Protein kinases of the chick oviduct: a study of the cytoplasmic and nuclear enzymes.

Subcellular fractionation of oviduct tissue from estrogen-treated chicks indicated that the bulk of the protein kinase activity of this tissue is located in the cytoplasmic and nuclear fractions, DEAE-cellulose chromatography of cytosol revealed a major peak of cAMP stimulatable activity eluting at 0.2 M KCl. This peak was further characterized and found to exhibit properties consistent with cytoplasmic cAMP dependent protein kinases isolated from other tissues; it had a Km for ATP of 2 X 10(-5) M, preferred basic proteins such as histones, as substrate, and had a M of 165 000. Addition of 10(-6) M cAMP caused the holoenzyme to dissociate into cAMP binding regulatory subunit and a protein kinase catalytic subunit. Extraction of purified oviduct nuclei with 0.3 M KCl released greater than 80% of the kinase activity in this fraction. Upon elution from phospho-cellulose, the nuclear extract was resolved into two equal peaks of kinase activity (designated I and II). Peak I had a sedimentation coefficient of 3S and a Km for ATP of 13 muM. while peak II had a sedimentation coefficient of 6S and a Km for ATP of 9 muM. Both enzymes preferred alpha-casein as a substrate over phosvitin or whole histone, although they exhibited different salt-activity profiles. The cytoplasmic and nuclear enzymes were well separated on phospho-cellulose and this resin was used to quantitate the amount of cAMP dependent histone kinase activity in the nucleus and the amount of casein kinase activity in the cytosol. Protein kinase activity in nuclei from estrogen-stimulated chicks was found to be 40% greater than hormone-withdrawn animals. This increase in activity was not due to translocation of the cytoplasmic protein kinase in response to hormone, but to an increase in nuclear (casein) kinase activity. During the course of this work, we observed small but significant amounts of cAMP binding activity very tightly bound to the nuclear fraction. Solubilization of the binding activity by sonication in high salt allowed comparison studies to be performed which indicated that the nuclear binding protein is identical with the cytoplasmic cAMP binding regulatory subunit. The possible role of the nuclear binding activity is discussed.     

Casein kinases I and II bound to pig brain microtubules

1. Microtubules prepared from pig brain by two cycles of assembly-disassembly comprise cyclic nucleotide-independent protein kinase activity with phosvitin and troponin T as substrates. 2. Phosphocellulose chromatography resolved two phosvitin kinase activity peaks, one of which coincided with the troponin T kinase peak. 3. The activity peak corresponding to troponin T kinase was inhibited by heparin (I50 = 0.06 micrograms/ml), whereas the other phosvitin kinase peak was unaffected. 4. Both kinase fractions phosphorylated tubulin and microtubule-associated protein (MAP-2). 5. It is concluded that pig brain microtubules contain bound casein kinases I and II. The association may target the action of these kinases toward microtubular proteins in vivo.     

Purification and characterization of a phosvitin kinase from the thyroid gland

1. Two cyclic AMP independent protein kinases phosphorylating preferentially acidic substrates have been identified in soluble extract from human, rat and pig thyroid glands. Both enzymes were retained on DEAE-cellulose. The first enzyme activity eluted between 60 and 100 mM phosphate (depending on the species), phosphorylated both casein and phosvitin and was retained on phosphocellulose; this enzyme likely corresponds to a casein kinase already described in many tissues. The second enzyme activity eluted from DEAE-cellulose at phosphate concentrations higher than 300 mM, phosphorylated only phosvitin and was not retained on phosphocellulose. These enzymes were neither stimulated by cyclic AMP, cyclic GMP and calcium, nor inhibited by the inhibitor of the cyclic AMP dependent protein kinases. 2. The second enzyme activity was purified from pig thyroid gland by the association of affinity chromatography on insolubilized phosvitin and DEAE-cellulose chromatography. Its specific activity was increased by 8400. 3. The purified enzyme (phosvitin kinase) was analyzed for biochemical and enzymatic properties. Phosvitin kinase phosphorylated phosvitin with an apparent Km of 100 micrograms/ml; casein, histone, protamine and bovine serum albumin were not phosphorylated. The enzyme utilized ATP as well as GTP as phosphate donor with an apparent Km of 25 and 28 microM, respectively. It had an absolute requirement for Mg2+ with a maximal activity at 4 mM and exhibited an optimal activity at pH 7.0. The molecular weight of the native enzyme was 110 000 as determined by Sephacryl S300 gel filtration. The analysis by SDS-polyacrylamide gel electrophoresis revealed a major band with a molecular weight of 35000 suggesting a polymeric structure of the enzyme.     

