Phosphorylation of thylakoid proteins by a purified kinase

A simplified method is given for the purification of a 64-kilodalton protein kinase from spinach or pea thylakoid membranes (Coughlan, S., and Hind, G. (1986) J. Biol. Chem. 261, 11378-11385). In a heterogeneous reconstitution system comprised of purified kinase and washed thylakoids (having their intrinsic kinase inactivated or removed), endogenous light-harvesting pigment protein of photosystem II could serve as a substrate. Its phosphorylation did not require rebinding of kinase to the thylakoid membrane and, like the phosphorylation of solubilized pigment protein, was not under redox control. No reconstitution was observed upon replacing 64-kilodalton protein kinase with 25-kilodalton protein kinase (Coughlan, S., and Hind, G. (1986) J. Biol. Chem. 261, 14062-14068). Tryptic digestion of phosphorylated membranes removed the site of phosphorylation; the phosphorylated amino acid present in light-harvesting pigment protein and its tryptic peptide was threonine. Immunoglobulin from a polyclonal antiserum, raised against the purified enzyme, fully inhibited kinase activity toward solubilized and endogenous pigment protein. At higher titers, the antibody was effective in totally inhibiting the redox-sensitive phosphorylation of thylakoid proteins by endogenous kinase; inhibition profiles for phosphorylation of pigment protein and thylakoid proteins of 32, 16, and 9 kilodaltons were essentially identical. The 64-kilodalton protein kinase would thus appear to be responsible for all of the observed phosphorylation of thylakoid phosphoproteins.     

Phosphatidylglycerol-modulated protein kinase activity from human spleen. II. Interaction with phospholipid vesicles

Phosphorylation of endogenous and artificial protein substrates by protein kinase P is stimulated by phosphatidylinositol or phosphatidylglycerol (D. J. Klemm, and L. Elias (1987) J. Biol. Chem. 262, 7580-7585; L. Elias and A. Davis (1985) J. Biol. Chem. 260, 7023-7028). Stimulation of protein kinase P activity required phospholipid vesicles rather than free phospholipid molecules. Protein kinase P activity increased as the phosphatidylinositol content of the vesicles was raised from 20 to 100%; no stimulation was detected below 20% phosphatidylinositol. This suggests that a vesicle surface rich in phosphatidylinositol is required for enzyme activation. Maximum activation of protein kinase P activity showed an optimum value with respect to phospholipid concentration, with both endogenous and artificial protein substrates. The phospholipid concentration at which optimal enzyme activity occurred shifted in response to the concentration of protein substrate, but not enzyme concentration. Therefore, the density of substrate molecules on the surface of phospholipid vesicles is a critical feature of protein kinase P stimulation. Binding of protein kinase P to vesicles was independent of micelle composition, but the binding of the artificial substrate, histone H2B, was specific for vesicles containing phosphatidylinositol or phosphatidylglycerol, and increased as the content of phosphatidylinositol was increased. Thus, an important feature of protein kinase P activation appeared to be the specific binding of protein substrate to phospholipid vesicles.     

The catalytic requirements for reduction and acetylation of protein X and the related regulation of various forms of resolved pyruvate dehydrogenase kinase

The pyruvate dehydrogenase kinase consists of a catalytic subunit (Kc) and a basic subunit (Kb) which appear to be anchored to the dihydrolipoyl transacetylase core component (E2) by another subunit, referred to as protein X (Rahmatullah, M., Jilka, J. M., Radke, G. A., and Roche, T. E. (1986) J. Biol. Chem. 261, 6515-6523). We determined the catalytic requirements for reduction and acetylation of the lipoyl moiety in protein X and linked those changes in protein X to regulatory effects on kinase activity. Using fractions prepared by resolution and proteolytic treatments, we evaluated which subunits are required for regulatory effects on kinase activity. With X-KcKb fraction (treated to remove the mercurial agent used in its preparation), we found that the resolved pyruvate dehydrogenase component, the isolated inner domain of E2 (lacking the lipoyl-bearing region of E2), and the dihydrolipoyl dehydrogenase component directly utilize protein X as a substrate. The resulting reduction and acetylation of protein X occurs in association with enhancement of kinase activity. Following tryptic cleavage of E2 and protein X into subdomains, full acetylation of the lipoyl-bearing subdomains of these proteins is retained along with the capacity of acetylating substrates to stimulate kinase activity. All kinase-containing fractions, including those in which the Kb subunit was digested, were inhibited by pyruvate or ADP, alone, and synergistically by the combination suggesting that pyruvate and ADP bind to Kc. Our results suggest that the Kb subunit of the kinase does not contribute to the observed regulatory effects. A dynamic role of protein X in attenuating kinase activity based on changes in the mitochondrial redox and acetylating potentials is considered.     

