A Retinal Calmodulin-Dependent Kinase: Calcium/ Calmodulin-Stimulated and -Inhibited States

Abstract: A calcium/calmodulin-dependent protein kinase was isolated from retina. The retinal enzyme is composed exclusively of 50-kilodalton (kD) subunits and has a molecular mass of approximately 275 kD, in contrast to forebrain calmodulin kinase II, which is composed of 50-kD and 60-kD subunits in a 3:1 ratio and has a molecular mass of approximately 520 kD. Similar substrate specificities, kinetic properties, capacity to bind calmodulin, and immunoreactivity suggest that the retinal kinase is an isoenzyme of forebrain calmodulin kinase II. Both kinases autophosphorylate in an intramolecular manner; however, auto-phosphorylation has different effects on the activities of the two enzymes. Autophosphorylation of retinal calmodulin kinase converts the enzyme from a calcium/calmodulin-dependent to a calcium/calmodulin-inhibited kinase, with high activity in the absence of calcium, whereas autophosphorylation of the forebrain kinase results in a less active, calcium/calmodulin-independent enzyme. These properties of calmodulin kinase may play an important role in retinal function.     

Phosphoglycerate kinase B from ram testis. Purification, characterisation and comparison with the muscle isoenzyme

1. The testis-specific isoenzyme of phosphoglycerate kinase (phosphoglycerate kinase B) has been isolated from ram testes using a procedure which separates it from 'normal' phosphoglycerate kinase which is also present in testis tissue. The purification procedure is described. 2. The best preparations had no detectable impurity on electrophoresis, and had specific activities comparable with the same enzyme from other sources. 3. Kinetic studies indicated that the two isoenzymes have identical properties, within experimental error, for substrate affinity (for MgATP, 3-phosphoglycerate and MgADP), energy of activation and thermal denaturation. 4. The molecular weights of both isoenzymes were not distinguishably different from those previously reported, as measured by polyacrylamide/dodecylsulphate electrophoresis. The amino acid compositions showed only slight differences, and tryptic peptide maps showed that there was close homology of sequence. Starch gel electrophoresis at pH 6.5 indicates that the B isoenzyme has 1--2 more positive charges than the A. 5. Phosphoglycerate kinase A isolated from sheep muscle was shown, within experimental error, to be identical to the phosphoglycerate kinase A isolated from testis. 6. The results further substantiate the suggestion that the B isoenzyme is coded by a gene which was duplicated from the phosphoglycerate kinase A gene.     

Protein kinases from liver mitochondria of tumour-bearing rats.

The mitochondria of liver of Yoshida ascites tumour-bearing rats contained two forms of protein kinase distinguishable on the basis of their kinetic properties, substrate specificity and responses to cyclic adenosine 3',5'-monophosphate (cAMP). One of these (kinase I) was activated 2-3 fold by cAMP while the other form (kinase II) was insensitive to the action of cAMP. Kinase I which was selective towards histone F1 as substrate was obtained as a homogeneous preparation and was observed to have a molecular weight of 170 000 by Sephadex G-150 gel filtration. Protein kinase II appeared to be a smaller protein with molecular weight of 54 000 and was specific towards acidic proteins namely casein and phosvitin. Protein kinases isolated from liver mitochondria of normal rats showed variations in respect to elution profile of DEAE-cellulose and electrophoretic mobility. The preparation corresponding to kinase I did not show stimulatory responses to cAMP.     

Cyclic AMP-stimulated phosphorylation of bovine tracheal smooth muscle contractile and non-contractile proteins.