Purification and characterization fo a phosvitin kinase from the thyroid gland

1. Two cyclic AMP independent protein kinases phosphorylating preferentially acidic substrates have been identified in soluble extract from human, rat and pig thyroid glands/ Both enzymes were retained on DEAE-cellulose. The first enzyme activity eluted between 60 and 100 mM phosphate (depending on the species), phosphorylated both casein and phosvitin and was retained on phosphocellulose; this enzyme likely corresponds to a casein kinase already described in many tissues. The second enzyme activity eluted from DEAE-cellulose at phosphate concentrations higher than 3000 mM, phosphorylated only phosvitin and was not retained on phophocellulose. These enzymes were neither stimulated by cyclic AMP, cyclic GMP and calcium, nor inhbiited by the inhibitor of the cyclic AMP dependent protein kinases. 2. The second enzyme activity was purified from pig thyroid gland by the association of affinity chromatography on insolubilized phosvitin and DEAE-cellulose chromatography. Its specific activity was increased by 8400. 3. The purified enzyme (phosvitin kinase) was analyzed for biochemical and enzymatic properties. Phosvitin kinase phosphorylated phosvitin with an apparent K_m of 100 μg/ml; casein, histone, protamine and bovine serum albumin were not phosphorylated. The enzyme utilized ATP as well as GTP as phosphate donor with an apparent K_m of 25 and 28 μM, respectively. It had an absolute requirement for Mg²⁺ with a maximal activity at 4 mM and exhibited an optimal activity at pH 7.0. The molecular weight of the native enzyme was 110 000 as determined by Sephacryl S300 gel filtration. The analysis by SDS-polyacrylamide gel electrophoresis revealed a major band with a molecualr weight of 35 000 suggesting a polymeric structure of the enzyme.     

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.     

Purification and characterization of two casein kinases from ejaculated bovine spermatozoa.

Two protein kinases active on casein and phosvitin were partially purified from the soluble fraction of ejaculated bovine spermatozoa. They were operationally termed casein kinase A and B based on the order of their elution from a phosphocellulose column. CK-A showed an approximate molecular mass of 38 kDa, and it phosphorylated serine residues of casein and phosvitin utilizing ATP as a phosphate donor (Km 19 microM). Enzyme activity was maximal in the presence of 10 mM MgCl2, whereas it decreased in the presence of spermine, polylysine, quercetin, and NaCl (20-250 mM). CK-B seemed to have a monomeric structure of about 41 kDa; it underwent autophosphorylation and cross-reacted with polyclonal antibodies raised against recombinant alpha, but not beta, subunit of human type 2 casein kinase. It phosphorylated both serine and threonine residues of casein and phosvitin, utilizing ATP (Km 12 microM) but not GTP as a phosphate donor. Threonine was more affected in the phosphorylated phosvitin than in the partially dephosphorylated substrate. CK-B was active toward the synthetic peptide Ser-(Glu)5 and calmodulin (in the latter case, in the presence of polylysine), and it was activated by spermine, polylysine, MgCl2 (30 mM), and NaCl (20-400 mM). The activity of the enzymes was not affected by cAMP, or the heat-stable inhibitor of the cAMP-dependent protein kinase, or calcium.     

Protein kinases in rat testes: evidence for different fractions of the enzyme.

Protein kinase activity of rat testis homogenate was separated into five fractions by means of pH 4.8 acidification and DEAE-cellulose chromatography. The five fractions showed a peculiar pattern of activity and cAMP dependency with the substrates used: casein, protamine, histone mixture, arginine-rich histone, lysine-rich histone, and phosvitin. The casein-sepharose substrate affinity column separated two fractions from the pH 4.8 precipitate. Peak number one phosphorylates histone preferently and is cAMP-dependent, while peak number tow has a strong affinity toward casein as substrate and is non cAMP-dependent.     

