Distribution of protein kinase activities in subcellular fractions of rat brain.

The subcellular distribution of histone and phosvitin kinase activities in brain has been studied and the ability of the various fractions to catalyse the phosphorylation of their endogenous proteins (intrinsic protein kinase activity) also examined. Synaptosome membrane fragments have little or no histone or phosvitin kinase activity but contain the highest concentration of cyclic AMP-stimulated intrinsic protein kinase activity. Homogenisation of the membrane fragments in Triton X-100 increased the histone kinase activity but on centrifugation it was all recovered in the supernatant, while the insoluble material contained all the intrinsic protein kinase activity. These results indicate that the intrinsic protein kinase activity of cerebral membrane fragments is due to the presence of a kinase enzyme which is specific to certain membrane proteins. The intrinsic protein kinase activity of synaptosome membrane fragments is a rather slow reaction which takes several minutes to saturate all the acceptor proteins.     

Adenosine 3',5'-monophosphate-dependent protein kinases of myocardial non-histone nuclear proteins.

Myocardial acidic non-histone nuclear proteins (NHPs) contain endogenous protein kinase activity. Phosphocellulose chromatography of purified NHPs identifies nine separate peaks of protein kinases which can phosphorylate both endogenous and exogenous substrates to a variable degree; endogenous NHPs are the best substrates. Cyclic AMP-stimulated protein kinase induced phosphorylation of endogenous and exogenous substrates; the extent of this stimulation varied according to the protein kinase fraction and substrate used. Cyclic AMP also enhanced NHP-induced stimulation of RNA polymerase activity. This enhancement was dependent on protein kinase-induced phosphorylation of NHPs since it was prevented by alkaline phosphatase pretreatment. It is concluded that nuclear protein kinases regulate myocardial RNA synthesis by enhancing phosphorylation of NHPs and that this regulation is under cyclic AMP control.     

Cyclic AMP-stimulated protein kinases at brain synaptic junctions.

We have examined endogenous cyclic AMP-stimulated phosphorylation of subcellular fractions of rat brain enriched in synaptic plasma membranes (SPM), purified synaptic junctions (SJ), and postsynaptic densities (PSD). The analyses of these fractions are essential to provide direct evidence for cyclic AMP-dependent endogenous phosphorylation at discrete synaptic junctional loci. Protein kinase activity was measured in subcellular fractions using both endogenous and exogenous (histones) proteins as substrates. The SJ fraction possessed the highest kinase activity toward endogenous protein substrates, 5-fold greater than SPM and approximately 120-fold greater than PSD fractions. Although the kinase activity as measured with histones as substrates was only slightly higher in SJ than SPM fractions, there was a marked preference of kinase activity toward endogenous compared to exogenous substrates in SJ fractions but in SPM fractions. Although overall phosphorylation in SJ fractions was increased only 36% by 5 micron cyclic AMP, there were discrete proteins of Mr = 85,000, 82,000, 78,000, and 55,000 which incorporated 2- to 3-fold more radioactive phosphate in the presence of cyclic AMP. Most, if not all, of the cyclic AMP-independent kinase activity is probably catalyzed by catalytic subunit derived from cyclic AMP-dependent kinase, since the phosphorylation of both exogenous and endogenous proteins was greatly decreased in the presence of a heat-stable inhibitor protein prepared from the soluble fraction of rat brain. The specific retention of SJ protein kinase(s) activity during purification and their resistance to detergent solubilization was achieved by chemical treatments which produce interprotein cross-linking via disulfide bridges. Two SJ polypeptides of Mr = 55,000 and 49,000 were photoaffinity-labeled with [32P]8-N3-cyclic AMP and probably represent the regulatory subunits of the type I and II cyclic AMP-dependent protein kinases. The protein of Mr = 55,000 was phosphorylated in a cyclic AMP-stimulated manner suggesting autophosphorylation as previously observed in other systems.     

Distribution of protein kinase activities in subcellular fractions of rat brain

The subcellular distribution of histone and phosvitin kinase activities in brain has been studied and the ability of the various fractions to catalyse the phosphorylation of their endogenous proteins (intrinsic protein kinase activity) also examined. Synaptosome membrane fragments have little or no histone or phosvitin kinase activity but contain the highest concentration of cyclic AMP-stimulated intrinsic protein kinase activity. Homogenisation of the membrane fragments in Triton X-100 increased the histone kinase activity but on centrifugation it was all recovered in the supernatant, while the insoluble material contained all the intrinsic protein kinase activity. These results indicate that the intrinsic protein kinase activity of cerebral membrane fragments is due to the presence of a kinase enzyme which is specific to certain membrane proteins. The intrinsic protein kinase activity of synaptosome membrane fragments is a rather slow reaction which takes several minutes to saturate all the acceptor proteins.     

Tyrosine-protein kinase inhibition in human erythrocytes by polyphosphoinositides (PIP and PIP2).

