Casein kinase I is regulated by phosphatidylinositol 4,5-bisphosphate in native membranes.

Casein kinase I activity is present in cells as a cytosolic and a membrane-bound enzyme. Previously, the erythroid membrane-bound casein kinase I was shown to associate with purified integral membrane proteins; this association and protein kinase activity was regulated by phosphatidylinositol 4,5-bisphosphate (PIP2) (Bazenet, C.E., Brockman, J.L., Lewis, D., Chan, C., and Anderson, R.A. (1990) J. Biol. Chem. 265, 7369-7376). Here we show that both the membrane-bound and the cytosolic casein kinase interact with native membranes and that this interaction is regulated by the membrane content of PIP2. On native membranes, casein kinase I activity is potently inhibited by small increases (10-20%) in the membrane content of either exogenously added or intrinsic PIP2. However, the majority of the intrinsic content of PIP2 in isolated membranes does not inhibit casein kinase, suggesting that this PIP2 is not accessible. Regulation of the casein kinases on membranes is sensitive to detergents and to chymotrypsin treatment of membranes.     

Characterization and comparison of membrane-associated and cytosolic cAMP-dependent protein kinases. Studies on human erythrocyte protein kinases.

Cyclic AMP-dependent protein kinase from human erythrocyte plasma membranes was solubilized with Triton X-100, partially purified, and systematically characterized by a series of physicochemical studies. Sedimentation and gel filtration experiments showed that the 6.6 S holoenzyme had a Stokes radius (a) of 5.7 nm and was dissociated into native 4.8 S cAMP-binding (a = 4.5 nm) and 3.2 S catalytic (a = 2.6 nm) subunits. A minimum subunit molecular weight of 48,000 was established for the regulatory subunit by photoaffinity labeling with 8-azido[32P]cAMP, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and autoradiography. These data suggest an asymmetric tetrameric (R2C2) structure (Mr approximately equal to 160,000) for the membrane-derived enzyme. Membrane-derived protein kinase was characterized as a type I enzyme on the basis of its R subunit molecular weight, pI values (R, 4.9; holoenzyme, 5.75 and 5.95), dissociation by 0.5 M NaCl and 50 microgram/ml of protamine, 20-fold reduced affinity for cAMP in the presence of 0.3 mM MgATP, elution from DEAE-cellulose at low ionic strength, and kinetic and cAMP-binding properties. The physicochemical properties of the membrane protein kinase closely parallel the characteristics of erythrocyte cytosolic protein kinase I but are clearly dissimilar from those of the soluble type II enzyme. Moreover, regulatory subunits of the membrane-associated and cytosolic type I kinases were indistinguishable in size, shape, subunit molecular weight, charge, binding and reassociation properties, and peptide maps of the photoaffinity-labeled cAMP-binding site, suggesting a high degree of structural and functional homology in this pair of enzymes. In view of the predominant occurrence of particulate type II protein kinases in rabbit heart and bovine cerebral cortex, the present results suggest that the distribution of membrane-associated protein kinases may be tissue- or species-specific, but not isoenzyme-specific.     

Interrelationships between protein kinases and spectrin phosphorylation in human erythrocytes

Casein kinase and histone kinase(s) are solubilized from human erythrocyte membranes by buffered ionic solutions (0.1 mM EDTA and subsequent 0.8 M NaCl, pH 8) containing 0.2% Triton X-100. Casein kinase is separated from histone kinase(s) by submitting the crude extracts directly to chromatography on a phosphocellulose column, eluted with a continuous linear gradient of potassium phosphate buffer, pH 7.0, containing 0.2% Triton X-100. Under these conditions, the membrane-bound casein kinase activity is almost completely recovered into a quite stable preparation, free of histone kinase activity. In contrast, it undergoes a dramatic loss of activity when the extraction and the subsequent phosphocellulose chromatography are carried out with buffers which do not contain Triton X-100. Isolated spectrin, the most abundant membrane protein, is phosphorylated, in the presence of [gamma-32P]ATP, only by casein kinase while histone kinase is ineffective. Only the smaller subunit (band II) of isolated spectrin (and not the larger one (band I) is involved in such a phosphorylation process, as in the endogenous phosphorylation occurring in intact erythrocytes.     