Membrane-associated protein kinase activities in seminal plasma from vasectomized men

Differential centrifugation was used to prepare heavy and light membrane fractions from the seminal plasma of vasectomized men. The two membrane fractions combined contained half of the phosvitin and histone kinase activities but only 7% of the total protein content in vasectomy semen. These two kinase activities as well as phosphorylation of endogenous membrane proteins were optimally stimulated by Mg2+; Mn2+ could effectively substitute for Mg2+ only in endogenous phosphorylation reactions. Neither the phosvitin nor histone kinase responded to cAMP or cGMP, but the histone kinase was strongly inhibited by the heat-stable cAMP-dependent protein kinase inhibitor. The phosvitin kinase was not affected by this inhibitor. The phosphorylation of endogenous proteins in the heavy membrane fraction was not affected by the protein kinase inhibitor but protein phosphorylation in the light membrane fraction was partly (45%) inhibited. The differential effects of increased ionic strength, sulphydryl protecting agents, and the protein kinase inhibitor on protein kinase activity towards lysine-rich histones, phosvitin and endogenous proteins, as well as differential extractability and binding to an anion exchange column of histone kinase and phosvitin kinase activities, indicate that more than one kinase activity is present in these membrane subfractions. Electron microscopic examination showed that there are several kinds of membrane-limited components in vasectomy seminal fluid that vary in size, density, and ultrastructure. The association of type(s) of protein kinase to individual membrane components remains to be established.     

Purification and properties of cAMP independent glycogen synthase kinase and phosvitin kinase from human leukocytes

Summary cAMP independent glycogen synthase kinase and phosvitin kinase activity was purified from the 180 000 × g supernatant of human polymorphonuclear leukocytes by ammonium sulphate precipitation and phosphocellulose chromatography. The cAMP independent glycogen synthase kinase eluted from the phosphocellulose at 0.54 m NaCl (peak A) separate from the major phosvitin kinase eluting at 0.68 m NaCl (peak B). The kinase activity of both peaks tended to form aggregates, but in the presence of 0.6 m NaCl, the peak B enzyme had M_r 250 000, 7.2S and the peak A enzyme M_r 38 000, 3.8S. The ratio between synthase kinase and phosvitin kinase activity in peak A was 1:3.2 and in peak B 1:31.4. In addition the kinase activities differed with respect to sensitivity to temperature, ionic strength and CaCl₂. It is suggested that the peak A enzyme represents the cAMP independent glycogen synthase kinase of leukocytes, whereas the peak B enzyme is a phosvitin kinase, which is insignificantly contaminated with some synthase kinase (peak A) and contains a separate, second synthase kinase.Synthase kinase had K _m ^app 4.2 m for muscle glycogen synthease I and K _m ^app 45 m for ATP. GTP was a poor substrate. The activity was not influenced by cyclic nucleotides, Ca²⁺, or glucose-6-P. Synthase I from muscle and leukocytes was phosphorylated to a ratio of independence of less than 0.05.Abbreviations cAMP adenosine cyclic 3:5-monophosphate - DTT dithiothreitol - EGTA ethylene glycol-bis-(-amino-ethylether)-N,N-tetraacetic acid - PMSF phenylmethylsulfonylfluoride - PKI protein kinase inhibitor - RI ratio of independence for glycogen synthase - SDS sodium dodecyl sulphate     

Adenosine 3′:5′-cyclic phosphate-dependent and -independent protein kinases of renal brush border membranes: Solubilization,separation, and characterization of multiple forms

Multiple protein kinase activities were found in the luminal segment of the renal proximal tubule cell plasma membrane (brush border membrane). Membranes were extracted with Lubrol, with no loss in activity, and the extract was chromatographed on diethylaminoethyl cellulose with a salt gradient. With protamine as substrate, activity eluted in two peaks, designated I and IIb, and was cyclic AMP independent. With histone VII-S, one peak, designated IIa, appeared, which eluted slightly ahead of IIb and was cyclic AMP dependent. The three activities eluted in their original patterns following rechromatography. Histone kinase activity in the combined IIa+b fraction was stimulated threefold by cyclic nucleotides (K_a = 0.013 and 0.94 μM for cyclic AMP and cyclic GMP, respectively) by increasing V. Cyclic AMP binding activity eluted with histone kinase activity. Rechromatography of IIa+b on diethylaminoethyl cellulose containing 1 μm cyclic AMP resulted in passage through the column of most of the histone kinase activity (IIa) prior to the salt gradient, but retention of kinase IIb, which again eluted in its original position. Characterization of the separated enzymes revealed that kinase I was highly specific for protamine and totally insensitive to cyclic AMP and a specific protein inhibitor of cyclic AMP-dependent kinases. Kinase IIa was relatively specific for histones and was completely inhibited by the protein inhibitor. Kinase IIb was nonspecific, catalyzing phosphorylation of protamine, casein, histones, and phosvitin in decreasing order of activity, and was insensitive to cyclic AMP and the protein inhibitor. Exposure of intact brush border membranes to elevated temperatures revealed that phosphorylation of intrinsic membrane proteins and protamine was thermolabile, whereas cyclic AMP-dependent histone kinase activity was relatively thermostable. These findings implicate cyclic AMP-independent protamine kinases in the cyclic AMP-independent autophosphorylation of the brush border membrane.     

