Protein kinases in brown adipose tissue of developing rats II. Two soluble kinase activities and their affinities for nucleotide and protein substrates

A heat-stable, soluble component of brown adipose tissue from newborn rats was found to be readily phosphorylated by protein kinase of the same subcellular fraction. The concentration of this component in brown fat decreased with the age of the animals. A boiled crude microsomal preparation from rat liver was also phosphorylated by brown fat protein kinase. The GTP-linked phosphorylation of the endogenous heat-stable protein was not stimulated by ATP (in contrast to phosphorylation of histone). The maximum velocity of phosphorylation achieved with GTP was about 2.5 times higher than that with ATP as nucleotide substrate. This difference was not due to ATPase activity in the assay. With histone as the protein acceptor both activities were the same. The affinity of protein kinase(s) for ATP was lower with the endogenous heat-stable brown-fat protein and with boiled microsomes (K_m of 0.21 mM and 0.17 mM, respectively) than with histone (K_m of 0.05 M). No detecable ATPase activity was present in either acceptor protein. It is concluded that the 100 000 × g supernatant fraction from brown fat of infant rats contains two protein kinase activities. One preferentially uses ATP and histone as substrates and the other uses endogenous heat-stable soluble proteins and either ATP or GTP.     

Protein kinases in brown adipose tissue of developing rats. I. GTP as nucleotide substrate for histone phosphorylation.

100 000 times g soluble extracts from interscapular brown adipose tissue catalyzed the transfer of the terminal phosphoryl group from GTP to histone. Maximal velocity was achieved only with both cyclic AMP and ATP present. The cyclic AMP dose-response curve was the same as for the ATP-utilizing enzyme, with maximum stimulation at 0.5 muM. ATP (1--100muM) increased the rate of histone phosphorylation with GTP as the radioactive substrate. Higher concentrations had a dilution effect similar to that of GTP on the ATP-utilizing enzyme. Similar effects were observed with ADP and AMP. The apparent Km values for histone were the same with both GTP and ATP as nucleotide substrates. The effects of pH, purified beef muscle kinase inhibitor and of NaCl were also the same. Maximum velocities of histone phosphorylation from ATP and those from GTP were almost the same in brown fat of all age groups testes, Separated on histone-Sepharose, the GTP-utilizing activity was absolutely dependent on the re-addition of the ATP-utilizing enzyme (a linear relationship with a slope of approx. 0.95). An extremely active nucleotide phosphotransferase activity was found in the same subcellular fraction. The rate of equilibration of the gamma-32-P between GTP and ATP could account for all the histone phosphorylation with [gamma-32-P] GTP. It is concluded that, in spite of the presence of nucleotide phosphotransferase and ATP-protein kinase activities, a direct transfer from GTP to a protein substrate cannot be excluded. Also, histone may not be the natural protein acceptor for GTP-linked phosphorylation.     

Protein kinases and their substrates in brown adipose tissue from newborn rats.

The 10000 X g supernatant fraction of brown fat from newborn rats catalyzed the cyclic AMP-dependent phosphorylation of both histone and a preparation of proteins from the same subcellular fraction (endogenous proteins). The apparent affinity for ATP was lower for the phosphorylation of the endogenous proteins than for the phosphorylation of histone. In order to discover whether the phosphorylation of histone and the endogenous proteins were catalyzed by different enzymes, the 100000 X g supernatant was fractionated by ion-exchange and adsorption chromatography. Three different cyclic AMP-dependent protein kinases and one cyclic AMP-independent protein kinase were separated and partially purified. Each of these enzymes catalyzed the phosphorylation of both substrates, and the difference in apparent Km for ATP remained. Neither affinity chromatography on histone-Sepharose, nor electrophoresis on polyacrylamide gels resulted in the separation of the phosphorylation of histone from that of the endogenous proteins of any of the partially purified kinases. Moreover, experiments in which the phosphorylated substrates were separated by differential precipitation with trichloroacetic acid showed that the endogenous proteins competitively inhibited the phosphorylation of lysine-rich histone. It is concluded that each of the partially purified kinase preparations contains protein kinase, which catalyzes the phosphorylation of both substrates. The difference in apparent Km for ATP was found to be due to the presence in the endogenous protein preparation of a low molecular weight compound which competes with ATP. This was not ATP nor the modulator protein. The ratio of the phosphorylation of endogenous proteins to that of histone was much higher for the cyclic AMP-independent kinase preparation than for the other enzymes. Electrophoresis of the endogenous substrates in the presence of sodium dodecyl sulphate showed that the enzyme phosphorylated a greater number of proteins than did the cyclic AMP-dependent kinases. The phosphorylation of endogenous proteins relative to that of histone was significantly lower for one of the cyclic AMP-dependent kinases than for the other two. This difference was not reflected in a different pattern of phosphorylation of the individual proteins of the endogenous mixture.     