Erythroid membrane-bound protein kinase binds to a membrane component and is regulated by phosphatidylinositol 4,5-bisphosphate

In the erythrocyte, a membrane-bound serine/threonine protein kinase (a casein kinase) has been shown to phosphorylate a number of membrane proteins, modulating their function. Here we report that the membrane-bound protein kinase binds to membranes by an association with a minor membrane component contained in preparations of glycophorin (possibly a minor glycophorin). The binding of the kinase to glycophorins does not significantly modify kinase activity. However, upon binding, the kinase activity is potently inhibited by phosphatidylinositol 4,5-bisphosphate, and the affinity of the kinase for the glycophorins is increased. Other phospholipids or polyanions such as inositol 1,4,5-trisphosphate or 2,3-diphosphoglycerate do not affect protein kinase activity when the kinase is bound to membranes but do inhibit the solubilized membrane-bound kinase. In the erythrocyte, there is a cytosolic form of the casein kinase which is very similar, having the same molecular weight and substrate specificity as the membrane-bound casein kinase. The cytosolic casein kinase is inhibited by 2,3-diphosphoglycerate but much less so by glycophorin preparations containing phosphoinositol 4,5-bisphosphate. When the sequences of both casein kinases were compared by two-dimensional peptide mapping, it was found that the two kinases were very similar but not identical.     

Two different mechanisms for activation of cyclic PIP synthase: by a G protein or by protein tyrosine phosphorylation

The biosynthesis of the functional, endogenous cyclic AMP antagonist, prostaglandylinositol cyclic phosphate (cyclic PIP) is performed by the plasma membrane-bound enzyme cyclic PIP synthase, which combines prostaglandin E (PGE) and activated inositol phosphate (n-IP) to cyclic PIP. The Km values of the enzyme for the substrates PGE and n-IP are in the micromolar range. The plasma membrane-bound synthase is activated by fluoride, by the stable GTP analog GMP-PNP, by protamine or biguanide, by noradrenaline, and by insulin. The activation by protamine or biguanide and fluoride (10 mM) is additive, which may indicate the presence of two different types of enzyme, comparable to phospholipase Cbeta and phospholipase Cgamma. Plasma membrane-bound cyclic PIP synthase is inhibited by the protein tyrosine kinase inhibitor tyrphostin B46 with an IC50 of 1.7 microM. However, the solubilized and gel-filtrated enzyme is no longer inhibited by tyrphostin, indicating that the activity of cyclic PIP synthase is connected with the activity of a membrane-bound protein tyrosine kinase. Cyclic PIP synthase activity of freshly prepared plasma membranes is unstable. Upon freezing and rethawing of liver plasma membranes, this instability is increased about 2-fold. Protein tyrosine phosphatase inhibitors [vanadate, fluoride (50-100 mM)] stabilize the enzyme activity, but protease inhibitors do not, indicating that inactivation of the enzyme is connected with protein tyrosine dephosphorylation. Cyclic PIP synthase is present in all tissues tested, like brain, heart, intestine, kidney, liver, lung, skeletal muscle, spleen, and testis. Apart from liver, cyclic PIP synthase activity in most tissues is rather low, but it can be increased up to 5-fold when protein tyrosine phosphatase inhibitors like vanadate are present in the homogenization buffer. Preincubation of cyclic PIP synthase of liver plasma membranes with the tyrosine kinase src kinase causes a 2-fold increase of cyclic PIP synthase activity, though this is certainly not the physiological role played by src kinase in intact cells. The data indicate that cyclic PIP synthase can be activated by two separate mechanisms: by a G protein or by protein tyrosine phosphorylation.     

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.     

Stimulation of ascites tumor RNA polymerase II by protein kinase.