Protein Kinase Activities in Neurospora crassa

Several protein kinase activities have been found in 105,000g supernatant of Neurospora crassa mycelia grown up to the logarithmic phase. By chromatography on DEAE-cellulose the following enzyme activities have been resolved: (i) a cyclic AMP-dependent protein kinase (peak I kinase) eluting at 0.20 m NaCl, more active with histone than with phosvitin (it was inhibited by both a thermolabile fraction having cyclic AMP-binding activity and a thermostable inhibitor isolated from 105,0005g mycelial supernates), (ii) a cyclic nucleotide-independent protein kinase (peak II kinase) eluting at 0.35 m NaCl, also more active with histone than with phosvitin (this kinase was not inhibited by the fraction having cyclic AMP-binding activity but it was sensitive to the thermostable inhibitor); and finally, (iii) a protein kinase eluting at 0.43 m NaCl (peak II kinase), with similar activity toward histone and phosvitin, insensitive to cyclic nucleotides and to fractions carrying cyclic AMP-binding capacity (this kinase activity also resulted insensitive to the thermostable inhibiting factor).     

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

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

Soluble protein kinase fractions from DEAF-cellulose chromatography

Summary The cytosol fraction from rat midbrain was chromatographed on DEAE-cellulose with a linear NaCl gradient (0–0.3 M). Two peaks of protein kinase activity were obtained when assayed with either historic or casein. A similar elution profile of the kinase activity was obtained from rat heart. The first peaks from midbrain and heart were compared in terms of their dependency upon cAMP and sensitivity to the endogenous protein kinase inhibitor. Neither of the two substances had an effect on the activity of the brain kinase. Furthermore, the dissociability of the midbrain and heart enzymes in the presence of cAMP or histone was compared by DEAE-cellulose chromatography. The heart enzyme was dissociated into a catalytic subunit characteristic of a cAMP-dependent protein kinase, whereas the brain kinase was totally unaffected by the cAMP or histone. The results of these tests indicate that although the elution profiles from DEAE-cellulose are similar between midbrain and heart, the first peak from brain contains a protein kinase that appears to be cAMP independent.     

Studies on protein kinases in two human rectocolic cell lines by polyacrylamide gel electrophoresis.Effect of the vasoactive intestinal peptide

Electrophoresis and subsequent assay of the enzyme directly onto the gel has allowed a rapid and quantitative characterization of the cyclic AMP-dependent and -independent histone kinases, protamine, phosvitin and casein kinases in HT 29 and HRT 18 cells. The technique has been applied to soluble extracts from cytoplasmic and nuclear fraction prepared in the presence and absence of neutral detergent. A more precise identification of these enzymes has been possible by analysing enzyme fractions obtained after ion-exchange chromatography of the above extracts. The protein kinase equipment of both cell lines was found to be identical (11 major components) but with different relative proportions of several enzymes. In cytoplasmic extracts: VIP activates only the type I, cytosolic, (band 4) and the type II, membrane-bound, (bands 6 and 8) cyclic AMP-dependent histone kinases. These enzymes account, respectively, for 34 and 55% of the total histone kinases in HT 29 and HRT 18 cells. The cyclic AMP-independent histone kinases (band 1,2,5 and 7) also phosphorylate protamine; band 5 was found 3o be much higher (4-fold) in HT 29 cells. In addition, two casein/phosvitin kinases have been identified in both cell lines with phosphorylating activity similar to the total histone kinases. In the nuclear extract two cyclic AMP-independent histone kinases have been found with electrophoretic mobility differing from the cytoplasmic enzymes. Also, two phosvitin/casein kinases specifically nuclear, due to their chromatographical and electrophoretical behaviour, have been characterized.     