1. Various proteins isolated from bovine tracheal smooth muscle were examined as phosphate acceptor substrates for a cyclic AMP-dependent protein kinase isolated from the same tissue. A fraction prepared in a manner similar to that of skeletal muscle troponin was the best substrate of the presumptive contractile proteins isolate. Actomyosin and tropomyosin were relatively poor substrates. 2. An assay was developed for the rapid detection in a large number of samples of the muscle specific substrate for the protein kinase on which we reported previously. 3. Using this assay, the muscle specific substrate found in bovine tracheal smooth muscle was partially purified resulting in a preparation which when resolved by polyacrylamide gel electrophoresis showed a single peak of 32P incorporated, and which could be further characterized. 4. Our findings suggest that the substrate contains a protein subunit of molecular weight 19 000, which can be phosphorylated at serine and threonine residues, in the presence of cyclic AMP and protein kinase. The phosphate is in a covalent ester linkage with these residues. 5. A phosphoprotein phosphatase was isolated from the bovine tracheal smooth muscle. 6. Bovine tracheal smooth muscle contains cyclic AMP dependent protein kinase and phosphoprotein phospahatase activity as well as the muscle specific substrate, suggesting that these elements may be part of a mechanism which regulates smooth muscle tone.     

Evidence for the identity of nuclear and cytoplasmic adenosine-3':5'-monophosphate-dependent protein kinase from porcine ovaries and nuclear translocation of the cytoplasmic enzyme.

Protein phosphokinase activity from a 0.5 M NaCl extract of purified porcine ovary nuclei has been resolved by Sephadex G-200 gel filtration into three forms of kinase, protein kinase I and III, both independent of adenosine 3':5'-monophosphate (cyclic AMP), and cyclic-AMP-dependent protein kinase II. Cyclic AMP-binding activity was associated with protein kinase II but not with protein kinases I and III. Protein kinases I, II, and III exhibited different cyclic nucleotide dependency and substrate specificity. Protein kinase II was inhibited by a heat-stable protein from rabbit skeletal muscle, whereas protein kinases I and III were not inhibited. According to previously established criteria [Traugh, J.A., Ashby, C.D. and Walsh, D.A. (1974) nuclear protein kinase II can be classified as cyclic-AMP-dependent protein kinase consisting of regulatory and catalytic subunits. Nuclear protein kinases I and III are cyclic-AMP-independent enzymes. Evidence for the identity of nuclear cyclic-AMP-dependent protein kinase II with cytosol (105 000 X g supernatant fraction) cyclic-AMP-dependent protein kinase was obtained in several ways. Nuclear and cytosol cyclic-AMP-dependent protein kinases exhibited identical elution characteristics on DEAE-cellulose and Sephadex G-200 indicating that both kinases are of similar molecular size and possess similar ionic charge. Both kinases exhibited an identical Km for ATP of 8 muM, showed similar substrate specificity, and revealed similar antigenic properties. Cyclic-AMP-dependent protein kinase II was also identified in nuclei isolated in nonaqueous media, eliminating the possibility that the cyclic-AMP-dependent protein kinase activity identified in nuclei isolated in aqueous media may have arisen as the result of cytoplasmic contamination. After incubation of neonatal porcine ovaries which lack nuclear cyclic-AMP-dependent protein kinase with 0.1 muM 8-p-chlorophenylthio cyclic AMP, considerable cyclic-AMP-dependent protein kinase II activity was identified in nuclei isolated in nonaqueous media. From these data it is concluded that the nuclear cyclic-AMP-dependent protein kinase II is related to or identical with the ovary cytoplasmic cyclic-AMP-dependent protein kinase, supporting the concept that nuclear cyclic-AMP-dependent protein kinase is of cytoplasmic origin.     

Unique substrate specificity and regulatory properties of PKC-epsilon: a rationale for diversity

PKC-epsilon was isolated from a murine brain cDNA library. The clone, lambda 61PKC-epsilon, encoded a polypeptide of 737 amino acids that is homologous to other PKCs. Northern analysis showed that the 7 kb mRNA for this cDNA is widely expressed. The protein when expressed in COS-1 cells displayed phorbol ester-binding activity. However in order to detect the kinase activity of PKC-epsilon, it was necessary to employ a synthetic peptide substrate based upon the pseudosubstrate site. Subsequent analysis demonstrated that PKC-epsilon, while showing certain properties characteristic of the PKC family, has a quite distinct substrate specificity and is independent of Ca2+.     