Protamine kinase from yeast.

A protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) which preferentially phosphorylates protamine is purified about 250-fold from the soluble fraction of baker's yeast (Saccharomyces cerevisiae). This enzyme is not sensitive to activation by cyclic nucleotides. Histone is about 5% as active as protamine in the reaction rate. Neither casein, phosvitin nor glycogen phosphorylase is active as substrate. The enzyme is distinguishable from casein kinase of the classical type (Rabinowitz, M. and Lipmann, F. (1960) J. Biol. Chem. 235, 1043-1050) and from adenoshine 3', 5'-monophosphate-dependent protein kinase described earlier (Takai, Y., Yamamura, H. and Nishizuka, Y. (1974) J. Biol. Chem. 249,530-535).     

Identification of an endogenous protein kinase C activity and its intrinsic 15-kilodalton substrate in purified canine cardiac sarcolemmal vesicles

The cardiac sarcolemmal 15-kDa protein, previously shown to be the principal sarcolemmal substrate phosphorylated in intact heart in response to beta-adrenergic stimulation (Presti, C. F., Jones, L. R., and Lindemann J. P. (1985) J. Biol. Chem. 260, 3860-3867), was demonstrated to be the major substrate phosphorylated in purified canine cardiac sarcolemmal vesicles by an intrinsic protein kinase C activity. The intrinsic protein kinase C, detected by its ability to phosphorylate H1 histones, was most concentrated in cardiac sarcolemmal vesicles and absent from sarcoplasmic reticulum membranes. Unmasking techniques localized the intrinsic protein kinase activity and its principal endogenous substrate, the 15-kDa protein, to the cytoplasmic surfaces of sarcolemmal vesicles; phospholamban contaminating the sarcolemmal preparation was not significantly phosphorylated. The intrinsic protein kinase C required micromolar Ca2+ for activity, but not calmodulin. Half-maximal phosphorylation of the 15-kDa protein occurred at 10 microM Ca2+; optimal phosphorylation of the 15-kDa protein by protein kinase C and Ca2+ was additive to that produced by cAMP-dependent protein kinase. Exogenous phospholipids were not required to activate endogenous protein kinase C. However, heat-treated sarcolemmal vesicles, in which intrinsic protein kinase activities were inactivated, were sufficient to maximally activate soluble protein kinase C prepared from rat brain, suggesting that all the necessary phospholipid cofactors were already present in sarcolemmal vesicles. Of the many proteins present in sarcolemmal vesicles, only the 15-kDa protein was phosphorylated significantly in heat-inactivated sarcolemmal vesicles by soluble protein kinase C, confirming that the 15-kDa protein was a preferential substrate for this enzyme. Consistent with a protein kinase C activity in sarcolemmal vesicles, the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate stimulated 15-kDa protein phosphorylation severalfold, producing approximately 70% of the maximal phosphorylation even in the absence of significant ionized Ca2+. The results are compatible with an intrinsic protein kinase C activity in sarcolemmal vesicles whose major substrate is the 15-kDa protein.     