Protein kinases in brown adipose tissue of developing rats I. GTP as nucleotide substrate for histone phosphorylation

100 000 × g soluble extracts from interscapular brown adipose tissue catalyzed the transfer of the terminal phosphoryl group from GTP to histone. Maximal velocity was achieved only with both cyclic AMP and ATP present. The cyclic AMP dose-response curve was the same as for the ATP-utilizing enzyme, with maximum stimulation at 0.5 μM. ATP (1–100 μM) increased the rate of histone phosphorylation with GTP as the radioactive substrate. Higher concentrations had a dilution effect similar to that of GTP on the ATP-utilizing enzyme. Similar effects were observed with ADP and AMP. The apparent K_m values for histone were the same with both GTP and ATP as nucleotide substrates. The effects of pH, purified beef muscle kinase inhibitor and of NaCl were also the same. Maximum velocities of histone phosphorylation from ATP and those from GTP were almost the same in brown fat of all age groups tested. Separated on histone-Sepharose, the GTP-utilizing activity was absolutely dependent on the re-addition of the ATP-utilizing enzyme (a linear relationship with a slope of approx. 0.95). An extremely active nucleotide phosphotransferase activity was found in the same subcellular fraction. The rate of equilibration of the γ-³² P between GTP and ATP could account for all the histone phosphorylation with [γ-³² P] GTP. It is concluded that, in spite of the presence of nucleotide phosphotransferase and ATP-protein kinase activities, a direct transfer from GTP to a protein substrate cannot be excluded. Also, histone may not be the natural protein acceptor for GTP-linked phosphorylation.     

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.     

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.     

Legionella micdadei protein kinase catalyzes phosphorylation of tubulin and phosphatidylinositol.

Legionella micdadei, a pathogen which enters into host phagocyte phagolysosomal structures, contains at least two protein kinases. We have purified to homogeneity the predominant, nucleotide-independent protein kinase and examined its ability to catalyze the transfer of phosphate from ATP to acceptors in human neutrophils. The L. micdadei protein kinase catalyzed the phosphorylation of proteins of 11.5, 14, 19, 23, 28, 34, and 38 kilodaltons (kDa) present in a Triton X-100 extract of neutrophil membranes and of 11.5, 13.5, 25, and 38 kDa in the neutrophil cytosol. Tubulin was a good substrate for the L. micdadei protein kinase in vitro. The bacterial kinase also catalyzed the phosphorylation of phosphatidylinositol (PI) at about half the rate at which histones were phosphorylated; phosphatidylinositol-4-phosphate (PIP) was not phosphorylated by the kinase. The PI kinase activity of the L. micdadei enzyme was optimum at pH 7.0, and the divalent cation requirement was satisfied best by Mg2+ and Ca2+. The maximum rate of PI phosphorylation was obtained with 0.6 mM PI; in the presence of MgCl2 (10 mM), the Km for PI was 0.9 mM and the Km for ATP was 1.5 mM. The detergents octyl-beta-D-glucoside (10 to 20 mM) and Triton X-100 (0.5%) stimulated kinase activity twofold when PI was the phosphate acceptor; however, only octyl glucoside stimulated histone kinase activity. Various membrane phospholipids inhibited PI kinase activity. The most potent phospholipid inhibitor was the product of the PI kinase reaction, PIP, which at a 0.6 mM concentration inhibited both PI and tubulin phosphorylation by 80%. The inhibition of kinase activity by PIP when histone served as the acceptor was noncompetitive in character. The L. micdadei kinase also phosphorylated PI in intact. (3H)inositol-labeled neutrophils. The PI kinase and histone kinase activities of teh L. micdadei kinase copurified and cofucused (pI, 5.8) when subjected to isoelectric focusing, suggesting that the two enzymatic activities reside in a single protein.     

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.     

Actin kinase: a protein kinase that phosphorylates actin of fragmin-actin complex.