The plasma membrane H+‐ATPase from maize roots is phosphorylated in the C‐terminal domain by a calcium‐dependent protein kinase

A protein kinase activity associated with maize root plasma membranes was partially purified and characterized. Biochemical properties, such as calcium dependence, inhibition by calmodulin antagonists, and absence of calmodulin stimulation, indicated that the enzyme belongs to the calcium‐dependent protein kinase (CDPK) family. By means of an in‐gel phosphorylation assay the molecular mass of active polypeptides was determined: two bands of 55 and 51 kDa became labelled. The same proteins were also immunodecorated by monoclonal antibodies against soybean CDPK. Results from in vitro assays demonstrated that maize H⁺‐ATPase was a suitable substrate for this protein kinase and that the phosphorylation site was located at the C‐terminal domain of the enzyme. This result was confirmed by using as substrate in phosphorylation assays the isolated C‐terminal domain of the H⁺‐ATPase expressed in Escherichia coli as a glutathione‐transferase fusion protein.     

Plasma membrane protein kinase activity in normal and Rous sarcoma virus-transformed chick embryo fibroblasts.

A preliminary study has been carried out to investigate the effect of Rous sarcoma virus transformation on plasma membrane protein kinase activity in chick embryo fibroblasts. Enzyme activity was measured using an in vitro phosphorylation method employing [gamma-32P]ATP with isolated plasma membranes serving as the source of both protein kinase and protein substrate. In general, the enzymatic properties observed were similar to those of other known protein kinases. However, for maximal activity a marked dependence on high Mg2+ concentrations was noted. Evidence was obtained which showed that cyclic nucleotide-dependent protein kinases were present in membranes from normal cells, but none could be measured in preparations from transformed cells. In addition, transformation appeared to result in a slight increase in basal plasma membrane protein kinase activity.     

Isolation of a major human placental substrate for the epidermal growth factor (urogastrone) receptor kinase: immunological cross-reactivity with transducin and sequence homology with lipocortin

Using as a starting material either a detergent extract or a protein fraction eluted from membranes with ethylene glycol bis (beta-aminoethyl ether)-N,N'-tetraacetic acid, we have isolated from human placental membranes a major substrate for the epidermal growth factor (urogastrone) receptor kinase (EGF kinase). The substrate was isolated both in an intact form, having a molecular mass of approximately 38-kDa (p38), and in a 35-kDa form (p35) representing a proteolytic cleavage product of p38. Both p38 and p35 cross-reacted with antibodies directed against bovine retinal transducin, but did not cross-react with antibodies directed against the 35-kDa beta subunit of human placental G-protein. Antisera directed against the placental EGF kinase substrate failed to react with either bovine or human placental src kinase substrate, p36. Conversely, antisera directed against p36 reacted only poorly with placental p38 or p35. Although p38 had a blocked amino terminus that precluded sequence analysis, p35 yielded an N-terminal sequence that was identical with residues 13-36 of human lipocortin. Our data clearly distinguish p38 from the previously described intestinal calcium binding protein calpactin I or p36 that is also a tyrosine kinase substrate, and our work points to a close relationship (if not identity) between p35 and a 35-kDa EGF receptor kinase substrate previously characterized in A431 cells. We conclude that p38 and p35, which very likely represent human placental lipocortin, may share only limited epitope homology with transducin alpha subunit; however, the possibility that p38, along with intestinal p36 and with a family of related calcium binding proteins, may, like transducin, play a role in receptor-mediated transmembrane signaling is discussed.     

Cloning and sequencing of a gene encoding pyruvate kinase from Schizosaccharomyces pombe; implications for quaternary structure and regulation of the enzyme

Abstract A cDNA encoding pyruvate kinase from Schizosaccharomyces pombe has been isolated from a lambda ZAPII library. This cDNA was sequenced and found to contain an open reading frame of 1524 nucleotides, giving a predicted protein subunit M _r of 55 470. The sequence shows a high degree of identity with other pyruvate kinase sequences, with residues implicated in the binding of substrate and metal ion co-factors conserved. However, there are significant differences in the putative subunit interface and effector binding regions which may account for the unusual quaternary structure and regulatory properties of the S. pombe enzyme.     

