Protein phosphorylation during Rhodnius prolixus embryogenesis: protein kinase casein kinase II activity.

Protein kinase casein kinase II (CK II) activity was assayed during Rhodnius prolixus embryogenesis. Vitellin (VT) is the main endogenous substrate during the whole development. It is maximally phosphorylated at the third day of embryogenesis by CK II and then its phosphorylation decreases to a basal level by the time of first instar eclosion. When dephosphorylated casein was used as an exogenous substrate a different profile of enzyme activity was obtained. CK II activity increases on day 1 after fertilization and reaches a plateau on day 7 and its activity remains elevated until eclosion. Extracts obtained from oocytes or from 3-day old eggs were fractionate through gel filtration chromatography. CK II activity was assayed in each fraction and the enzyme obtained from the 3-day old eggs was shown to be three times more active than that obtained from oocytes, although the amount of enzyme present in the fractions was the same. These enriched CK II fractions were assayed against different effectors, such as: cAMP, H-8, H-89, calphostin C, sphingosine, polylysine and heparin. Heparin was the most effective one. When CK II activity was assayed in non-fertilized eggs, no activation of the enzyme was observed when compared to fertilized eggs. These data indicate that CK II is activated in a fertilization dependent process. The decrease in CK II activity against VT coincides with the beginning of VT proteolysis processing suggesting a possible relationship between protein phosphorylation and yolk degradation.     

Cyclic nucleotide-independent phosphorylation of vitellin by casein kinase II purified from Rhodnius prolixus oocytes

In this study we show that Vitellin (VT) phosphorylation in chorionated oocytes of Rhodnius prolixus is completely inhibited by heparin (10 microg/ml), a classical casein kinase II (CK II) inhibitor. VT phosphorylation is not affected by modulators of cyclic nucleotide-dependent protein kinases such as c-AMP (10 microM), H-8 (1 microM) and H-89 (0.1 microM). We have obtained a 3000-fold VT-free enriched preparation of CK II. Autophosphorylation of this enzyme preparation in the presence of (32)P-ATP demonstrated that it lacks any endogenous substrates. Rhodnius CK II is strongly inhibited by heparin (Ki = 9 nM) and uses ATP (Km = 36 microM) or GTP (Km = 86 microM) as phosphate donors. Incubation of VT with purified Rhodnius CK II and (32)P-ATP led to the incorporation of 2 mols of phosphate/mol VT. However, the total number of phosphorylation sites available can be altered by previous incubation of VT with alkaline phosphatase. These data show that an insect yolk protein contain phosphorylation sites for a cyclic nucleotide-independent protein kinase such as CK II.     

Lysophosphatidylcholine metabolism and lipoprotein secretion by cultured rat hepatocytes deficient in choline.

A protein kinase activity was identified in pig brain that co-purified with microtubules through repeated cycles of temperature-dependent assembly and disassembly. The microtubule-associated protein kinase (MTAK) phosphorylated histone H1; this activity was not stimulated by cyclic nucleotides. Ca2+ plus calmodulin, phospholipids or polyamines. MTAK did not phosphorylate synthetic peptides which are substrates for cyclic AMP-dependent protein kinase, cyclic GMP-dependent protein kinase. Ca2+/calmodulin-dependent protein kinase II, protein kinase C or casein kinase II. MTAK activity was inhibited by trifluoperazine [IC50 (median inhibitory concn.) = 600 microM] in a Ca2+-independent fashion. Ca2+ alone was inhibitory [IC50 = 4 mM). MTAK was not inhibited by heparin, a potent inhibitor of casein kinase II, nor a synthetic peptide inhibitor of cyclic AMP-dependent protein kinase. MTAK demonstrated a broad pH maximum (7.5-8.5) and an apparent Km for ATP of 45 microM. Mg2+ was required for enzyme activity and could not be replaced by Mn2+. MTAK phosphorylated serine and threonine residues on histone H1. MTAK is a unique cofactor-independent protein kinase that binds to microtubule structures.     

Affinity purification and biochemical characterization of histolysin, the major cysteine proteinase of Entamoeba histolytica.