Actin of fragmin-actin complex is phosphorylated by an endogenous kinase from plasmodium of Physarum polycephalum. The phosphorylation abolishes the nucleation and capping activities of fragmin-actin complex. The kinase has been purified and termed actin kinase [Furuhashi, K. & Hatano, S. (1990) J. Cell Biol. 111, 1081-1087]. Enzymatic properties of the purified actin kinase were studied in detail. Actin kinase exhibited the highest activity under conditions physiological for the plasmodium (30 mM KCl, 6 mM MgCl2, pH 7.0). The Vmax and the Km of the enzyme for ATP were about 83 mumol/min/mg and 25 microM, respectively. The Km for fragmin-actin complex was 190 nM. The purified actin kinase phosphorylated actin of fragmin-actin complex at a constant rate regardless of Ca2+ concentration. Similarly, 2 microM cAMP, 2 microM cGMP, 2 micrograms/ml calmodulin in the presence of Ca2+ or 1 mM GTP showed no effect on the activity of the purified enzyme. Actin kinase did not phosphorylate histone H1, H2B, alpha-casein, or beta-casein, suggesting that actin kinase is a new kind of protein kinase which specifically phosphorylates actin of the fragmin-actin complex.     

Phosphorylation of rabbit and human erythrocyte membranes by soluble adenosine 3':5'-monophosphate-dependent and -independent protein kinases.

Previous reports from this laboratory and others have established that both the rabbit and human erythrocyte membranes contain multiple protein kinase and phosphate acceptor activities. We now report that these membranes also contain phosphoryl acceptor sites for the soluble cyclic AMP-dependent and -independent protein kinases from rabbit erythrocytes. The rabbit erythrocyte membrane, which does not contain a cyclic AMP-dependent protein kinase, has at least four polypeptides (Bands 2.1, 2.3, 4.5, and 4.8) which are phosphorylated in the presence of the soluble cyclic AMP-dependent protein kinases I, IIa, and IIb isolated from rabbit erythrocyte lysates. The resulting phosphoprotein profile is very similar to that obtained for the cyclic AMP-mediated autophosphorylation of human erythrocyte membranes. The activities of the soluble cyclic AMP-dependent protein kinases toward the membranes have been studied at several pH values. Although the substrate specificity of the three kinases is similar, polypeptide 2.3 appears to be phosphorylated to a greater extent by kinase IIa than by I or IIb. This occurs at all pH values studied. Also apparent is that the pH profile for membrane phosphorylation is different from that of histone phosphorylation. The phosphorylation of membrane proteins can also be catalyzed by the soluble erythrocyte casein kinases. These enzymes are not regulated by cyclic nucleotides and can use either ATP or GTP as their phosphoryl donor. Polypeptides 2.1, 2.9, 4.1, 4.5, 4.8, and 5 of both human and rabbit erythrocyte membranes are phosphorylated in the presence of GTP and the casein kinases. This reaction is optimal at pH 7.5. Experiments were performed to determine whether the phosphorylation of the membranes by the soluble and membrane-bound kinases is additive or exclusive. Our results indicate that after maximal autophosphorylation of the erythrocyte membranes, phosphoryl acceptor sites are available to the soluble cyclic AMP-dependent and -independent protein kinases. Furthermore, after maximal phosphorylation of the membranes with one type of soluble kinase, further 32P incorporation can occur as a result of exposure to the other type of soluble kinase.     

Characterization of maturation-activated histone H1 and ribosomal S6 kinases in sea star oocytes

DEAE-Sephacel chromatography of cytosolic extracts from sea star oocytes resolved at least two distinct peaks of maturation-activated protein kinase activity, each of which catalyzed the phosphorylation of histone H1, ribosomal protein S6, and Arg-Arg-Leu-Ser-Ser-Leu-Arg-Ala (RRLSSLRA), a synthetic peptide based on the sequence of a phosphorylation site in the latter protein. The first peak (elution conductivity approximately equal to 6 mmho) contained the major activated kinase with respect to the phosphorylation of histone H1, and the second peak (elution conductivity approximately equal to 10.5 mmho) contained the major activated kinase with respect to the phosphorylation of S6 and RRLSSLRA. These kinase activities were barely detectable in extracts from immature oocytes. The major stimulated histone H1 kinase exhibited an apparent Mr of approximately 90 000 on Sephacryl S-300 but eluted from TSK-400 with an apparent Mr of approximately 10 000. After DEAE-Sephacel fractionation, this kinase was shown to utilize both ATP (apparent Km approximately equal to 45 microM) and GTP (apparent Km approximately equal to 10 microM), although the Vmax was 8-fold higher with ATP than with GTP. The enzyme phosphorylated histone H1 with an apparent Km approximately equal to 50 micrograms/mL. Its properties resembled those of the growth-associated histone kinase. The major stimulated RRLSSLRA kinase had an apparent Mr of approximately 84 000 on Sephacryl S-300 and approximately 40 000 on TSK-400. After DEAE-Sephacel chromatography, this kinase selectively utilized ATP (apparent Km approximately equal to 25 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of a specific histone H1 protein kinase from mouse plasmacytoma