The activity of purified RNA polymerase II from Novikoff ascites tumor cells is stimulated 5-7-fold by a purified protein factor. This protein factor, designated HLF2, has extensive protein kinase activity and catalyzed the incorporation of gamma-32G from ATP into protein under normal RNA polymerase assay conditions. Protein phosphorylation is totally dependent on the presence of HLF2 and is stimulated 2-3-fold by the presence of highly purified RNA polymerase II. The purification procedure developed for the isolation of the polymerase stimulatory factor resulted in a 4000-fold purification of a protein kinase. Chromatography on carboxymethylcellulose, phosphocellulose, and Sephadex G-100 did not resolve polymerase stimulatory activity from protein kinase activity. Adenylimidodiphosphate (AMP-PNP), an inhibitor of protein kinases, inhibited the stimulatory activity of purified factor by 80%. The heat denaturation profile of protein kinase was paralleled by the loss of polymerase stimulatory activity. Concentrations of (NH4)2SO4 which are known to inhibit polymerase stimulation (Lee and Dahmus, 1973) also inhibit protein kinase activity. The protein kinase activity associated with stimulatory factor catalyzes the phosphorylation of basic proteins such as protamine or histone. The protein kinase is not stimulated by cyclic 3', 5'-AMP or -GMP over a concentration range of 10(-6)-10(-4)M. Furthermore, protein kinase activity is not inhibited by either the regulatory subunit of rabbit muscle protein kinase or by the heat-stable inhibitor of cyclic 3', 5'-AMP-dependent protein kinases. Protein kinase activity is stimulated by KCl or NH4Cl and is inhibited by MnCl2. The apparent Km values, determined in the presence of 4 mM Mg2+, are 0.02 mM for ATP, and 4.1 mM for GTP.     

Altered cyclic AMP-dependent protein kinase activity in a mutant adrenocortical tumor cell line

A somatic cell genetic approach has been used to evaluate the role of cyclic AMP-dependent protein kinase in ACTH action on adrenal steroidogenesis. A mutant clone, 8BrcAMP^r-1, previously was isolated from an ACTH-sensitive adrenocortical tumor cell line (clone Y1) following mutagenesis and selective growth in 8-bromoadenosine 3′, 5′-monophosphate. This study demonstrates that the 8BrcAMP⁴-1 cells have an altered cyclic AMP-dependent protein kinase. The protein kinase in the cytosol of the mutant characteristically requires, for half-maximal activity, concentrations of cyclic AMP 7-fold higher than those required by the enzyme in preparations from the parent. The cytosolic cyclic AMP-dependent protein kinases of Y1 and 8BrcAMP^r-1 cells chromatograph similarly on columns of DEAE-cellulose. From each cell line, a major peak of activity (≥ 70% of recovered activity), designated as Peak I, elutes with 0.04–0.06 M NaCl; a second peak of activity, designated as Peak II, elutes with 0.12–0.14 M NaCl. Protein kinase activity in the Peak I fraction of mutant cells has a decreased apparent affinity (4-fold) for cyclic AMP relative to the corresponding fraction of parental Y1 cells. The protein kinase activities present in Peak II fractions from Y1 and mutant cells are indistinguishable. The protein kinase mutant exhibits poor steroidogenic responses to added ACTH and cyclic AMP; and as shown previously does not display the growth arrest and morphological changes produced in Y1 by these agents. These results suggest that cyclic AMP-dependent protein kinase is important in the regulation of adrenal steroidogenesis, morphology and growth by ACTH.     

Purification and properties of cyclic AMP-independent glycogen synthase kinase 1 from rabbit skeletal muscle.

A cyclic AMP-independent casein (phosvitin) kinase eluted from a phosphocellulose column with 0.35 M KCl also possesses glycogen synthase kinase activity. This kinase, designated synthase kinase 1, is separable from other cyclic AMP-independent protein kinases, which also contain glycogen synthase kinase activity, by chromatography on a phosphocellulose column. This kinase was purified 15,000-fold from the crude extract. Synthase kinase activity co-purifies with casein and phosvitin kinase activities. Heat inactivation of these three kinase activities follow similar kinetics. It is suggested that these three kinase activities reside in a single protein. This kinase has a molecular weight of approximately 34,000 as determined by glycerol density gradient centrifugation and by gel filtration. The Km values for the synthase kinase-catalyzed reaction are 0.12 mg/ml (0.35 micronM) for synthase, 12 micronM for ATP, and 0.15 mM for Mg2+. The phosphorylation of glycogen synthase by the kinase results in the incorporation of 4 mol of phosphate/85,000 subunit; however, only two of the phosphate sites predominantly determine the glucose-6-P dependency of the synthase. Synthase kinase activity is sensitive to inhibition by NaCl or KCl at concentrations encountered during purification. Synthase kinase activity is insensitive to the allosteric effector (glucose-6-P) or substrate (UDP-glucose) of glycogen synthase at concentrations usually found under physiological condition.     