Insulin receptor was co-purified from human placenta together with insulin-stimulated kinase activity that phosphorylates the insulin receptor on serine residues. By using this 'in vitro' system, the mechanism of activation of the serine kinase by insulin was explored. Peptide 1150, histone, poly(Glu-Tyr), eliminating Mn2+ (Mg2+ only), treatment at 37 degrees C (1 h), N-ethylmaleimide, phosphate, beta-glycerol phosphate and anti-phosphotyrosine antibody all inhibited insulin-receptor tyrosine kinase activity and the ability of insulin to stimulate phosphorylation of the insulin receptor on serine. Additionally, direct stimulation of the receptor tyrosine kinase by vanadate increased serine phosphorylation of the insulin receptor. Insulin-stimulated tyrosine phosphorylation preceded insulin-stimulated serine phosphorylation of the insulin receptor. The activity of the insulin-sensitive receptor serine kinase was not augmented by cyclic AMP, cyclic GMP, Ca2+, Ca2+ + calmodulin, Ca2+ + phosphatidylserine + diolein or spermine, or inhibited appreciably by heparin. Additionally, the serine kinase phosphorylated casein or phosvitin poorly and was active with Mn2+. This indicates that it is distinct from Ca2+, Ca2+/phospholipid, Ca2+/calmodulin, cyclic AMP- and cyclic GMP-dependent protein kinases, casein kinases I and II and insulin-activated ribosomal S6 kinase. Taken together, these data indicate that a novel species of serine kinase catalyses the insulin-dependent phosphorylation of the insulin receptor and that activation of this receptor serine kinase by insulin requires an active insulin-receptor tyrosine kinase.     

Effects of cationic polypeptides on the activity, substrate interaction, and autophosphorylation of casein kinase II: a study with calmodulin.

The effects of basic polypeptides on the ability of casein kinase II to phosphorylate an exogenous substrate (calmodulin) are correlated with steady-state autophosphorylation of the alpha- and beta-subunits of casein kinase II. Polylysine and polyarginine increase autophosphorylation of the alpha-subunit with a concomitant decrease in beta-subunit phosphorylation, while enhancing casein kinase II-stimulated phosphorylation of calmodulin over 100-fold. The highly basic carboxyl terminal segment of the endogenous p21c-Ki-ras has similar effects on the phosphorylation of calmodulin and the alpha- and beta-subunits of casein kinase II. Altering the concentration of cationic polypeptides produces a biphasic effect on the phosphorylation of both calmodulin and the alpha-subunit, which correlate positively with each other but do not correlate with beta-subunit phosphorylation. When the KCl concentration is changed, casein kinase II activity correlates positively only with alpha-subunit phosphorylation. In contrast, the biphasic response of calmodulin phosphorylation by casein kinase II at different Ca2+ concentrations correlates positively with both alpha- and beta-subunit phosphorylation. Therefore, in the presence of basic protein activators, the rate of phosphorylation of a substrate, calmodulin, correlates with steady-state phosphorylation of the alpha-subunit, but not with the beta-subunit under all conditions tested. Endogenous cationic factors may modulate the in vivo activity of casein kinase II and alter the interaction of the enzyme with specific intracellular substrates.     

Phosphorylation of vimentin in mitotically selected cells. In vitro cyclic AMP-independent kinase and calcium-stimulated phosphatase activities

The phosphorylation of the intermediate filament protein vimentin was examined under in vitro conditions. Cell cytosol and Triton-insoluble cytoskeleton preparations from nonmitotic and mitotically selected mouse L-929 cells exhibited vimentin kinase activity that is apparently cAMP and Ca2+ independent. The level of vimentin kinase activity was greater in preparations from mitotically selected cells than nonmitotic cells. Addition of Ca2+ to mitotic cytosol decreased net vimentin phosphorylation. Dephosphorylation experiments indicated that there is phosphatase activity in these preparations which is stimulated by addition of Ca2+. Fractionation of cytosol from nonmitotic cells on DEAE-Sephacel and phosphocellulose revealed a single major vimentin kinase activity (peak I). Fractionation of cytosol from mitotically selected cells yielded a similar activity (peak I) and an additional vimentin kinase activity (peak II) that was not found in nonmitotic preparations. Based on substrate specificity and lack of inhibition to characteristic inhibitors, the semipurified peak I and II vimentin kinase activities appear to be cAMP-independent enzymes that are distinct from casein kinases I and II. Phosphopeptide mapping studies indicated that both peak I and peak II vimentin kinases phosphorylate tryptic peptides in the NH2-terminal region of vimentin that are phosphorylated in intact cells. Electron microscopic examination of reconstituted vimentin filaments phosphorylated with both semipurified kinases indicated that phosphorylation induced filament disassembly. These experiments indicate that the increased phosphorylation of vimentin during mitosis may be catalyzed by a discrete cAMP-independent protein kinase. In addition, preparations from mitotic cells exhibited a Ca2+-stimulated phosphatase activity, suggesting that Ca2+ may play a regulatory role in vimentin dephosphorylation during mitosis.     