A protein kinase with high specificity for histone H1 was purified from a plasmacytoma microsomal fraction by a high-salt wash, ammonium sulfate precipitation, chromatography on DEAE-cellulose, hydroxyapatite and Sephadex G-200 columns, and the main properties of this kinase were studied. A sulfhydryl compound, such as 2-mercaptoethanol or dithiothreitol, was necessary for full activity. The optimum pH was 7.4-7.8. After purification, the histone H1 kinase was not stimulated by cAMP or cGMP. It was not inhibited by the heat-stable cAMP-dependent protein kinase inhibitor from beef heart. It utilized preferentially GTP over ATP as phosphate donor. Km values for ATP and GTP were 58 microM and 1.4 microM respectively; the Km for histone H1 was 14 microgram ml-1. The molecular weight was approximately 90 000 by gel-exclusion chromatography. Analysis of the purified H1-specific protein kinase by polyacrylamide gel electrophoresis in dodecylsulfate showed two bands having molecular weights of approximately 64 000 and 54 000. Many characteristics of this kinase were similar to those of the chromatin-bound protein kinase reported by other workers in rapidly proliferating cells.     

Identification of casein kinase 1, casein kinase 2, and cAMP-dependent protein kinase-like activities in Trypanosoma evansi

Trypanosoma evansi contains protein kinases capable of phosphorylating endogenous substrates with apparent molecular masses in the range between 20 and 205 kDa. The major phosphopolypeptide band, pp55, was predominantly localized in the particulate fraction. Anti-alpha and anti-beta tubulin monoclonal antibodies recognized pp55 by Western blot analyses, suggesting that this band corresponds to phosphorylated tubulin. Inhibition experiments in the presence of emodin, heparin, and 2,3-bisphosphoglycerate indicated that the parasite tubulin kinase was a casein kinase 2 (CK2)-like activity. GTP, which can be utilized instead of ATP by CK2, stimulated rather than inactivated the phosphorylation of tubulin in the parasite homogenate and particulate fraction. However, GTP inhibited the cytosolic CK2 responsible for phosphorylating soluble tubulin and other soluble substrates. Casein and two selective peptide substrates, P1 (RRKDLHDDEEDEAMSITA) for casein kinase (CK1) and P2 (RRRADDSDDDDD) for CK2, were recognized as substrates in T. evansi. While the enzymes present in the soluble fraction predominantly phosphorylated P1, P2 was preferentially labeled in the particulate fractions. These results demonstrated the existence of CK1-like and CK2-like activities primarily located in the parasite cytosolic and membranous fractions, respectively. Histone II-A and kemptide (LRRASVA) also behaved as suitable substrates, implying the existence of other Ser/Thr kinases in T. evansi. Cyclic AMP only increased the phosphorylation of histone II-A and kemptide in the cytosol, demonstrating the existence of soluble cAMP-dependent protein kinase-like activities in T. evansi. However, no endogenous substrates for this enzyme were identified in this fraction. Further evidences were obtained by using PKI (6-22), a reported inhibitor of the catalytic subunit of mammalian cAMP-dependent protein kinases, which specifically hindered the cAMP-dependent phosphorylation of histone II-A and kemptide in the parasite soluble fraction. Since the sum of the values obtained in the parasite cytosolic and particulate fractions were always higher than the values observed in the total T. evansi lysate, the kinase activities examined here appeared to be inhibited in the original extract.     