Characteristics of the protein kinase activity associated with rat neurofilament preparations

Some properties of the protein kinase activity associated with neurofilaments isolated from the brain stem and spinal cord of rats have been investigated. The activity had an apparent Km for ATP of 20 microM, a pH optimum of 8.0 and phosphorylated both serine and threonine residues in neurofilament proteins. Cyclic AMP had no effect on the in vitro reaction and casein was a preferred exogenous substrate in comparison to histone. Phosphopeptide mapping of the 145 kDa subunit from neurofilaments phosphorylated in the presence and absence of microtubule proteins indicated that the neurofilament-associated activity was distinct from the microtubule-associated protein kinase. Limited proteolysis of neurofilaments with chymotrypsin indicated that the enzyme activity was not associated with a domain of the 200 kDa subunit which may form the side-arm projections on neurofilaments.     

Developmental Changes in Calmodulin-Kinase II Activity at Brain Synaptic Junctions: Alterations in Holoenzyme Composition

Synaptic junctions (SJs) from rat forebrain were isolated at increasing postnatal ages and examined for endogenous protein kinase activities. Our studies focused on the postnatal maturation of the multifunctional protein kinase designated Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II). This kinase is comprised of a major 50-kilodalton (kDa) and a minor 60-kDa subunit. Experiments examined the developmental properties of CaM-kinase II associated with synaptic plasma membranes (SPMs) and synaptic junctions (SJs), as well as the holoenzyme purified from cytosolic extracts. Large developmental increases in CaM-kinase II activity of SJ fractions were observed between postnatal days 6 and 20; developmental changes were examined for a number of properties including (a) autophosphorylation, (b) endogenous substrate phosphorylation, (c) exogenous substrate phosphorylation, and (d) immunoreactivity. Results demonstrated that forebrain CaM-kinase II undergoes a striking age-dependent change in subunit composition. In early postnatal forebrain the 60-kDa subunit constitutes the major catalytic and immunoreactive subunit of the holoenzyme. The major peak of CaM-kinase II activity in SJ fractions occurred at approximately postnatal day 20, a time near the end of the most active period of in vivo synapse formation. Following this developmental age, CaM-kinase II continued to accumulate at SJs; however, its activity was not as highly activated by Ca2+ plus calmodulin.     

Microsomal cAMP-independent histone H1 kinase activity in plasmacytoma, Morris hepatoma and normal liver

A protein kinase activity with high specificity for histone H1 was isolated from mouse plasmacytoma, Morris hepatoma and normal mouse liver and compared by ion exchange chromatography after DEAE-cellulose, hydroxylapatite and Sephadex G-200 chromatography. This cAMP-independent histone H1 kinase is not affected by the heat-stable cAMP-dependent protein kinase inhibitor. It has the following particular properties: it prefers GTP to ATP as substrate and was found to be present with a great activity only in neoplastic tissues. No phosphatase activity was detected in the partially purified histone H1 kinase fraction from normal and neoplastic cells. These results suggest either an increase amount of histone H1 kinase and/or of its activator in neoplastic cells, or the presence of a strong inhibitor in normal cells. This histone H1 kinase appears to be analogous to the chromatin bound kinase which phosphorylates histone H1 at the NH2 and COOH terminal regions. We might suggest an implication of this kinase in the regulation of cell division.     

