kinases, with each exhibiting its own unique subcellular topography. The preferential expression of PIP-kinase specific activity in the synaptic membranes is suggestive of the involvement of PIP₂ in synaptic function, while the expression of PI-kinase specific activity in the microsomal fraction suggests additional, yet unknown, functions for PIP in these membranes.     

Kinetics of regulatory serine variants of tyrosine hydroxylase with cyclic AMP-dependent protein kinase and extracellular signal-regulated protein kinase 2

Rat tyrosine hydroxylase is phosphorylated at four serine residues, at positions 8, 19, 31, and 40 in its amino terminal regulatory domain by multiple protein kinases. Cyclic AMP-dependent protein kinase phosphorylates S40, which results in alleviation of inhibition by dopamine. Extracellular signal-regulated protein kinase 2 phosphorylates S8 and S31. Site-directed serine-to-glutamate mutations were introduced into tyrosine hydroxylase to mimic prior phosphorylation of the regulatory serines; these proteins were used as substrates for cAMP-dependent kinase and extracellular signal-regulated kinase 2. The activity of cAMP-dependent kinase was unaffected by the substitution of serines 8, 19 or 31 with glutamate and the activity of extracellular signal-regulated kinase 2 was unaffected by substitution of serines 19 or 40 with glutamate. Cyclic AMP-dependent kinase was less active in phosphorylating S40 if dopamine was bound to tyrosine hydroxylase, but extracellular signal-regulated kinase 2 phosphorylation at S31 was unaffected by the presence of dopamine.     

Interleukin 2 and diacylglycerol stimulate phosphorylation of 40 S ribosomal S6 protein. Correlation with increased protein synthesis and S6 kinase activation

Interleukin 2 (IL-2) and the synthetic diacylglycerol, 1-oleoyl-2-acetylglycerol (OAG), a direct activator of protein kinase C, induce phosphorylation of the ribosomal S6 protein in a murine IL-2-dependent lymphocyte clone. The phosphorylation of S6 protein was correlated with increased protein synthesis in this cell line. Using cell-free assay systems, two unique kinases capable of phosphorylating the S6 protein were identified, namely, a calcium/phospholipid-dependent phosphotransferase, protein kinase C, and a second phospholipid-independent kinase detected in crude cytosolic fractions. Peptide mapping of the S6 protein demonstrated that the degree of S6 phosphorylation stimulated by IL-2 and OAG was similar to that achieved using the second (calcium/phospholipid-independent) kinase but not to the level of phosphorylation achieved with protein kinase C. The kinase responsible for phosphorylating S6 was soluble in stimulated cells and was induced in a time-dependent manner by either IL-2 or diacylglycerol treatment of intact cells. These data support the notion that, although protein kinase C is activated by IL-2 or OAG, subsequent events such as S6 phosphorylation may be the result of the activation of secondary phosphotransferase systems regulated by protein kinase C.     

Calcium . calmodulin-dependent protein kinase II and calcium . phospholipid-dependent protein kinase activities in rat tissues assayed with a synthetic peptide

Rat tissue levels of Ca2+ . calmodulin-dependent protein kinase II (protein kinase II) and Ca2+ . phospholipid-dependent protein kinase (protein kinase C) were selectively assayed using the synthetic peptide syntide-2 as substrate. The sequence of syntide-2 (pro-leu-ala-arg-thr-leu-ser-val-ala-gly-leu-pro-gly-lys-lys) is homologous to phosphorylation site 2 in glycogen synthase. The relative Vmax/Km ratios of the known Ca2+-dependent protein kinases for syntide-2 were determined to be as follows: protein kinase II, 100; protein kinase C, 22; phosphorylase kinase, 2; myosin light chain kinase, 0.005. Levels of protein kinase II were highest in cerebrum (3.36 units/g tissue) and spleen (0.85 units/g) and lowest in testis (0.05 units/g) and kidney (0.04 units/g). Protein kinase II activity was localized predominantly in the 100,000g particulate fraction of cerebrum and testis, in the supernatant fraction of heart, liver, adrenal, and kidney, and about equally distributed between particulate and supernatant in spleen and lung. Likewise, protein kinase C activity was highest in cerebrum (0.56 units/g) and spleen (0.47 units/g), and the majority of activity was present in the cytosolic fraction for all tissues measured except for cerebrum and testis in which the kinase activity was equal in both fractions. Finally, the ratios of protein kinase II to protein kinase C were different in various rat tissues and between particulate and supernatant fractions. These results suggest somewhat different functions for these two Ca2+-regulated, multifunctional protein kinases.     

cAMP,not Ca2+/calmodulin,regulates the phosphorylation of acetylcholine receptor in Torpedo californica electroplax

The regulation of the phosphorylation of the acetylcholine receptor in electroplax membranes from Torpedo californica and of purified acetylcholine receptor was investigated. The phosphorylation of the membrane-bound acetylcholine receptor was not stimulated by Ca²⁺/calmodulin, nor was it inhibited by EGTA, but it was stimulated by the catalytic subunit of cAMP-dependent protein kinase, and was blocked by the protein inhibitor of cAMP-dependent protein kinase. Purified acetylcholine receptor was not phosphorylated by Ca²⁺/calmodulin-dependent protein kinase activity in electroplax membranes, nor by partially purified Ca²⁺/calmodulin-dependent protein kinases from soluble or particulate fractions from the electroplax. Of the four acetylcholine receptor subunits, termed α, β, γ and δ, only the γ- and δ-subunits were phosphorylated by the cAMP-dependent protein kinase (+cAMP), or by its purified catalytic subunits.     

Protein phosphatases active on acetyl-CoA carboxylase phosphorylated by casein kinase I, casein kinase II and the cAMP-dependent protein kinase

The protein phosphatases in rat liver cytosol, active on rat liver acetyl-CoA carboxylase (ACC) phosphorylated by casein kinase I, casein kinase II and the cAMP-dependent protein kinase, have been partially purified by anion-exchange and gel filtration chromatography. The major phosphatase activities against all three substrates copurify through fractionation and appear to be identical to protein phosphatases 2A1 and 2A2. No unique protein phosphatase active on 32P-ACC phosphorylated by the casein kinases was identified.     

cAMP-dependent protein kinase isozymes with preference for histone H2B as substrate in mitochondria of bovine heart

Mitochondria from bovine hearts were fractionated by three different procedures and the fractions were characterized by marker enzymes. Highly purified outer membranes, membrane vesicles, and inner membranes, as well as two high-speed soluble fractions, were obtained. Azide (or oligomycin) resistant ATPase was not found to be a marker for outer membranes. The data were consistent with the association of the protein kinase activity with the soluble matrix of the mitochondria. Activity was highest with histone H2B as the substrate, with histone H1 next in preference. In contrast to the mitochondrial protein kinases studied previously, protamine, casein, and phosvitin were very poor substrates and there was no detectable phosphorylation of pyruvate dehydrogenase. Activity was stimulated by cAMP but not by cGMP, calmodulin, or phosphatidylserine--diolein, with or without Ca2+. Two cAMP-dependent isozymes were separated from the soluble fraction of the mitochondria by chromatography on DE-52 columns. Phosphorylation of histone H2B by the isozymes was inhibited by 98% by Kemptide.