Protein kinase C from chicken gizzard: characterization and detection of an inhibitor and endogenous substrates

Protein kinase C was purified from the cytosolic fraction of chicken gizzard by Ca2+ -dependent hydrophobic interaction chromatography, anion-exchange chromatography, and hydrophobic chromatography. The molecular weight was estimated as 61,500 by gel filtration and 80,000 by denaturing gel electrophoresis, indicating that the native enzyme is a monomer. Using the mixed micellar assay, with histone III-S as the substrate, protein kinase C required Ca2+, phospholipid, and diacylglycerol for activity, with half-maximal activation at approximately 5 x 10(-7) M Ca2+ in the presence of L-alpha-phosphatidyl-L-serine and 1,2-diolein. No activation by Ca2+ was observed in the absence of diacylglycerol. Protein kinase C requires free Mg2+, in addition to the MgATP2- substrate, for activity. The Km for ATP was determined to be 20 microM. Activity was sensitive to ionic strength, with half-maximal inhibition at 70 mM NaCl. Using the liposomal assay, phosphorylation of platelet P47 protein and smooth muscle vinculin was more strongly dependent on Ca2+ and lipids than was histone phosphorylation. Partial digestion of protein kinase C with trypsin yielded a constitutively active fragment. A heat-stable inhibitor and three major endogenous protein substrates of protein kinase C were also detected in chicken gizzard smooth muscle.     

A novel protein kinase activity in rabbit gastric gland cytosol

A novel protein kinase activity was characterized from the cytosolic fraction of isolated rabbit gastric glands. The kinase phosphorylated a major 33,000 Da endogenous protein (pp33) and was stimulated by Zn2+ and Mn2+ with Kact of 1.0 and 7.5 mM, respectively. Mg2+ and Ca2+ failed to stimulate any pp33 kinase activity. The kinase utilized both ATP and GTP as phosphate donors with a Km of 10 microM for both. The pp33 protein displayed an isoelectric point of 7.5 to 7.8 and was phosphorylated predominantly on threonine residues. The kinase activity is clearly differentiable from all reported kinase activities and appeared to be enriched in rabbit gastric fundic mucosa. The results indicate that gastric fundic mucosa contains a novel protein kinase activity.     

Phosphorylation of a 100 000 dalton component and its relationship to calcium transport in sarcoplasmic reticulum from rabbit skeletal muscle

Sarcomplasmic reticulum from rabbit fast skeletal muscle contains intrinsic protein kinase activity (ATP:protein phosphotransferase, EC 2.7.1.37) and a substrate. The protein kinase activity was Mg2+ dependent and could also phosphorylate exogenous protein substrates. Autophosphorylation of sarcoplasmic reticulum vesicles was not stimulated by cyclic AMP, neither was it inhibited by the heat-stable protein kinase inhibitor protein. The phosphorylated membranes had the characteristics of a protein with a phosphoester bond. An average of 73 pmol Pi/mg protein were incorporated in 10 min at 30 degrees C. Addition of exogenous cyclic AMP-dependent protein kinase increased the endogenous level of phosphorylation by 25-100%. Sarcoplasmic reticulum membrane phosphorylation, mediated by either endogenous cyclic AMP-independent or exogenous cyclic AMP-dependent protein kinase, occurred on a 100 000 dalton protein and both enzyme activities resulted in enhanced calcium uptake and Ca2+-dependent ATPase (ATP phosphohydrolase, EC 3.6.1.3), in a manner similar to cardiac microsomal preparations. Regulation of Ca2+ transport in skeletal sarcoplasmic reticulum may be mediated by phosphorylation of a 100 000 dalton component of these membranes.     

Cyclic adenosine monophosphate-dependent and -independent protein kinase activity of renal brush border membranes.

