[46] Cytoplasmic hepatic protein kinases

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HEPATIC PROTEIN KINASES

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C y t o p l a s m i c H e p a t i c Protein Kinases

B y LEE-JINO CHEN and DONAL A. WALSH1

Studies of the protein kinase activity of liver have been approached from several points of view and in consequence have led to the description of several potentially different~ protein kinase activities. However, comparative and detailed data on the various protein kinases is still sparse, and so it is not possible to clearly identify each of the activities as separate molecular entities. In particular, the exact correlation between the nuclear and cytoplasmic activities is uncertain. Properties of some of the former are described by Kish and Kleinsmith? This presentation is restricted to the enzymes that are found principally in the cytoplasm.

I. Cytosol Cyclic AMP-Regulated Protein Kinase

Mechanism of Cyclic AMP Action Cyclic AMP (cAMP) activates cAMP-dependent protein kinase according to the mechanism delineated for the enzyme from many tissues, Eq. (1), and described elsewhere in this volume? RC (inactive) -4- cAMP ~- R • cAMP + C (active)

(1)

In consequence, this discussion of cAMP-dependent protein kinase includes not only the properties of the holoenzyme (RC) but also of the active catalytic fraction (C) and of the cAMP binding unit (R).

Assay Method The procedure is essentially that of Reimann et al? for rabbit muscle protein kinase with a slight modification. Reagents

Assay buffer: 50 mM 2-(N-morpholino)ethanosulfonate, 50 mM magnesium chloride, 50 mM sodium chloride, 10 mM aminophylline, pH 5.9 1Established Investigator of American Heart Association. 2V. Kish and L. Kleinsmith, J. Biol. Chem. g49, 750 (1974) ; this series Vol. 40, [16]. 3j. A. Beavo, P. J. Bechtel, and E. G. Krebs, this volume [43]. "E. M. Reimann, D. A. Walsh, and E. G. Krebs, J. Biol. Chem. 9.46, 1986 (1971).

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Histone f2b, prepared by the methods of Johns, ~ 1 mg/ml [V-32P]Adenosine triphosphate, 10 raM, specific activity 4-9 X 109 cpm/mmole, pH 7.0 cAMP, 10 ~M (when added) Procedure. A cocktail is prepared fresh before each assay containing equal amounts of the above reagents. An aliquot of 100 tL1 is pipetted to a test tube (12 X 75 mm). The reaction is started by adding 20 td of enzyme preparation and incubated for 25 minutes at 30 ° in a water bath. The reaction is terminated by removing 50 ~l of the mixture with an Eppendorf pipette onto a filter paper (2.0 X 2.0 cm of Whatman No. 3lET). The filter paper is dropped immediately into cold 10% TCA solution with a ratio of one paper per 10 ml of TCA solution. The paper is washed in cold 10% TCA for 30 minutes, cold 5% TCA for 30 minutes, and twice in 5% TCA for 30 minutes at room temperature. The paper is washed with 95% ethanol (enough to cover the papers) 5 minutes and rinsed with ether, dried, and transferred to a toluene-based scintillation fluid for counting. Purification

A. Purification of Holoenzyme 6 Procedure 1 Step 1. Preparation of Tissue Extract. All purification procedures are carried out at 4 ° unless otherwise stated. Minced rat liver (4 g) is homogenized with 20 ml of 0.25 M sucrose containing 5 mM Tris" C1 (pH 7.5), 1 mM EDTA, and 10 mM fl-mercaptoethanol, utilizing a PotterElvehjem homogenizer with a Teflon pestle. The homogenate is centrifuged at 105,000 g for 35 minutes and the resultant supernatant solution is passed through two layers of cheesecloth to remove lipid. Step 2. Fractionation on DEAE-Sephadex A-25. The filtrate (12 ml) is applied to a 1.8 X 22 cm column of DEAE-Sephadex A-25, which has been previously equilibrated with 5 mM Tris, C1 buffer (pH 7.5), containing 1 mM EDTA, and 10 mM fl-mercaptoethanol (TME buffer). The column is eluted with a linear gradient of NaC1 in the same buffer. The reservoir contains 50 ml of 0.5 M NaCl in T M E buffer, and the mixing flask contains 50 ml of the same buffer without NaCl. Fractions of 2 ml are collected at a rate of 1 ml per minute. Three enzymatically active fractions are resolved by this procedure. The activity of fraction I, eluted between 0 and 0.06 M NaC1, is not stimulated by cAMP, and is free E. W. Johns, Biochem. J. 9, 55 (1964). L. J. Chert and D. A. Walsh, Biochemistry 10, 3614 (1972).