Purification and characterization of a multisubunit phosphatase from turkey gizzard smooth muscle. The effect of calmodulin binding to myosin light chain kinase on dephosphorylation

A phosphatase that is active in dephosphorylating the isolated 20,000-Da light chain of myosin, as well as the enzyme myosin light chain kinase, has been purified to apparent homogeneity from turkey gizzards. The enzyme has a molecular weight of 165,000 by sedimentation-equilibrium centrifugation under nondenaturing conditions and is composed of three subunits (Mr = 60,000, 55,000, and 38,000) in a 1:1:1 molar ratio. The properties of the holoenzyme, as well as the purified catalytic subunit (Mr = 38,000) were compared using myosin light chains, intact myosin, and myosin light chain kinase as substrates. Although the holoenzyme is active in dephosphorylating the isolated myosin light chains and the enzyme myosin light chain kinase, the holoenzyme does not dephosphorylate myosin. On the other hand, the catalytic subunit of the holoenzyme dephosphorylates all three substrates. When myosin light chain kinase, which has been phosphorylated at two sites is used as substrate, both sites are rapidly dephosphorylated by the phosphatase in the absence of bound calmodulin. If calmodulin is bound to the diphosphorylated kinase, only one site is dephosphorylated. Interestingly, the single site dephosphorylated when calmodulin is bound to myosin light chain kinase is the site that is not phosphorylated when the calmodulin-myosin kinase complex is phosphorylated by cAMP-dependent protein kinase.     

Plasma membrane protein kinase activity in normal and Rous sarcoma virus-transformed chick embryo fibroblasts

A preliminary study has been carried out to investigate the effect of Rous sarcoma virus transformation on plasma membrane protein kinase activity in chick embryo fibroblasts. Enzyme activity was measured using an in vitro phosphorylation method employing [γ-³²P]ATP with isolated plasma membranes serving as the source of both protein kinase and protein substrate. In general, the enzymatic properties observed were similar to those of other known protein kinases. However, for maximal activity a marked dependence on high Mg²⁺ concentrations was noted. Evidence was obtained which showed that cyclic nucleotide-dependent protein kinases were present in membranes from normal cells, but none could be measured in preparations from transformed cells. In addition, transformation appeared to result in a slight increase in basal plasma membrane protein kinase activity.     

Cyclic AMP-dependent histone-specific nucleoplasmic protein kinase from rat liver.

A nucleoplasmic histone kinase activity was isolated from livers of adult rats and purified 39-fold compared with whole nuclei by ultracentrifugation of the nuclear extract and Sephadex G-200 gel filtration in the presence of cyclic AMP. Analysis by polyacrylamide-gel electrophoresis as well as by gel filtration indicates a mol.wt. of approx. 60,000 for the catalytic subunit and 130000-150000 for the cyclic AMP-binding activity. The purified enzyme displays a 20-fold greater preference for histone fractions 1 and 2b than for any non-histone substrate, including alpha-casein. Endogenous protein in the preparation is not appreciably phosphorylated. The unfractioned enzyme is stimulated significantly by cyclic GMP, cyclic IMP and dibutyryl cyclic AMP as well as by cyclic AMP. The catalytic reaction requires Mg2+ (Km 1.9mM) and ATP (Km 15.4 micron). Half-maximal activity of the enzyme is observed with histone 2b at 12micron and histone 1 at a higher substrate concentration. The pH optima are 6.1 and 6.5 with histones 2b and 1 respectively. This nuclear protein kinase appears to be distinct from other nuclear enzymes that have been reported, on the basis of histone specificity, univalent-salt-sensitivity, pH optima and nuclear location. However, the enzyme possesses many properties similar to those of the cytoplasmic kinases, including cyclic AMP-dependence, Mg2+ and ATP affinities and pH optima. It differs from cytoplasmic protein kinase type I, the major form in the liver, with respect to bivalent-cation effects and response to the heat-stable protein kinase inhibitor protein isolated from ox heart.     