Kinase(s) in brush border membranes, isolated from rabbit renal proximal tubules, phosphorylated proteins intrinsic to the membrane and exogenous proteins. cAMP stimulated phosphorylation of histone; phosphorylation of protamine was cAMP independent. cAMP-dependent increases in phosphorylation of endogenous membrane protein were small, but highly reproducible. Most of the ³²P incorporated into membranes represented phosphorylation of serine residues, with phosphorylthreonine comprising a minor component. cAMP did not alter the electrophoretic pattern of ³²P-labeled membrane polypeptides. The small cAMP-dependent phosphorylation of brush border membrane proteins was not due to membrane phosphodiesterase or adenylate cyclase activities. Considerable cAMP was found “endogenously” bound to the membranes as prepared. However, this did not result in preactivation of the kinase since activity was not inhibited by a heat-stable protein inhibitor of cAMP-dependent protein kinases. With intrinsic membrane protein as phosphate acceptor, the relationship between rate of phosphorylation and ATP concentration appeared to follow Michaelis-Menton kinetics. With histone the relationship was complex. cAMP did not affect the apparent K_m for histone. One-half maximal stimulation of the rate of histone phosphorylation was obtained with 7 × 10^?8m cAMP. The K_a values for dibutyryl cAMP, cIMP, and cGMP were one to two orders of magnitude greater. Treatment of brush border membranes with detergent greatly increased the dependency of histone phosphorylation on cAMP. Phosphorylations of intrinsic membrane protein and histone were nonlinear with time, due in part to the lability of the protein kinase, the hydrolysis of ATP, and minimally to the presence of phosphoprotein phosphatase in the border membrane. The membrane phosphoprotein phosphatase was unaffected by cyclic nucleotides. Protein kinase activity was also found in cytosolic and crude particulate fractions of the renal cortex. Activity was enriched in the brush border membrane relative to that in the crude membrane preparation. The kinase activities in the different loci were distinct both in relative activities toward different substrates and in responsiveness to cAMP.     

The ribosomal proteins phosphorylated in vitro by protein kinase activities from Krebs II ascites cells

Studies were performed to identify in cytoplasmic extracts of Krebs II ascites cells protein kinase activities that might be responsible for the phosphorylation of the ribosomal proteins previously identified as phosphoproteins in these cells in vivo. Column chromatography resolved a casein kinase activity that could use ATP or GTP as a phosphoryl donor to phosphorylate, in ribosomes, exclusively the acidic 60S phosphoprotein(s) phosphorylated in vivo. A second casein kinase fraction could use ATP, only, in a similar reaction, but also contained protein kinase activity with respect to other ribosomal proteins, including the basic ribosomal protein phosphorylated in vivo, ribosomal protein S6. This latter was also among several proteins phosphorylated by an activity in the cyclic AMP-independent histone kinase fraction.     

Some properties of the reaction catalyzed by protein kinase bound to cardiac sarcolemma

A highly purified sarcolemmal fraction from rat heart consisted of closed inside-out oriented vesicles and possessed high activities of Na+, K+-ATPase, adenylate cyclase and creatine kinase. Contaminations of sarcolemmal preparation by other membranous fractions were practically absent. This sarcolemmal fraction contained protein kinase tightly bound to the membrane. Substrates of the phosphorylation reaction catalyzed by this protein kinase were either endogenous sarcolemmal protein (proteins) with molecular weight of 11500 or exogenous protein--histone, type II. Phosphorylation of the endogenous but not of the exogenous substrate was completely independent of cyclic AMP. A kinetic analysis of the sarcolemmal protein kinase reaction with Mg[gamma-32P]ATP and histone as substrates revealed that the kinetic mechanism of this reaction is characterized by the following kinetic parameters: Km (Mg-ATP) = 12.1 microM; Km (histone) = 0.47 mg/ml; Ki (Mg-ADP) = 15.6 microM. A comparison of experimental results to literary data allows to suggest that the sarcolemmal enzyme is virtually soluble protein kinase tightly bound to the sarcolemma.     

Characterization of Ca2+/Calmodulin-Dependent Protein Kinase Associated with Rat Cerebral Synaptic Junction: Substrate Specificity and Effect of Autophosphorylation

The Ca2+/calmodulin (CaM)-dependent protein kinase associated with rat cerebral synaptic junction (SJ) was characterized, using the SJ fraction as the enzyme preparation, to clarify the functional significance of the enzyme in situ. The protein kinase was greatly activated in the presence of micromolar concentrations of both Ca2+ and calmodulin (EC50 for Ca2+, 1.0 microM; that for CaM, 100 nM). The Km for ATP was 150 microM. SJ proteins were phosphorylated without a lag time, and the phosphorylation reached its maximum within 2-10 min at 25 degrees C. The endogenous substrates consisted of four major (160K, 120K, 60K, and 51K Mr) and 10 minor proteins. Compared with the endogenous substrate phosphorylation, the phosphorylation of exogenously added proteins (myosin light chains from chicken muscle, casein, arginine-rich histone, microtubule-associated protein-2, tau-protein, and tubulin) was weak, although they are expected to be good substrates for the soluble form of the Ca2+/CaM-dependent protein kinase. Autophosphorylation of the enzyme in SJ inhibited its activity and did not alter the subcellular distribution of the enzyme.