Identification of myosin II kinase from sea urchin eggs as protein kinase CK2

Here we purified and identified a myosin II kinase from sea urchin eggs. The activity of this myosin II kinase in the egg extract was not significantly affected by Ca(2+)/calmodulin (CaM). Using sequential column chromatographies, we purified the myosin II kinase from the egg extract as a complex composed of 36- (p36) and 28-kDa (p28) proteins. Partial amino acid sequences of these two components were highly coincident with those of the alpha and beta subunits of protein kinase CK2 (formerly casein kinase II) in sea urchin eggs, respectively. To confirm that the purified myosin II kinase was CK2, we obtained a cDNA which encodes p36 from a cDNA library of sea urchin eggs. The amino acid sequence derived from the obtained cDNA showed over 70% homology to CK2 from various eukaryotes. Furthermore, recombinant p36, as well as the purified myosin II kinase, phosphorylated MRLC. One dimensional phosphopeptide mapping revealed that the phosphorylation site(s) of MRLC by both recombinant p36 and the purified myosin II kinase was identical. These clearly showed that the Ca(2+)/CaM-independent myosin II kinase activity in sea urchin eggs was identical to CK2.     

Purification and characterization of two wheat-embryo protein phosphatases.

Two protein phosphatases (enzymes I and II) were extensively purified from wheat embryo by a procedure involving chromatography on DEAE-cellulose, phenyl-Sepharose CL-4B, DEAE-Sephacel and Ultrogel AcA 44. Preparations of enzyme I (Mr 197,000) are heterogeneous. Preparations of enzyme II (Mr 35,000) contain only one major polypeptide (Mr 17,500), which exactly co-purifies with protein phosphatase II on gel filtration and is not present in preparations of enzyme I. However, this major polypeptide has been identified as calmodulin. Calmodulin and protein phosphatase II can be separated by further chromatography on phenyl-Sepharose CL-4B. Protein phosphatases I and II do not require Mg2+ or Ca2+ for activity. Both enzymes catalyse the dephosphorylation of phosphohistone H1 (phosphorylated by wheat-germ Ca2+-dependent protein kinase) and of phosphocasein (phosphorylated by wheat-germ Ca2+-independent casein kinase), but neither enzyme dephosphorylates a range of non-protein phosphomonoesters tested. Both enzymes are inhibited by Zn2+, Hg2+, vanadate, molybdate, F-, pyrophosphate and ATP.     

Effect of Ethanol on Nuclear Casein Kinase II Activity in Brain

Casein kinase II (CK II) plays an important role in serine/threonine dependent protein phosphorylation. In brain it is associated with long term potentiation besides its involvement in DNA, RNA and protein metabolism. Ethanol has been shown to induce cognitive impairment and affects DNA, RNA and protein metabolism at various steps. Since CK II is central in all these events, which are specifically affected by ethanol, the role of nuclear CK II is investigated in the present study. Total nuclear casein kinase activity was unaffected while heparin sensitive nuclear casein kinase II activity showed a 30% decrease in the brain from chronic alcohol fed rats. Cytosolic CK II activity was also unaffected. Immunological detection by western analysis using CK II antibodies showed no alteration in the quantity of enzyme. The decrease in nuclear casein kinase II might be responsible for ethanol induced cognitive impairment in the brain.     

Characterization of a cyclic nucleotide- and calcium-independent neurofilament protein kinase activity in axoplasm from the squid giant axon