Protein kinases of the thylakoid membrane

The claim of Racker and co-workers (Lin, Z. F., Lucero, H. A., and Racker, E. (1982) J. Biol. Chem. 257, 12153-12156 and Lucero, H. A., Lin, Z. F., and Racker, E. (1982) J. Biol. Chem. 257, 12157-12160) that two protein kinases, designated CPK1 (25 kDa) and CPK2 (38 kDa), are present in spinach thylakoid membranes was investigated in light of results from this laboratory (Coughlan, S. J., and Hind, G. (1986) J. Biol. Chem. 261, 11378-11385) showing that 75-80% of the measurable protein kinase activity of isolated thylakoids is attributable to a protein kinase of 64 kDa apparent molecular mass. Extraction of thylakoid membranes with octyl glucoside/cholate according to the procedure of Lin et al. (Lin, Z. F., Lucero, H. A., and Racker, E. (1982) J. Biol. Chem. 257, 12153-12156) released proteins assignable to CPK1 and CPK2 on the basis of photoaffinity labeling with 8-azido-[32P]ATP. The 64-kDa protein kinase was present in this extract and accounted for greater than 80% of the total phosphotransferase activity toward lysine-rich histone as substrate; it was not labeled by the photoaffinity reagent. The three presumptive kinases were purified by ammonium sulfate precipitation, sucrose density gradient centrifugation, hydroxylapatite chromatography, and affinity chromatography. CPK1 was specifically eluted from Cibacron blue-Sepharose by 10 mM ATP; it electrophoresed on denaturing polyacrylamide gels as a single band with apparent molecular mass of 25 kDa. Its specific activity toward lysine-rich histone as substrate was approximately 250 pmol of phosphate transferred (mg protein)-1 min-1. The 64-kDa protein kinase was eluted from the affinity column by 1% (w/v) lithium dodecyl sulfate or from a histone IIIs-Sepharose affinity column by 0.25 M NaCl. Its specific activity towards lysine-rich histone was 100-200 times greater than that of CPK1. CPK2 eluted from the Cibacron blue affinity column in 10 mM NADP+; it had an apparent molecular mass of 38 kDa, possessed NADPH-dependent diaphorase activity (specific activity: 225 nmol of ferricyanide reduced (mg protein)-1 min-1), and cross-reacted with immunoglobulin raised against purified ferredoxin:NADP+ oxidoreductase, with which it was thus identified. Kinase activity was not detectable in CPK2 or in reductase isolated by conventional procedures.     

Regulation of calcium transport by protein phosphatase activity associated with cardiac sarcoplasmic reticulum

Canine cardiac sarcoplasmic reticulum is phosphorylated by an endogenous calcium-calmodulin-dependent protein kinase on a 22,000 proteolipid, called phospholamban. Phosphorylation by the calcium-calmodulin-dependent protein kinase is associated with stimulation of the initial rates of calcium transport (Davis, B. A., Schwartz, A., Samaha, F. J., and Kranias, E. G. (1983) J. Biol. Chem. 258, 13587-13591). The present study shows that protein phosphatase activity, associated with canine cardiac sarcoplasmic reticulum vesicles, can catalyze dephosphorylation of the calcium-calmodulin-dependent sites on phospholamban. The activity was maximally stimulated by manganese; fluoride was inhibitory, but its effect was reversible. Dephosphorylation of phospholamban, which was prephosphorylated by calcium-calmodulin-dependent protein kinase, resulted in a reduction of the stimulation on calcium transport rates, particularly at submaximal calcium concentrations. The decrease in calcium transport was associated with a statistically significant decrease in the apparent affinity (EC50) for calcium. Rephosphorylation of phospholamban by the endogenous calcium-calmodulin-dependent protein kinase caused full recovery of the stimulation on calcium transport rates and reversal of the effects mediated by the protein phosphatase. Thus, the calcium pump in cardiac sarcoplasmic reticulum appears to be under reversible regulation mediated by endogenous calcium-calmodulin-dependent protein kinase and protein phosphatase. Such regulation may represent an important control mechanism for the myocardium.     

Sphingosine inhibition of protein kinase C activity and of phorbol dibutyrate binding in vitro and in human platelets