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catalytic subunit. Fractions II and III are eluted between 0.08 and 0.25 M, and between 0.27 and 0.36 M NaCl, respectively. The activities of each of these two fractions are dependent on cAMP. Fraction II consisted of more than a single enzymatic component (see below). Step 3. Isoelectro]ocusing Electrophoresis. Technique of isoeleetrofocusing electrophoresis is performed according to the method originally described by Svensson 7 using a ll0-ml column (LKB Instruments, Inc.) maintained at 0 ° by a circulating water bath. A 2% carrier ampholyte with a pH range of 5 to 8 is used. The pH gradient is.established during electrophoresis following the sequential addition of ampholyte solution in a 0 to 47% (w/v) sucrose gradient. Fractions II and III separated by chromatography on the DEAE-Sephadex A-25 column are each dialyzed against i0 volumes of T M E buffer with two changes for 3 hours. The dialyzed solution (II or III) is applied in the center of the column. Electrophoresis is initiated at 200 V, increased to 800 V over a period of 24 hours, and continued at this voltage for at least another 16 hours for equilibration. Upon completion of the electrophoresis, fractions of 2 ml are collected and assayed for protein kinase activity and pH determination. Fractions II and III are each eluted as single peaks of enzymatic activity at pH of 5.2. They may be stored at 4 ° for a few days following dialysis against 0.25 M sucrose in T M E buffer.

Procedure 2 The method described here is an alternative method originally described by Kumon et al2 and Yamamura et alY Step 1. Tissue Extract. The rat liver (12 g) is homogenized with 5 volumes of 0.25 M sucrose containing 6 mM fl-mercaptoethanol and 3.3 mM CaCl~ utilizing a Potter-Elvehjem homogenizer. Diisopropyl fluorophosphate, suspended in 5 volumes of isopropanol, is added to the homogenate to give a final concentration of 10 mM, and the mixture is stirred for 20 minutes at 0 °. The solution is then centrifuged at 20,000 g for 20 minutes. The supernatant (50 ml) is brought to 70% saturation with 23.6 g of ammonium sulfate. The precipitate is collected by centrifugation and dissolved in 20 ml of 20 mM Tris.Cl buffer, pH 7.5, containing 6 mM fl-mercaptoethanol. The solution is dialyzed against 2 liters of the same buffer for 15 hours with two changes. Step 2. DEAE-Sephadex A-50 Chromatography. The dialyzate is apH. Svensson, Acta Chem. Scand. 16, 456 (1962). A. Kumon, H. Yamamura, and Y. Nishizuka, Biochem. Biophys. Res. Commun. 41, 1290 (1970). H. Yamamura, Y. Inoue, R. Shimomura, and Y. Nishizuka, Biochem. Biophys. Re~. Commun. 46, 589 (1972).

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plied to a DEAE-Sephadex A-50 column (2 X 35 cm) that is equilibrated with 20 mM Tris. C1, pH 7.5, containing 50 mM NaC1 and 6 mM fl-mercaptoethanol. After the column is washed with 250 ml of the same buffer, enzyme elution is carried out with a linear concentration gradient of NaC1. The mixing flask and reservoir each contains 200 ml of 50 mM and 0.6 M NaC1, respectively, in 20 mM Tris.C1, pH 7.5, containing 6 mM fl-mereaptoethanol. Fractions of 5 ml are collected. Three fractions with protein kinase activities are resolved by this procedure. Fraction A, eluted between fractions 21 and 36, fraction B eluted between fractions 40 and 54, the activity of each was stimulated by cAMP. Fraction C, eluted between tubes 56 and 70, is not stimulated by cAMP and is more active with casein or phosvitin as substrates than with histone. The last enzyme is possibly the same species as phosvitin kinase which is described in the following section. Step 3. Hydroxyapatite Chromatography. Each of the first two fractions (A and B) is further purified on a column of hydroxyapatite (2 X 5 cm). The column is equilibrated with 30 mM potassium phosphate, pH 7.5, containing 6 mM fl-mercaptoethanol. After the column is washed with 100 ml of the same buffer, elution is carried out with a linear gradient of potassium phosphate. The mixing chamber contains 200 ml of 50 mM potassium phosphate, pH 7.5, in 6 m M fl-mercaptoethanol, and the reservoir contains 0.25 M potassium phosphate, pH 7.5, and in 6 mM fl-mercaptoethanol. Fractions of 4 ml are collected. Fraction A is resolved into two enzymatically active fractions. The activity in the first, peak (tubes 20 through 45) is cAMP dependent, whereas the second peak (tubes 46 through 50) is cAMP independent. The chromatogram of fraction B exhibits a broad peak (tubes 51-75) which does not correspond to either peak of fraction I. The identity of these fractions with those prepared by procedure 1 has not been determined.