The phosphorylation activity associated with a neurofilament-enriched cytoskeletal preparation isolated from the squid giant axon has been studied and compared to the phosphorylation activities in intact squid axoplasm. The high molecular weight (greater than 300 kDa) and 220-kDa neurofilament proteins are the major endogenous substrates for the kinases in the axoplasm and the neurofilament preparation, whereas 95- and less than 60-kDa proteins are the major phosphoproteins in the ganglion cell preparation. The squid axon neurofilament (SANF) protein kinase activity appeared to be both cAMP and Ca2+ independent and could phosphorylate both casein (Km = 40 microM) and histone (Km = 180 microM). The SANF protein kinase could utilize either ATP or GTP in the phosphotransferase reaction, with a Km for ATP of 58 microM and 129.4 microM for GTP when casein was used as the exogenous substrate; and 25 and 98.1 microM for ATP and GTP, respectively, when the endogenous neurofilament proteins were used as substrates. The SANF protein kinase activity was only slightly inhibited by 2,3-diphosphoglycerate and various polyamines at high concentrations and was poorly inhibited by heparin (34% inhibition at 100 micrograms/ml). The failures of heparin to significantly inhibit and the polyamines to stimulate the SANF protein kinase indicate that it is not a casein type II kinase. The relative efficacy of GTP as a phosphate donor indicates that SANF protein kinase differs from known casein type I kinases. Phosphorylated (32P-labeled) neurofilament proteins were only slightly dephosphorylated in the presence of axoplasm or stellate ganglion cell supernatants, and the neurofilament-enriched preparation did not dephosphorylate 32P-labeled neurofilament proteins. The axoplasm and neurofilament preparations had no detectable protein kinase inhibitor activity, but a strong inhibitor activity, which was not dialyzable but was heat inactivatable, was found in ganglion cells. This inhibitor activity may account for the low phosphorylation activity found in the stellate ganglion cells and may indicate inhibitory regulation of SANF protein kinase activity in the ganglion cell bodies.     

Nuclear protein phosphorylation in isolated nuclei from HeLa cells. Evidence that 32P incorporation from [gamma-32P]GTP is catalyzed by nuclear kinase II

A nuclear system for studying nuclear protein phosphorylation is characterized, using as phosphate donor either low levels of [gamma-32P]GTP, low levels of [gamma-32P]ATP, or low levels of labeled ATP plus excess unlabeled GTP. Since nuclear casein kinase II is the only described nuclear protein kinase to use GTP with high affinity, low levels of GTP should specifically assay this enzyme. ATP should measure all kinases, and ATP plus unlabeled GTP should measure all kinases except nuclear casein kinase II (ATP-specific kinases). The results are consistent with these predictions. In contrast with the ATP-specific activity, endogenous phosphorylation with GTP was enhanced by 100 mM NaCl, inhibited by heparin and quercetin, stimulated by polyamines, and did not use exogenous histone as substrate. The GTP- and ATP-specific kinases phosphorylated different subsets of about 20 endogenous polypeptides each. Addition of purified casein kinase II enhanced the GTP-supported phosphorylation of the identical proteins that were phosphorylated by endogenous kinase. These results support the hypothesis that activity measured with GTP is catalyzed by nuclear casein kinase II, though other minor kinases which can use GTP are not ruled out. Preliminary observations with this system suggest that the major nuclear kinases exist in an inhibited state in nuclei, and that the effects of polyamines on nuclear casein kinase II activity are substrate specific. This nuclear system is used to determine if the C-proteins of hnRNP particles, previously shown to be substrates for nuclear casein kinase II in isolated particles, is phosphorylated by GTP in intact nuclei. The results demonstrate that the C-proteins are effectively phosphorylated by GTP, but in addition they are phosphorylated by ATP-specific kinase activity.     

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.     

Phosphorylation of conserved casein kinase sites regulates cAMP-response element-binding protein DNA binding in Drosophila

The Drosophila homolog of cAMP-response element-binding protein (CREB), dCREB2, exists with serine 231, equivalent to mammalian serine 133, in a predominantly phosphorylated state. Thus, unlike the mammalian protein, the primary regulation of dCREB2 may occur at a different step from serine 231 phosphorylation. Although bacterially expressed dCREB2 bound cAMP-response element sites, protein from Drosophila extracts was unable to do so unless treated with phosphatase. Phosphorylation of recombinant protein by casein kinase (CK) I or II, but not calcium-calmodulin kinase II or protein kinase A, inhibited DNA binding. Up to four conserved CK sites likely to be phosphorylated in vivo were responsible for this effect, and these sites were phosphorylated by a kinase present in Drosophila cell extracts that biochemically resembles CKII. We propose that the relative importance of different signaling pathways in regulating CREB activity may differ between Drosophila and mammals. In Drosophila, the dephosphorylation of CK sites appears to be the major regulatory step, while phosphorylation of serine 231 is necessary but secondary.     