Sphingosine inhibited protein kinase C activity and phorbol dibutyrate binding. When the mechanism of inhibition of activity and phorbol dibutyrate binding was investigated in vitro using Triton X-100 mixed micellar methods, sphingosine inhibition was subject to surface dilution; 50% inhibition occurred when sphingosine was equimolar with sn-1,2-dioleoylglycerol (diC18:1) or 40% of the phosphatidylserine (PS) present. Sphingosine inhibition was modulated by Ca2+ and by the mole percent of diC18:1 and PS present. Sphingosine was a competitive inhibitor with respect to diC18:1, phorbol dibutyrate, and Ca2+. Increasing levels of PS markedly reduced inhibition by sphingosine. Since protein kinase C activity shows a cooperative dependence on PS, the kinetic analysis of competitive inhibition was only suggestive. Sphingosine inhibited phorbol dibutyrate binding to protein kinase C but did not cause protein kinase C to dissociate from the mixed micelle surface. Sphingosine addition to human platelets blocked thrombin and sn-1,2-dioctanoylglycerol-dependent phosphorylation of the 40-kDa (47 kDa) dalton protein. Moreover, sphingosine was subject to surface dilution in platelets. The mechanism of sphingosine inhibition is discussed in relation to a previously proposed model of protein kinase C activation. The possible physiological role of sphingosine as a negative effector of protein kinase C is suggested and a plausible cycle for its generation is presented. The potential physiological significance of sphingosine inhibition of protein kinase C is further established in accompanying papers on HL-60 cells (Merrill, A. H., Jr., Sereni, A. M., Stevens, V. L., Hannun, Y. A., Bell, R. M., Kinkade, J. M., Jr. (1986) J. Biol. Chem. 261, 12010-12615) and human neutrophils (Wilson, E., Olcott, M. C., Bell, R. M., Merrill, A. H., Jr., and Lambeth, J. D. (1986) J. Biol. Chem. 261, 12616-12623). These results also suggest that sphingosine will be a useful inhibitor for investigating the function of protein kinase C in vitro and in living cells.     

Pro-Leu-Ser/Thr-Pro is a consensus primary sequence for substrate protein phosphorylation. Characterization of the phosphorylation of c-myc and c-jun proteins by an epidermal growth factor receptor threonine 669 protein kinase.

A growth factor-stimulated (MAP2-related) protein kinase, ERT, that phosphorylates the epidermal growth factor receptor at Thr669 has been purified from KB human tumor cells by Northwood and co-workers (Northwood, I. C., Gonzalez, F. A., Wartmann, M., Raden, D. L., and Davis, R. J. (1991) J. Biol. Chem. 266, 15266-15276). The ERT protein kinase has a restricted substrate specificity, and the structural determinants employed for substrate recognition by this enzyme have not been defined. As an approach toward understanding the specificity of substrate phosphorylation, we have used an in vitro assay to identify additional substrates for the ERT protein kinase. In this report we describe two novel substrates: (a) the human c-myc protein at Ser62 and (b) the rat c-jun protein at Ser246. Alignment of the primary sequences surrounding the phosphorylation sites located within the epidermal growth factor receptor (Thr669), Myc (Ser62), and Jun (Ser246) demonstrated a marked similarity. The observed consensus sequence was Pro-Leu-Ser/Thr-Pro. We propose that this sequence forms part of a substrate structure that is recognized by the ERT protein kinase.     

Mechanism of self-phosphorylation of adenosine 3':5'-monophosphate-dependent protein kinase from bovine cardiac muscle.

The adenosine 3':5'-monophosphate (cAMP)-dependent protein kinase purified from bovine cardiac muscle catalyzes the transfer of up to 2 mol of 32P from [lambda-32P]ATP to seryl residues in its cyclic nucleotide-binding protein component (Erlichman, J., Rosenfeld, R., and Rosen, O. M. (1974) J. Biol. Chem. 249, 5000-5003). We now present three lines of evidence to support our conclusions that the undissociated holoenzyme does not catalyze the phosphorylation of exogenous substrates but can undergo self-phosphorylation by an intramolecular reaction: (a) addition of either cAMP-binding protein or the protein kinase inhibitor (Walsh, D. A., Ashby C. D., Gonzales, C., Calkins, D., Fischer, E. H., and Krebs, D. G. (1971) J. Biol. Chem. 241, 1977-1985) does not inhibit self-phosphorylation as it does phosphorylation of exogenous substrates in the presence or absence of cAMP; (b) addition of catalytic subunit to an excess of cyclic nucleotide-binding protein results in phosphorylation equivalent to the amount of holoenzyme so generated; (c) the rate of self-phosphorylation is not affected by dilution of the holoenzyme.     