B. Preparation o] Catalytic Subunit Modification of procedure 1 for the preparation of holoenzyme can yield 3 catalytic subunits that are distinct on the basis of isoelectric point. Fraction I obtained from chromatography on DEAE-Sephadex A-25 has an isoelectric point of 8.2. Isoelectrofocusing electrophoresis of fraction II, after preincubation with cAMP, yields two catalytic subunits (IIA and IIB) of pI 7.6 and &5, respectively. These various forms of catalytic subunits (I, IIA and IIB) can be identified as such on the basis of interaction with regulatory subunit or the heat stable inhibitor protein, l° ~J. A. Traugh, C. D. Ashby, and D. A. Walsh, this volume [42].

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C. Preparation o] Regulatory Subunit Several methods are available for preparation of protein kinase regulatory subunit. 6'8'11 The procedure described here is that of Kumon et al. 8,1~ Rat liver (16 g) is homogenized in 90 ml of 0.25 M sucrose, pH 7.5, containing 3.3 mM CaCl~ with a Potter-E1vehjem homogenizer with a Teflon pestle. The homogenate is filtered through four layers of cheesecloth to remove lipid, and centrifuged at 105,000 g for 45 minutes. Fifteen grams of finely powdered (NH4)2SO4 is added slowly to 48 ml of the above supernatant. The precipitate is collected by centrifugation after stirring for 30 minutes, and then dissolved in 7.5 ml of 10 mM Tris.Cl buffer, pH 7.5, containing 10% glycerol and 6 mM fl-mercaptoethanol (TMG buffer). After dialysis against 5 liters of TMG buffer overnight, 8.8 ml of dialyzate is added to 24 ml calcium phosphate gel suspension (27.3 mg/ml in TMG buffer). The gel is washed three times with 24 ml TMG buffer, and the regulatory subunit is eluted from the gel with 56 ml of 0.2 M potassium phosphate buffer, pH 8.1, containing 10% glycerol and 6 mM fl-mercaptoethanol. The eluate is dialyzed against 3 liters of TMG buffer with 2 changes for 14-16 hours and applied to a DEAEcellulose column (2 X 18 cm), which has been previously equilibrated with TMG buffer. The column is initially washed with 100 ml of TMG buffer containing 50 mM NaC1 and subsequently eluted with a linear NaC1 concentration gradient (300 ml, 50 mM to 0.5 M). The flow rate is maintained at 18 ml per hour and fractions of 2.5 ml are collected. Fractions which contain protein kinase activity and regulatory subunit, tubes 100 through 150, are pooled and dialyzed against 5 liters of TMG buffer overnight with two changes. The solution is chromatographed on a hydroxyapatite column (1 X 4 cm) equilibrated with the same buffer. After the column is washed with 250 ml of 10 mM potassium phosphate, pH 7.5, containing 6 mM fl-mercaptoethanol and 10% glycerol, the regulatory protein is eluted as a sharp peak with 250 ml of 30 mM potassium phosphate, pH 7.5, containing 6 mM fl-mercaptoethanol and 10% glycerol. The regulatory protein thus obtained is free of protein kinase. Properties The various hepatic cAMP-dependent protein kinases have to date only been minimally characterized. To the limited extent investigated, hepatic protein kinase shows a rather broad substrate specificity catalyzing the phosphorylation of histone, rabbit skeletal muscle glycogen phos~1A. Kumon, K. Nishiyama, H. Yamamura, and Y. Nishizuka, J. Biol. Chem. 9.47, 3726 (1972).

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phorylase

b, 9 and ribosomal protein, 12 and converting muscle glycogen synthetase I to the D form2 Salmon sperm protamine and bovine casein are approximately 12% and 3% as active as calf thymus histone, respectively. Egg yolk phosvitin, bovine albumin, and human y-globulin are not substrates. The rate of phosphorylation of different histone fractions is in the following order: f~b ~ f2a, f3 ~ fl with a slightly different activity ratio between different catalytic subunits2 The different forms of catalytic subunits exhibit similar kinetic properties. The Km for ATP is 20 ~M and the optimum pH is 7.0. Each species exhibits an S~o.~ of 4.0. The forms of holoenzyme are identical in size (s~o,~ --- 6.8), charge (pI = 5.2), and apparent Ka for cyclic AMP (40 nM).