The distribution and dissociation of cyclic adenosine 3':5'-monophosphate-dependent protein kinases in adipose, cardiac, and other tissues.

In crude extracts of adipose tissue the protein kinase dissociates slowly at 30 degrees into regulatory and catalytic subunits in the presence of 700 mug per ml of histone or 0.5 M NaCl. If the kinase is first dissociated by adding 10 muM adenosine 3':5'-monophosphate (cAMP), reassociation occurs instantaneously after removal of the cAMP by Sephadex G-25 chromatography. In contrast, in crude xtracts of heart, the protein kinase dissociates rapidly in the presence of 700 mug per ml of histone or 0.5 M NaCl and reassociates slowly after removal of cAMP. These differences are accounted for by the existence of two types of protein kinases in these tissues, referred to as types I and II. DEAE-cellulose chromatography of extracts of adipose tissue produces only one peak of cAMP-dependent protein kinase activity (type II) which elutes between 0.15 and 0.25 M NaCl. Similar chromatography of heart extracts resolves enzyme activity into two peaks; a type I enzyme which elutes between 0.05 and 0.1 M and predominates (greater than 75% of total activity), and a type II enzyme which elutes between 0.15 and 0.25 M NaCl. The dissociation properties of the types I and II enzymes from heart and adipose tissue are retained after partial purification by DEAE-cellulose and Sepharose 6B chromatography. Rechromatography of the separated peaks of the cardiac enzymes does not change the elution pattern. Sucrose density gradient centrifugation and gel filtration studies indicate that the molecular weights of these enzymes are very similar. The type II enzyme isolated by DEAE-cellulose chromatography of heart extracts resembles the adipose tissue enzyme, i.e. it undergoes slow dissociation at 30 degrees in the presence of histone or 0.5 M NaCl. The adipose tissue kinase and the heart type II kinase are not identical, however, since they do not elute at exactly the same point on DEAE-cellulose columns. A survey of several tissues indicates the presence of type I and II protein kinases similar to the enzymes in adipose tissue and heart as determined by DEAE-cellulose chromatography of crude extracts and by dissociation of the enzymes with histone. The presence of MgATP prevents dissociation of type I enzyme from heart by 0.5 M NaCl or histone. The profile of the enzyme on DEAE-cellulose, however, is not changed...     

Protein kinases derived from the conditioned media of human peripheral blood mononuclear cells

Two classes of cyclic nucleotide-independent protein kinase from the conditioned media of human peripheral blood mononuclear cells were detected. The first one was specific for histone, and was not retained by the remazol blue-agarose column. The second one was specific for casein and phosvitin, and was retained by the remazol blue-agarose column. Histone kinase activity was elevated in Con A-conditioned media. These peripheral blood mononuclear cells were subsequently fractionated into adherent and nonadherent cell populations. It was clear that histone kinase was secreted by adherent cells while casein and phosvitin kinases were secreted by nonadherent cells.     

Effects of polyamines and polyanions on a cyclic nucleotide-independent and a cyclic AMP-dependent

The phosphorylation of phosvitin in vitro by a cyclic nucleotide-independent protein kinase (phosvitin kinase) derived from rooster liver is markedly stimulated by the divalent cation, Mg²⁺. In addition, the activity is further stimulated by low concentrations of the polyamines putrescine, spermidine and spermine leading to higher rates of phosphate incorporation than could be obtained at any concentration of Mg²⁺. Spermine is inhibitory at higher concentrations. The polyamines shift the Mg²⁺ requirement for maximal activity to lower concentrations. The activity of a cyclic AMP-dependent histone kinase from beef heart is not altered by the presence of polyamines. Heparin is a potent inhibitor of phosvitin kinase but has no effect on histone kinase. Polyribonucleotides (polyadenylic acid and transfer RNA) inhibit both types of kinases, but the degree of inhibition of phosvitin kinase is variable and depends upon the type of the polyanion present. Spermidine and spermine, but not Mg²⁺, efficiently counteract the inhibitory action of heparin and tRNA. The results suggest that, also in vivo, naturally occurring polyamines and polyanions such ass tRNA may have a regulatory function on protein kinases.     