Sphingosine-dependent protein kinase-1, directed to 14-3-3, is identified as the kinase domain of protein kinase C delta

Some protein kinases are known to be activated by d-erythro-sphingosine (Sph) or N,N-dimethyl-d-erythro-sphingosine (DMS), but not by ceramide, Sph-1-P, other sphingolipids, or phospholipids. Among these, a specific protein kinase that phosphorylates Ser60, Ser59, or Ser58 of 14-3-3beta, 14-3-3eta, or 14-3-3zeta, respectively, was termed "sphingosine-dependent protein kinase-1" (SDK1) (Megidish, T., Cooper, J., Zhang, L., Fu, H., and Hakomori, S. (1998) J. Biol. Chem. 273, 21834-21845). We have now identified SDK1 as a protein having the C-terminal half kinase domain of protein kinase Cdelta (PKCdelta) based on the following observations. (i). Large-scale preparation and purification of proteins showing SDK1 activity from rat liver (by six steps of chromatography) gave a final fraction with an enhanced level of an approximately 40-kDa protein band. This fraction had SDK1 activity approximately 50000-fold higher than that in the initial extract. (ii). This protein had approximately 53% sequence identity to the Ser/Thr kinase domain of PKCdelta based on peptide mapping using liquid chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry data. (iii). A search for amino acid homology based on the BLAST algorithm indicated that the only protein with high homology to the approximately 40-kDa band is the kinase domain of PKCdelta. The kinase activity of PKCdelta did not depend on Sph or DMS; rather, it was inhibited by these sphingoid bases, i.e. PKCdelta did not display any SDK1 activity. However, strong SDK1 activity became detectable when PKCdelta was incubated with caspase-3, which releases the approximately 40-kDa kinase domain. PKCdelta and SDK1 showed different lipid requirements and substrate specificity, although both kinase activities were inhibited by common PKC inhibitors. The high susceptibility of SDK1 to Sph and DMS accounts for their important modulatory role in signal transduction.     

Urate synthesis in the blood-sucking insect rhodnius prolixus. Stimulation by hemin is mediated by protein kinase C.

Hemin is a catalyst of the formation of reactive oxygen species. We proposed that hematophagous insects are exposed to intense oxidative stress because of hemoglobin hydrolysis in their midgut (Petretsky, M. D., Ribeiro, J. M. C., Atella, G. C., Masuda, H., and Oliveira, P. L. (1995) J. Biol. Chem. 270, 10893-10896). We have shown that hemin stimulates urate synthesis in the blood-sucking insect Rhodnius prolixus (Gra?a-Souza, A. V., Petretsky, J. H., Demasi, M., Bechara, E. J. H., and Oliveira, P. L. (1997) Free Radical Biol. Med. 22, 209-214). Once released by fat body cells, urate accumulates in the hemolymph, where this radical scavenger constitutes an important defense against blood-feeding derived oxidative stress. Incubation of Rhodnius fat bodies with okadaic acid raises the level of urate synthesis, suggesting that urate production can be controlled by protein phosphorylation/dephosphorylation. Urate synthesis is stimulated by dibutyryl cAMP and inhibited by N(2((p-bromocinnamil)amino)ethyl)-5-isoquinolinesulfonamide (H-89), an inhibitor of protein kinase A, as well as activated by the protein kinase C activator phorbol 12-myristate 13-acetate. In the presence of hemin, however, inhibition of urate synthesis by H-89 does not occur, suggesting that the hemin stimulatory effect is not mediated by protein kinase A. Calphostin C completely inhibits the hemin-induced urate production, suggesting that the triggering of urate antioxidant response depends on protein kinase C activation. This conclusion is reinforced by the observation that in fat bodies exposed to hemin, both protein kinase C activity and phosphorylation of specific endogenous polypeptides are significantly increased.     

Phosphorylation of a 22,000-dalton component of the cardiac sarcoplasmic reticulum by adenosine 3':5'-monophosphate-dependent protein kinase.