Purification of a Phosphatidylinositol 4-Phosphate Kinase from Bovine Brain Membranes

A phosphatidylinositol 4-phosphate (PIP) kinase (EC 2.7.1.68) was purified from bovine brain membranes in a six-step procedure involving solubilization of the enzyme with 170 mM NaCl followed by chromatography on diethylaminoethyl-cellulose, phosphocellulose, Ultrogel AcA44, hydroxylapatite, and ATP-agarose. The enzyme preparation was nearly homogeneous and was purified 5,600-fold with a final specific activity of 85 nmol/min/mg of protein and a yield of 20%. Its molecular mass was 110 kilodaltons, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was specific for PIP; phosphorylation of phosphatidylinositol and diacylglycerol was not observed.     

Properties of a membrane-bound tyrosine kinase phosphorylating the cytosolic fragment of the red cell membrane band 3 protein

Band 3 protein of human erythrocyte membrane is phosphorylated on a tyrosine residue located near the NH2 terminal by an endogenous tyrosine kinase activity (Dekowski, S., Rybicki, A. and Drickamer, K. (1983) J. Biol. Chem. 258, 2750-2753). A tyrosine kinase phosphorylating the band 3 protein in situ has been extracted from ghosts by non-ionic detergent and partially characterized (Phan-Dinh-Tuy, F., Henry, J. and Kahn, A. (1985) Biochem. Biophys. Res. Commun. 126, 304-312). We have studied the properties of the tyrosine kinase activity which remains bound to the ghosts after detergent extraction using the 43 kDa fragment of protein 3 as substrate. This activity, solubilized from the detergent-resistant material at 0.25 M NaCl and concentrated by phosphocellulose and tyrosine-agarose chromatographies, remains linked to high molecular weight complexes. It is specific for tyrosine. Assayed with the purified 43 kDa fragment it requires the presence of Mn2+ which cannot be replaced by Mg2+. Its affinity for 43 kDa fragment is very high with a Km of 3.3 microM. ATP acts as a phosphoryl donor with a Km of 0.55 microM. The tyrosine kinase activity was not modified by insulin, DMSO, phorbol ester and epidermal growth factor, vanadate and xanthine derivatives. Polyamines spermidine and the polylysine are inhibitors in the presence of Mn2+ but not in the presence of Mg2+. Heparin is a competitive inhibitor of ATP. 2,3-Diphosphoglycerate is an inhibitor at physiological concentrations (Ki = 2 mM). Purified red cell actin is not phosphorylated by the tyrosine kinase. These properties distinguish the red cell membrane-bound tyrosine kinase from other tyrosine kinases extracted from normal cells.     

Enhancement of carbonic anhydrase activity by erythrocyte membranes

The human erythrocyte membrane is an efficient enhancer of both high (CA II) and low (CA I) activity isozymes of red blood cell carbonic anhydrase. The presence of membrane increased CO2 hydration catalyzed by bovine CA II 1.6-fold, human CA II 3.5-fold, and human CA I 1.6-fold. With the high activity CA isozymes, maximal stimulation was observed in the presence of 1-3 micrograms membrane protein/ml. The Vmax for bovine CA II (4 nM) rose from 0.302 to 0.839 mM/s, while that for human CA II (6 nM) increased from 0.113 to 0.414 mM/s in the absence and presence of membrane, respectively. The apparent Km for CO2 increased from 13.2 to 51.2 mM for bovine CA II, and from 6.5 to 38.5 mM for human CA II. Mixtures of membrane plus enzyme, upon centrifugation through linear sucrose density gradients, displayed enhanced Ca activity only in membrane-containing gradient fractions, verifying the stimulatory ability of membranes on enzyme activity and indicating tight and stable complex formation. Membrane enhancement of CA activity appears to be a general phenomenon in that mouse hepatocyte membranes also stimulated CA activity, although less efficiently than erythrocyte membranes. Of the many soluble putative effectors assayed, only imidazole enhanced CA II activity to an extent comparable with erythrocyte membranes; imidazole did not, however, stimulate the activity of human CA I. The data are consistent with a model of CA II activation by membrane association that may effect a distortion of the enzyme conformation in such a way as to facilitate intra- and/or intermolecular proton transfer between membrane-bound and enzyme-bound proton shuttling residues (perhaps the imidazole moiety of histidine) and the Zn-bound hydroxide at the catalytic site of the enzyme.     

Partial purification and properties of a chromatin-associated phosphoprotein kinase from rat liver nuclei.