Characterization and androgenic regulation of soluble protein kinases and protein phosphorylation in rat ventral prostate gland

The soluble protein kinase activities for protamine and casein, the histone kinases modulated by cAMP or Ca2+ and phospholipid, as well as the phosphorylation patterns of endogenous proteins were measured in rat ventral prostates from normal adults, castrates, and dihydrotestosterone-treated castrates. In normal prostate, the ratio of cAMP-dependent type I and II kinases was approximately 1:5. After a 3-week period of castration-induced regression, the concentrations of both enzymes were increased, but on a total organ basis, type I was decreased to 56%, while type II was reduced to 20% of normal levels. Casein kinase activity in unfractionated cytosol was not significantly altered by castration but when partially resolved into type I and II enzymes, there appeared to be a selective reduction in the type I component. In contrast, the total organ activities of protamine kinase or Ca2+-activated, phospholipid-dependent kinase, two measures of protein kinase C enzyme, were significantly increased (64 and 71%, respectively) above sham controls in regressed organs of castrates. All of the castration-induced changes in protein kinases were restored toward normal by dihydrotestosterone treatment. Castration effects on protein kinase C and the cAMP-dependent kinases appeared to be manifest in the phosphorylation of endogenous proteins. Castration resulted in a qualitative shift in the cAMP-dependent phosphorylation patterns as measured by gel electrophoresis, with increases in four major bands and decreases in two others, whereas the Ca2+-activated, phospholipid-dependent phosphorylation patterns were all enhanced. It is concluded that the androgenic regulation of protein kinase C differed qualitatively from that of other kinases, and its activation upon withdrawal of the androgenic stimulus may be involved in autophagic mechanisms in the prostate.     

Nuclear protein phosphorylation in isolated nuclei from HeLa cells. Evidence that 32P incorporation from [γ-32P]GTP is catalyzed by nuclear kinase II

A nuclear system for studying nuclear protein phosphorylation is characterized, using as phosphate donor either low levels of [γ-³²P]GTP, low levels of [γ-³²P]ATP, or low levels of labeled ATP plus excess unlabeled GTP. Since nuclear casein kinase II is the only described nuclear protein kinase to use GTP with high affinity, low levels of GTP should specifically assay this enzyme. ATP should measure all kinases, and ATP plus unlabeled GTP should measure all kinases except nuclear casein kinase II (ATP-specific kinases). The results are consistent with these predictions. In contrast with the ATP-specific activity, endogenous phosphorylation with GTP was enhanced by 100 mM NaCl, inhibited by heparin and quercetin, stimulated by polyamines, and did not use exogenous histone as substrate. The GTP- and ATP-specific kinases phosphorylated different subsets of about 20 endogenous polypeptides each. Addition of purified casein kinase II enhanced the GTP-supported phosphorylation of the identical proteins that were phosphorylated by endogenous kinase. These results support the hypothesis that activity measured with GTP is catalyzed by nuclear casein kinase II, though other minor kinases which can use GTP are not ruled out. Preliminary observations with this system suggest that the major nuclear kinases exist in an inhibited state in nuclei, and that the effects of polyamines on nuclear casein kinase II activity are substrate specific. This nuclear system is used to determine if the C-proteins of hnRNP particles, previously shown to be substrates for nuclear casein kinase II in isolated particles, is phosphorylated by GTP in intact nuclei. The results demonstrate that the C-proteins are effectively phosphorylated by GTP, but in addition they are phosphorylated by ATP-specific kinase activity.     

Purification and identification of myosin heavy chain kinase from bovine brain

A high salt extract of bovine brain was found to contain a protein kinase which catalyzed the phosphorylation of heavy chain of brain myosin. The protein kinase, designated as myosin heavy chain kinase, has been purified by column chromatography on phosphocellulose, Sephacryl S-300, and hydroxylapatite. During the purification, the myosin heavy chain kinase was found to co-purify with casein kinase II. Furthermore, upon polyacrylamide gel electrophoresis of the purified enzyme under non-denaturing conditions, both the heavy chain kinase and casein kinase activities were found to comigrate. The purified enzyme phosphorylated casein, phosvitin, troponin T, and isolated 20,000-dalton light chain of gizzard myosin, but not histone or protamine. The kinase did not require Ca2+-calmodulin, or cyclic AMP for activity. Heparin, which is known to be a specific inhibitor of casein kinase II, inhibited the heavy chain kinase activity. These results indicate that the myosin heavy chain kinase is identical to casein kinase II. The myosin heavy chain kinase catalyzed the phosphorylation of the heavy chains in intact brain myosin. The heavy chains in intact gizzard myosin were also phosphorylated, but to a much lesser extent. The heavy chains of skeletal muscle and cardiac muscle myosins were not phosphorylated to an appreciable extent. Although the light chains isolated from brain and gizzard myosins were efficiently phosphorylated by the same enzyme, the rates of phosphorylation of these light chains in the intact myosins were very small. From these results it is suggested that casein kinase II plays a role as a myosin heavy chain kinase for brain myosin rather than as a myosin light chain kinase.     