Cardiac microsomes were incubated with [gamma-32P]ATP and a cardiac adenosine 3':5'-monophosphate (cyclic AMP)-dependent protein kinase in the presence of ethylene glycol bis(bets-aminoethyl ether)-N,N'-tetraacetic acid. After solubilization in sodium dodecyl sulfate and fractionation by polyacrylamide gel electrophoresis, a single microsomal protein component of approximately 22,000 daltons was found to bind most of the 32P label. The 32P labeling of this component increased several fold when NaF was included in the incubation medium. No other component of cardiac microsomes, including sarcoplasmic reticulum ATPase protein, contained significant amounts of 32P label. This 22,000-dalton phosphoprotein formed by cyclic AMP-dependent protein kinase had stability characteristics of a phosphoester rather than an acyl phosphate. Washing of microsomes with buffered KCl did not decrease the amount of 32P labeling to the 22,000-dalton protein, suggesting that this protein is associated with the membranes of sarcoplasmic reticulum rather than being a contaminant from other soluble proteins. The 22,000-dalton protein was susceptible to trypsin. Brief digestion with trypsin in the presence of 1 M sucrose did not significantly affect microsomal calcium transport activity, but prevented both subsequent phosphorylation of the 22,000-dalton protein and stimulation of calcium uptake by cyclic AMP-dependent protein kinase, suggesting that this protein is a modulator of the calcium pump. These results are consistent with previous findings (Kirchberger, M.A., Tada, M., and Katz, A.M. (1974) J. Biol. Chem. 249, 6166-6173; Tada, M., Kirchberger, M.A., Repke, D.I., and Katz, A.M. (1974) J. Biol. Chem. 249, 6174-6180) that cyclic AMP-dependent protein kinase-catalyzed phosphorylation is associated with stimulation of calcium transport in the cardiac sarcoplasmic reticulum, and further indicate that this phosphorylation occurs at a component of low mass (22,000 daltons) of the cardiac sarcoplasmic reticulum which, while separable from the calcium transport ATPase protein (100,000 daltons) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, has the ability to regulate calcium transport by the cardiac sarcoplasmic reticulum.     

Phosphorylation by cyclin-dependent protein kinase 5 of the regulatory subunit of retinal cGMP phosphodiesterase. I. Identification of the kinase and its role in the turnoff of phosphodiesterase in vitro

Cyclic GMP phosphodiesterase (PDE) is an essential component in retinal phototransduction. PDE is regulated by Pgamma, the regulatory subunit of PDE, and GTP/Talpha, the GTP-bound alpha subunit of transducin. In previous studies (Tsuboi, S., Matsumoto, H. , Jackson, K. W., Tsujimoto, K., Williamas, T., and Yamazaki, A. (1994) J. Biol. Chem. 269, 15016-15023; Tsuboi, S., Matsumoto, H., and Yamazaki, A. (1994) J. Biol. Chem. 269, 15024-15029), we showed that Pgamma is phosphorylated by a previously unknown kinase (Pgamma kinase) in a GTP-dependent manner in photoreceptor outer segment membranes. We also showed that phosphorylated Pgamma loses its ability to interact with GTP/Talpha, but gains a 10-15 times higher ability to inhibit GTP/Talpha-activated PDE than that of nonphosphorylated Pgamma. Thus, we propose that the Pgamma phosphorylation is probably involved in the recovery phase of phototransduction through shut off of GTP/Talpha-activated PDE. Here we demonstrate that all known Pgammas preserve a consensus motif for cyclin-dependent protein kinase 5 (Cdk5), a protein kinase believed to be involved in neuronal cell development, and that Pgamma kinase is Cdk5 complexed with p35, a neuronal Cdk5 activator. Mutational analysis of Pgamma indicates that all known Pgammas contain a P-X-T-P-R sequence and that this sequence is required for the Pgamma phosphorylation by Pgamma kinase. In three different column chromatographies of a cytosolic fraction of frog photoreceptor outer segments, the Pgamma kinase activity exactly coelutes with Cdk5 and p35. The Pgamma kinase activity ( approximately 85%) is also immunoprecipitated by a Cdk5-specific antibody, and the immunoprecipitate phosphorylates Pgamma. Finally, recombinant Cdk5/p35, which were expressed using clones from a bovine retina cDNA library, phosphorylates Pgamma in frog outer segment membranes in a GTP-dependent manner. These observations suggest that Cdk5 is probably involved in the recovery phase of phototransduction through phosphorylation of Pgamma complexed with GTP/Talpha in mature vertebrate retinal photoreceptors.     

Activation of rabbit skeletal muscle adenosine-3':5'-monophosphate-dependent protein kinase by agitation.

Protein kinase activity in a preparation from rabbit skeletal muscle (Wastila, W.B., Stull, J.T., Mayer, S.E., and Walsh, D.A. (1971)J. Biol. Chem. $\text{246}$, 1996–2003.) was increased appr. 15-fold after a 30 to 40 sec agitation of the incubation mixture on a Vortex mixer in either the presence or absence of the protein substrate, histone. Saturating concentrations of adenosine-3′:5′-monophosphate (cAMP) stimulated the activity appr. 23-fold. 0.1% Triton X-100, present during the agitation, completely prevented agitation-induced activation, but left cAMP-dependent activation unaffected.     