II. Cytoplasmic Phosvitin Kinase Assay Method Reagents Assay buffer: 0.1 M 2-(N-morpholino)ethanosulfonate, 0.1 M NaF, 0.1 M theophylline, pH 7.5 Phosvitin, 10 mg/ml {Nutritional Biochemicals, Cleveland) [~-a2P]Adenosine triphosphate, 6 raM, specific activity 4--8 X 10° cpm/mmole; magnesium acetate, 18 raM, adjusted to pH 7.0 Procedure. Each tube contains 10 ~l of MES buffer, 10 ~l of phosvitin solution, and 20 ~l of ATP/Mg 2÷. The reaction is initiated by adding 20 ~l of enzyme. The mixture is incubated at 30 ° for 10 min and the reaction is terminated by pipeting 50 ~l of the reaction mixture to a filter paper. The remainder of the procedure is as described in the above section.

Purification Procedure The procedure described here is that of Baggio et al. 13 Rat liver (80 g) is homogenized with 400 ml of 0.25 M sucrose with a Potter-Elvehjem homogenizer. The homogenate is centrifuged for 60 minutes at 105,000 g. The supernatant (about 500 ml) is dialyzed against 5 liters of 50 mM Tris.C1 buffer, pH 7.5, for 4 hours with two changes. The dialyzate is applied to a phosphocellulose column (4.5 X 15 cm) previously equilibrated with the same buffer. The column is washed with 150 ml of the same buffer containing 0.25 M NaC1 at a flow rate of 1.5 ml per minute. C. Ell and I. G. Wool, Biochem. Biophys. Res. Commun. 43, 1001 (1971). ~sB. Baggio, L. Pinna, V. Moret, and N. Siliprandi, Biochim. Biophys. Acta 212, 515

(1970).

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KINASES

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The bulk of protein is removed by this procedure, but the enzyme is retained on the column and is subsequently eluted by 300 ml of 0.75 M NaC1. The enzyme is purified more than 1000-fold with a nearly 100% recovery by this simple procedure. Properties

The properties of the enzyme have not yet been well characterized; the phosphorylation of several proteins has been described. The relative activity with different substrates is casein ( 1 . 0 ) > histone f:b (0.27) > histone f3 (0.12) > histone f2a (0.I0) > histone fl (0.08). The enzyme is not stimulated by cAMP nor is it inhibited by regulatory subunit of cAMP-dependent protein kinase nor by heat-stable protein inhibitor. Thus, according to the criteria presented elsewhere in this volume,TM this enzyme is classified as a type I I I protein kinase and is distinguished from the various forms of catalytic subunit of cAMP-dependent protein kinase.

Acknowledgments This work was supported by Grant A M Service,

13613 from the U.S. Public Health

[47] P u r i f i c a t i o n a n d C h a r a c t e r i z a t i o n o f C y c l i c GMP-Dependent Protein Kinases B y J. F. K~Jo and PAVL GREENGARD

After the discovery of cyclic AMP (cAMP)-dependent protein kinase in mammalian skeletal muscle,1 this class of enzyme was subsequently found in liver, 2 and in numerous other vertebrate 3 and invertebrate 3-'~ tissues. In addition, the occurrence of cyclic GMP (cGMP)-dependent protein kinases, which are activated by low concentrations of cGMP rather than by cAMP, was established in various lobster, 4 insectf and mammalian tissues2 In general, cAMP-dependent enzymes appear to be 1D. A. Walsh, J. P. Perkins, and E. G. Krebs, 3. Biol. Chem. 243, 3763 (1968). T. A. Langan, Science 162, 579 (1968). 8j. F. Kuo and P. Greengard,Proe. Nat. Acad. Sci. U.S. 64, 1349 (1969). 4J. F. Kuo and P. Greengard, J. Biol. Chem. 245, 2493 (1970). J. F. Kuo, G. R. Wyatt, and P. Greengard, J. Biol, Chem. 246, 7159 (1971). ~P. Greengard and J. F. Kuo, in "Role of Cyclic AMP in Cell Function" (P. Greengard and E. Costa, eds.), p. 287. Raven, New York, 1970.