Biochemical characterization of a calcium-sensitive protein kinase LeCPK2 from tomato

LeCPK2 (GenBank GQ205414), a versatile calcium-dependent protein kinase (CDPK or CPK) gene was isolated from tomato in our previous study. In this study, the biochemical properties of LeCPK2 were further investigated. To examine the role of the C-terminal calmodulin-like domain (CLD) of LeCPK2 with respect to Ca2+ activation, the kinase activities of recombinant full-length and truncated LeCPK2 were measured by Kinase-Glo Luminescent kinase assay (Promega). The results showed that LeCPK2 activity was Ca(2+)-dependent and the C-terminal CLD of 161 residues was essential for the activation of LeCPK2. The activity of LeCPK2 was sharply stimulated by Ca2+ with K0.5 (concentration of Ca2+ for half-maximal activity) of 48.8 and 45.5 nM with substrate histone IIIs and syntide 2, respectively. The optimal concentration of Mg2+ for LeCPK2 activity was 20 and 10 mM for substrate histone IIIs and syntide 2, respectively. The K(m) value of LeCPK2 towards histone IIIs and syntide 2 was 44.9 microg/ml and 89.52 microM, respectively. The determination of biochemical properties of LeCPK2 would provide some clues on how its activity was regulated in vivo.     

Myosin light chain kinase structure function analysis using bacterial expression

A 40-kDa fragment of chicken smooth muscle myosin light chain kinase was produced and partially purified from a bacterial expression system. This fragment exhibits calmodulin binding and substrate phosphorylation properties similar to those of the isolated chicken gizzard enzyme. A series of 3'-deletion mutants was prepared and used to produce proteins with the same NH2 terminus but with COOH termini varying over 180 amino acids. Results show that truncation of the enzyme at Ser-512 (based on the amino acid numbering system described for the partial cDNA clone by Guerriero, V., Jr., Russo, M. A., Olson, N. J., Putkey, J. A., and Means, A. R. (1986) Biochemistry 25, 8372-8381) does not alter calmodulin binding, calmodulin regulation, or enzymatic properties. Removal of an additional 5 residues from the COOH terminus completely inhibits calmodulin binding and results in an inactive kinase that can be fully activated by limited proteolysis. Site specific mutations within these 5 residues demonstrate that Gly-508 and Arg-509 are independently involved in calmodulin-dependent binding and activation of myosin light chain kinase. Truncation of the enzyme at residues within the protein kinase catalytic domain results in inactive protein that cannot be activated by proteolysis.     

Identification and cloning of Kidins220, a novel neuronal substrate of protein kinase D

Protein kinase D (PKD) is a serine/threonine kinase regulated by diacylglycerol signaling pathways with unique domain composition and enzymatic properties, still awaiting identification of its specific substrate(s). Here we have isolated, cloned, and characterized a novel protein from PC12 cells, termed Kidins220 (kinase D-interacting substrate of 220 kDa), as the first identified PKD physiological substrate. Kidins220 contains 11 ankyrin repeats and four transmembrane domains within the N-terminal region. We have shown that Kidins220 is an integral membrane protein selectively expressed in brain and neuroendocrine cells, where it concentrates at the tip of neurites. In PC12 cells, PKD co-immunoprecipitates and phosphorylates endogenous Kidins220. This phosphorylation is increased after stimulating PKD activity in vivo by phorbol-12, 13-dibutyrate treatment. A constitutively active PKD mutant (PKD-S744E/S748E) phosphorylates recombinant Kindins220-VSVG in vitro in the absence of phorbol-12,13-dibutyrate. Conversely, Kidins220-VSVG phosphorylation is abolished when a dominant negative mutant of PKD (PKD-D733A) is used. Moreover, a peptide within the Kidins220 sequence, containing serine 919 in a consensus motif for PKD-specific phosphorylation, behaved as the best peptide substrate to date. Substitution of serine 919 to alanine abrogated peptide phosphorylation. Furthermore, by generating an antibody recognizing Kidins220 phosphorylated on serine 919, we show that phorbol ester treatment causes the specific phosphorylation of this residue in PC12 cells in vivo. Our results provide the first physiological substrate for PKD and indicate that Kidins220 is phosphorylated by PKD at serine 919 in vivo.