Purification and properties of cAMP dependent and independent histone kinases from human leukocytes

Summary Histone kinase activity was purified from human polymorphonuclear leukocytes by ammonium sulphate precipitation of a 180 000 × g supernatant, followed by DEAF-cellulose chromatography and gelfiltration. On DEAE-cellulose cAMP dependent kinase activity eluted in two peaks, I and III, at 1.2 mmho and 6.5 mmho, respectively. Catalytic subunit (C) from both peaks had M_r 33 000, 3.0S. Regulatory subunit (R) from peak I and III both had M_r 33 000 upon gelfiltration, but sedimented at 2.8–3.0S and 3.0–3.2S, respectively. R₂ and R₄ subunits were identified. The R-C dimer from peak I and III sedimented at 4.8S and (4.8)–5.1S, respectively. The holoenzyme from peak I had M_r 165 000, 6.7S, which suggest a R₂C₂ structure, while that of peak III sedimented at 6.7S, but eluted at M_r 330 000 (2R₂C₂) by gelfiltration.The K _m ^app for peak I and III enzymes were, respectively: histone IIA 0.5 mg/ml (both forms), ATP 18 m and 23 m, and cAMP 5 × 10^–8 m and 6.3 × 10^–8 m. Both enzymes had pH optimum 6.7–6.9 and were equally sensitive to Ca²⁺ temperature and protein kinase inhibitor. The substrate specificity was histone VS histone IIA = histone VIS casein > phosvitin. Peak I enzyme, but not peak III enzyme, was dissociated by histone and high ionic strength and reassociation of R and C subunits were facilitated by ATP-Mg. It is concluded that peak I and III enzymes represent type I and II cAMP dependent protein kinases, respectively. Type I comprises 20–30% of cAMP dependent protein kinase activity and is absent from the 180 000 × g supernatant of gently disrupted cells.Purified catalytic subunit had K _m ^app (ATP) 20 m with rabbit muscle glycogen synthase I as substrates. Synthase I from rabbit muscle and human leukocytes were phosphorylated by catalytic subunit to synthase D (ratio of independence less than 0.07).cAMP independent histone kinase activity eluted in one peak (Peak II) at 3 mmho. The enzymatic activity sedimented at 3.4S and eluted from gelfiltration with M_r 78 000. K _m ^app for ATP was 78 m and for histone IIA 0.5 mg/ml. The enzyme was sensitive to temperature, but less sensitive than cAMP dependent protein kinase to Ca²⁺, and insensitive to protein kinase inhibitor. The substrate specificity was histone IIA > histone VS = histone VIS, while casein and phosvitin were poor substrates. Glycogen synthase I was not phosphorylated. The cAMP independent histone kinase activity comprised 15% of the total histone kinase activity in a crude homogenate of leukocytes. Its physiological substrate is unknown.Abbreviations AR activity ratio for cAMP dependent protein kinase - cAMP adenosine cyclic 3:5-monophosphate - cIMP inosine cyclic 3:5-monophosphate - cGMP guanosine cyclic 3:5-monophosphate - Glucose-6-P glucose-6-phosphate - DDT dithiothreitol - EGTA ethylene glycol-bis-(-aminoethylether)-N, N-tetraacetic acid - PMSF phenylmethylsulfonylfluoride - PKI protein kinase inhibitor - RI ratio of independence for glycogen synthase - SDS sodium dodecyl sulphate     

Characterization of microtubule-associated protein 1-associated protein kinases from rat brain

The microtubule-associated protein (MAP) 1 preparation, MAP1A and 1B, obtained from rat brain microtubules was associated with protein kinases that were insensitive to cAMP, cGMP, calcium, calcium/calmodulin and calcium/phosphatidylserine. The fractionation of highly purified MAP1 by phosphocellulose chromatography revealed that protein kinase activity to phosphorylate phosvitin was separated into three major peaks (MAP1 kinases A, B and C). MAP1 was recovered in the MAP1 kinase A fraction and phosphorylated by the contained kinase. MAP1 kinase A is a novel protein kinase that is remarkably activated by poly- -lysine and poly- -arginine, but very insensitive to heparin among the kinases. Photoaffinity labeling using [-³²P]8-azido ATP indicated that the e65 kDa polypeptide is identified as an ATP-binding protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the highly purified MAP1 and MAP1 kinase A fractions. MAP1 kinases B and C may be identified as casein kinase I- and II-like kinases. The present results show that MAP1 is associated with at least three kinases and provide an insight for understanding thoroughly the MAP1-mediated microtubule functions.