A phosphoprotein kinase (EC 2.7.1.37) KIVb, from rat liver nuclei, was purified 75-fold by phosphocellulose chromatography and gel filtration on Sephadex G-200. The enzyme, which has an apparent molecular weight of 55 000, phosphorylates casein and chromatin-bound nonhistone proteins more readily than histones or ribosomal proteins. It exhibits an absolute requirement for divalent cation with optimum activity at 15--20 mM Mg2+. Maximal kinase activity is achieved at 100 mM NaCl. The pH vs. activity curve is biphasic with optima at pH 6.5 and pH 8.0. The Km value for casein is 280 mug/ml and the Km for ATP is 6-10(-6) M. Kinase KIVb phosphorylates numerous nonhistone nuclear proteins as shown by electrophoretic analysis. The addition of kinase KIVb to reaction mixtures containing nonhistone proteins results in the phosphorylation of a spectrum of polypeptides similar to those that are phosphorylated by endogenous nuclear kinases. Nonhistone proteins bound to chromatin appear to be better substrates for KIVb than nonhistones dissociated from chromatin. A comparison of nuclear phosphoproteins phosphorylated either in the intact animal or in vitro (by the addition of kinase KIVb) indicates some differences and some similarities in the patterns of phosphorylation.     

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...     

Purification and characterization of phosphatidylinositol 4-kinase from human erythrocyte membranes.

Two species of PtdIns 4-kinase with molecular masses of 50 kDa and 45 kDa were detected in human erythrocyte membranes using SDS/PAGE. These enzymes were purified to near homogeneity and found to display very similar enzymatic characteristics. The purification scheme consisted of solubilization from erythrocyte membranes in the presence of Triton X-100, followed by Cibacron-blue-Sephadex, phosphocellulose and Mono Q anion-exchange chromatography. The final step in the purification protocol was preparative SDS/PAGE, followed by electroelution and renaturation of the enzyme. This procedure afforded an about 4000-fold purification of the enzyme from erythrocyte membranes. Characterization of the [32P]PtdInsP products formed by the purified PtdIns kinases indicated that these enzymes specifically phosphorylated the D-4 position of the inositol ring. The Km values of both PtdIns 4-kinase species for PtdIns and ATP were found to be 0.2 mM and 0.1 mM, respectively. The enzymes are both activated by Mg2+, and inhibited by Ca2+ and by adenosine. The potential importance of these effectors for the regulation of PtdIns phosphorylation in cells is discussed.     

Tissue cholinesterases. A comparative study of their kinetic properties

The substrate saturation and temperature-dependent kinetic properties of soluble and membrane-bound forms of acetylcholinestarase (AChE) from brain and butyrylcholinesterase (BChE) from heart and liver were examined. In simultaneous studies these parameters were also measured for AChE in erythrocyte membranes and for BChE in the serum from rat and humans. For both soluble and membrane-bound forms of the enzyme from the three tissues, two components were discernible. In the brain, Km of component I (high affinity) and component II (low affinity) was somewhat higher in membrane-bound form than that of the soluble form components, while the Vmax values were significantly higher by about five fold. In the heart, Km of component II was lower in membrane-bound form than in the soluble form, while Vmax for both the components was about four to six fold higher in the membrane-bound form. In the liver, Vmax was marginally higher for the two components of the membrane-bound enzyme; the Km only of component I was higher by a factor of 2. In the rat erythrocyte membranes three components of AChE were present showing increasing values of Km and Vmax. In contrast, in the human erythrocyte membranes only two components could be detected; the one corresponding to component II of rat erythrocyte membranes was absent. In the rat serum two components of BChE were present while the human serum was found to possess three components. Component I of the human serum was missing in the rat serum. Temperature kinetics studies revealed that the Arrhenius plots were biphasic for most of the systems except for human serum. Membrane binding of the enzyme resulted in decreased energy of activation with shift in phase transition temperature (Tt) to near physiological temperature.     

Stereo-specific substrate recognition by phosphatidylinositol phosphate kinases is swapped by changing a single amino acid residue.