Phosphorylation of neurofilament H subunit at the tail domain by CDC2 kinase dissociates the association to microtubules

We sought the mammalian neurofilament tail domain-specific kinase. Several well known kinases including cAMP-dependent protein kinase, protein kinase C, Ca(2+)-calmodulin-dependent protein kinase II, casein kinase I, and casein kinase II phosphorylated the high (NF-H) and middle molecular mass subunit (NF-M) of bovine neurofilaments, but they did not reduced the electrophoretic mobility of the dephosphorylated form of NF-M and NF-H by phosphorylation nor was the amount of phosphorylation increased by dephosphorylation of NF proteins, indicating that the phosphorylation sites by these kinases are not major in vivo phosphorylation sites at the tail domain. In contrast, cdc2 kinase phosphorylated specifically the dephosphorylated form of NF-H. 4 mol of phosphates were incorporated per mol of NF-H and this phosphorylation returned the electrophoretic mobility of the dephosphorylated form of NF-H to the position of the isolated, fully phosphorylated form of NF-H. Furthermore, the phosphorylation by cdc2 kinase dissociated the binding of dephosphorylated NF-H to microtubules. Phosphorylation sites were located at the carboxyl-terminal tail domain. The KSPXK motif, but not KSPXX, in the repetitive sequence was suggested to be the phosphorylation site by using synthetic peptides.     

Dietary and hypothyroid hypercholesterolemia induces hepatic apolipoprotein E expression in the rat: direct role of cholesterol

Ser-473 is solely phosphorylated in vivo in the tail region of neurofilament L (NF-L). With peptides including the native phosphorylation site, it was not possible to locate responsible kinases. We therefore adopted full-length dephosphorylated NF-L as the substrate, and employed MALDI/TOF (matrix-assisted laser desorption and ionization/time of flight) mass spectrometry and a site-specific phosphorylation-dependent antibody recognizing Ser-473 phosphorylation. The antibody showed that casein kinase I (CK I) as well as casein kinase II (CK II) phosphorylated Ser-473 in vitro, while neither GSK-3beta nor calcium/calmodulin-dependent protein kinase II did so. However, the mass spectra of the tail fragments of the phosphorylated NF-L indicated that CK II was the kinase mediating Ser-473 phosphorylation in vitro as opposed to CK I, because CK I phosphorylated another site as well as Ser-473 in vitro. The antibody also demonstrated that NF-L phosphorylated at Ser-473 was abundant in the neuronal perikarya of the rat cortex, indicating that phosphorylation of Ser-473 may take place there. This result may support the suggestion that CK II is the kinase responsible for Ser-473 phosphorylation. Despite many reports showing that CK I mediates phosphorylation of neurofilaments, CK II may phosphorylate NF-L in vivo.     

Saccharomyces cerevisiae protein kinase dependent on Ca2+ and calmodulin.

A Ca2+- and calmodulin (CaM)-dependent protein kinase of Saccharomyces cerevisiae was partially purified by CaM affinity chromatography of the soluble fraction, and the properties of the enzyme were investigated. The protein kinase activity of the affinity-purified preparation was stimulated at least eightfold by the simultaneous presence of Ca2+ and CaM. The enzyme stimulation was strongly inhibited by trifluoperazine (TFP), a CaM antagonist. When the kinase was incubated in the presence of ATP, Ca2+, and CaM before the assay, the enzyme showed activity even in the presence of the Ca2+ chelator ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) and TFP. The conversion to this Ca2+- and CaM-independent form occurred very rapidly under the incubation conditions required for protein phosphorylation by the kinase. At the highest level of conversion, Ca2+- and CaM-independent kinase activity, which was measured in the presence of EGTA and TFP, was nearly equal to the total kinase activity, which was measured in the presence of Ca2+ and CaM. A protein with a molecular weight of 58,000 was the major species that was phosphorylated in a Ca2+- and CaM-dependent manner by incubation of the CaM affinity-purified proteins with [gamma-32P]ATP. The protein kinase activity of the protein with the same molecular weight was demonstrated by in situ protein phosphorylation in sodium dodecyl sulfate-polyacrylamide gels by using casein as the substrate, after removal of the detergent from electrophoresed CaM-binding proteins. These data indicate that phosphorylation of the kinase is responsible for the conversion of enzyme activity. Enzyme regulation by this mode may play an important role in integrating cellular functions during the cell cycle. A possible role for the Ca2+-and CaM-dependent protein kinase in the signal transduction of the mating pheromone alpha factor is also discussed.