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.     

Isolation and characterization of two growth factor-stimulated protein kinases that phosphorylate the epidermal growth factor receptor at threonine 669.

A growth factor-stimulated protein kinase activity that phosphorylates the epidermal growth factor (EGF) receptor at Thr669 has been described (Countaway, J. L., Northwood, I. C., and Davis, R. J. (1989) J. Biol. Chem. 264, 10828-10835). Anion-exchange chromatography demonstrated that this protein kinase activity was accounted for by two enzymes. The first peak of activity eluted from the column corresponded to the microtubule-associated protein 2 (MAP2) kinase. However, the second peak of activity was found to be a distinct enzyme. We present here the purification of this enzyme from human tumor KB cells by sequential ion-exchange chromatography. The isolated protein kinase was identified as a 46-kDa protein by polyacrylamide gel electrophoresis and silver staining. Gel filtration chromatography demonstrated that the enzyme was functional in a monomeric state. A kinetic analysis of the purified enzyme was performed at 22 degrees C using a synthetic peptide substrate based on the primary sequence of the EGF receptor (KREL VEPLT669PSGEAPNQALLR). The Km(app) for ATP was 40 +/- 5 microM (mean +/- S.D., n = 3). GTP was not found to be a substrate for the purified enzyme. The Km(app) for the synthetic peptide substrate was 260 +/- 40 microM (mean +/- S.D., n = 3). The Vmax(app) for the isolated protein kinase was determined to be 400-900 nmol/mg/min. The purified enzyme was designated EGF receptor Thr669 (ERT) kinase. It is likely that the MAP2 and ERT kinases account for the phosphorylation of the EGF receptor at Thr669 observed in cultured cells. The marked stimulation of protein kinase activity caused by growth factors indicates that these enzymes may have an important function during signal transduction.     

Characterization of the interaction of 5'-p-fluorosulfonylbenzoyl adenosine with the epidermal growth factor receptor/protein kinase in A431 cell membranes

Treatment of membrane vesicles from A431 cells, a human epidermoid carcinoma line, with the affinity label 5'-p-fluorosulfonylbenzoyl [8-14C]adenosine (5'-p-FSO2Bz[14C]Ado) results in an inhibition of the epidermal growth factor (EGF)-stimulable protein kinase and in the modification of proteins having the same molecular weight (Mr = 170,000 and 150,000) as the receptor for EGF (Buhrow, S. A., Cohen, S., and Staros, J. V. (1982) J. Biol. Chem. 257, 4019-4022). Modification of the vesicles with 5'-p-FSO2BzAdo inhibits not only the EGF-stimulated phosphorylation of endogenous membrane proteins but also the EGF-stimulated phosphorylation of an exogenous synthetic tyrosine-containing peptide substrate. This indicates that the EGF-stimulable protein kinase is modified by 5'-p-FSO2BzAdo at a site affecting catalytic activity. Membrane vesicles were treated with 5'-p-FSO2Bz-[14C]Ado to affinity label the kinase, then the EGF receptor was purified by affinity chromatography on immobilized EGF. The EGF receptor thus purified contains the 5'-p-SO2Bz[14C]Ado moiety. These data strongly support our hypothesis that the EGF receptor and EGF-stimulable kinase are two parts of the same polypeptide chain.     

Internalization of functional epidermal growth factor:receptor/kinase complexes in A-431 cells

We have isolated and partially purified an intracellular vesicle fraction from A-431 cells that contains both epidermal growth factor (EGF) and enzymatically active EGF:receptor/kinase. Exposure of intact A-431 cells to EGF leads to an accumulation of both EGF and kinase activity in this vesicle fraction. The accumulation is time- and temperature-dependent and is blocked by inhibitors of energy production. The EGF receptor in internalized vesicles is capable of autophosphorylation and, in the presence of Ca2+, of phosphorylation of the previously isolated 35-kDa protein (Fava, R. A., and Cohen, S. (1984) J. Biol. Chem. 259, 2636-2645). The demonstration of an EGF-induced increase in kinase activity of an internalized vesicle fraction lends credence to the hypothesis that EGF-induced endocytosis of the receptor is of physiological significance in the response of cells to this ligand. In addition, these results are consistent with the suggestion that the phosphorylation of the 35-kDa protein is associated with internalization of the EGF:receptor/kinase complex.