Type I and type II phosphatidylinositol phosphate (PIP) kinases generate the lipid second messenger phosphatidylinositol (PtdIns) 4,5-bisphosphate and thus play fundamental roles in the regulation of many cellular processes. Although the two kinase families are highly homologous, they phosphorylate distinct substrates and are functionally non-redundant. Type I PIP kinases phosphorylate PtdIns 4-phosphate at the D-5 hydroxyl group and are consequently PtdIns 4-phosphate 5-kinases. By contrast, type II PIP kinases are PtdIns 5-phosphate 4-kinases that phosphorylate PtdIns 5-phosphate at the D-4 position. Type I PIP kinases, in addition, also phosphorylate other phosphoinositides in vitro and in vivo and thus have the potential to generate multiple lipid second messengers. To understand how these enzymes differentiate between stereoisomeric substrates, we used a site-directed mutagenesis approach. We show that a single amino acid substitution in the activation loop, A381E in IIbeta and the corresponding mutation E362A in Ibeta, is sufficient to swap substrate specificity between these PIP kinases. In addition to its role in substrate specificity, the type I activation loop is also key in subcellular targeting. The Ibeta(E362A) mutant and other mutants with reduced PtdIns 4-phosphate binding affinity were largely cytosolic when expressed in mammalian cells in contrast to wild-type Ibeta which targets to the plasma membrane. These results clearly establish the role of the activation loop in determining both signaling specificity and plasma membrane targeting of type I PIP kinases.     

Hormonal activation of the cAMP-dependent protein kinases in AtT20 cells. Preferential activation of protein kinase I by corticotropin releasing factor, isoproterenol, and forskolin

The hormonal regulation of adenylate cyclase, cAMP-dependent protein kinase activation, and adrenocorticotropic hormone (ACTH) secretion was studied in AtT20 mouse pituitary tumor cells. Corticotropin releasing factor (CRF) stimulated cAMP accumulation and ACTH release in these cells. Maximal ACTH release was seen with 30 nM CRF and was accompanied by a 2-fold rise in intracellular cAMP. When cells were incubated with both 30 nM CRF and 0.5 mM 3-methylisobutylxanthine (MIX) cAMP levels were increased 20-fold, however, ACTH release was not substantially increased beyond release seen with CRF alone. The activation profiles of cAMP-dependent protein kinases I and II were studied by measuring residual cAMP-dependent phosphotransferase activity associated with immunoprecipitated regulatory subunits of the kinases. Cells incubated with CRF in the absence of MIX showed concentration-dependent activation of protein kinase I which paralleled stimulation of ACTH release. Protein kinase II was minimally activated. When cells were exposed to CRF in the presence of 0.5 mM MIX there was still a preferential activation of protein kinase I, although 50% of the cytosolic protein kinase II was activated. Complete activation of both protein kinases I and II was seen when cells were incubated with 0.5 mM MIX and 10 microM forskolin. Under these conditions cAMP levels were elevated 80-fold. CRF, isoproterenol, and forskolin stimulated adenylate cyclase activity in isolated membranes prepared from AtT20 cells. CRF and isoproterenol stimulated cyclase activity up to 5-fold while forskolin stimulated cyclase activity up to 15-fold. Our data demonstrate that ACTH secretion from AtT20 cells is mediated by small changes in intracellular levels of cAMP and activation of only a small fraction of the total cytosolic cAMP-dependent protein kinase in these cells is required for maximal ACTH secretion.     

Purification and characterization of a cytosolic protein-tyrosine kinase from porcine spleen

A cytosolic protein-tyrosine kinase has been highly purified from porcine spleen using [Val5]angiotensin II as a substrate. The purification procedure involves sequential column chromatographies on phosphocellulose, Sephacryl S-200, casein-Sepharose 4B, heparin-Sepharose CL-6B and anti-(4-aminobenzyl phosphonic acid)--Sepharose 4B. Analysis of the most highly purified preparation by SDS/PAGE revealed a major silver-stained band of molecular mass 40 kDa. The 40-kDa cytosolic protein-tyrosine kinase was purified approximately 10,000-fold with an overall yield of about 7%. It had autophosphorylation activity which was carried out by intramolecular catalysis. The stoichiometry of phosphate incorporation was about 1 mol phosphate/mol enzyme. In the autophosphorylation reaction, the apparent Km value for ATP was relatively low, 0.35 microM; Mn2+ was slightly preferred to Mg2+ as divalent cation. [Val5]Angiotensin II phosphorylation activity of the 40-kDa kinase increased with the amount of phosphate incorporated into the enzyme. A phosphate exchange reaction was observed during the autophosphorylation. These results suggest that the 40-kDa kinase described here is a different type of protein-tyrosine kinase than the enzymes so far reported.