In human erythrocytes Ser/Thr- and Tyr-phosphorylations of cytoplasmic domain of band 3 are catalyzed by casein kinase I and Tyr-protein kinase respectively, both distributed between cytosol and membrane structures. The results reported here show that purified cytosolic Tyr-protein kinase activity, assayed on added substrates such as poly(Glu,Tyr)4:1 and isolated chymotryptic fragments of band 3 cytoplasmic domain (cdb3), is potently inhibited by PIP and even more by PIP2. Similar inhibitory effects are displayed by these polyphosphoinositides also on the endogenous Tyr-phosphorylation of band 3, when they are added to the isolated native membranes, thus suggesting their involvement in regulating in-vivo Tyr-phosphorylation of membrane proteins.     

The angiogenesis inhibitor beta-cyclodextrin tetradecasulfate inhibits ecto-protein kinase activity.

The growth of new blood vessels plays an important role in the pathogenesis of several diseases including cancer, diabetes, and arthritis. Beta-cyclodextrin tetradecasulfate, when administered with an appropriate steroid inhibits angiogenesis, and can stimulate angiogenesis when given alone. The regulation of angiogenesis is not well understood, and the mechanism of action of beta-cyclodextrin tetradecasulfate is similarly not well defined. Ecto-protein kinase activity that utilizes extracellular ATP has recently been reported on several types of cells. Human neutrophils appear to possess two distinct ecto-protein kinase activities; one that phosphorylates exogenous substrates including vitronectin and basic fibroblast growth factor, and one that phosphorylates endogenous cell-surface proteins. This report shows that beta-cyclodextrin tetradecasulfate inhibits the phosphorylation of the exogenous substrates casein, vitronectin (the major ecto-protein kinase substrate in serum), and basic fibroblast growth factor by human neutrophil ecto-protein kinase activity. In contrast, beta-cyclodextrin tetradecasulfate had no effect on the phosphorylation of endogenous cell-surface proteins by the neutrophil ecto-protein kinase activity. Ecto-protein kinase activity that was inhibited by beta-cyclodextrin tetradecasulfate was also detected on porcine aortic and human umbilical vein endothelial cells. The effects of beta-cyclodextrin tetradecasulfate on ecto-protein kinase activities may play a role in its effects on angiogenesis.     

Phosphorylation of cardiac sarcolemma by endogenous and exogenous protein kinases.

Sarcolemmal membranes isolated from guinea pig heart ventricles contained endogenous protein kinase activity and protein substrates for this enzyme. Phosphorylation of sarcolemma was modestly stimulated by cyclic AMP with the half-maximal stimulation at 0.5 μm cyclic AMP. The phosphorylation of sarcolemma due to endogenous kinase was dependent on Mg²⁺. The apparent affinity for Mg²⁺ was found to be 1.4 and 0.53 mm in the absence and presence of 1 μm cyclic AMP, respectively. The apparent affinity for ATP was 55 μm. Sarcolemmal membranes were also phosphorylated by exogenous (purified) cyclic AMP-dependent protein kinase(s). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of phosphorylated membranes, followed by slicing and determination of the radioactivity in the gel slices, showed that endogenous protein kinase activity promoted the phosphorylation of specific protein peaks, arbitrarily designated a–g in order of increasing relative mobility (relative molecular weights 125,000, 110,000, 86,000, 58,000, 48,000, 22,000, and 16,000, respectively); peak e (48,000) was the major phosphorylated band. Exogenous protein kinase stimulated the phosphorylation of all peaks. However, the degree of stimulation of the low molecular weight peaks f and g was more marked. Results obtained after treatment of phosphorylated membranes with hydroxylamine at acid pH indicated the absence of any significant amount of acyl phosphate-type incorporation of phosphate. Purified phosphoprotein phosphatase from rabbit liver effected dephosphorylation of previously phosphorylated sarcolemma; this treatment resulted in dephosphorylation of all peaks (a–g). Pretreatment of sarcolemma with trypsin (membrane to trypsin ratio of 100) was found to markedly reduce both the total membrane phosphorylation as well as relative phosphorylation of peaks c, f, and g. On the other hand, pretreatment of sarcolemma with phospholipase c slightly stimulated total membrane phosphorylation with nondiscriminatory enhancement of the phosphorylation of all peaks. Microsomal membrane vesicles (enriched in sarcoplasmic reticulum fragments) isolated from guinea pig heart ventricle also contained endogenous protein kinase activity. Cyclic AMP modestly increased the kinase. Polypeptides of molecular weights 56,000, 22,000, and 16,000 were found to be phosphorylated. Exogenous (purified) cyclic AMP-dependent protein kinase increased the phosphorylation of microsomes and of 22,000 and 16,000 molecular weight polypeptides.     

Characterization of human neutrophil ecto-protein kinase activity released by kinase substrates.

Although most studies of protein phosphorylation have focused on intracellular protein kinases, evidence for protein kinase activity on the surface of several types of cells has been described. Evidence was recently provided for the existence of ecto-protein kinase activity on the surface of human neutrophils. Evidence for three distinct ecto-protein kinase activities was detected, one that phosphorylates endogenous surface proteins, one that phosphorylates exogenous substrates in a cAMP-independent manner and is released in the presence of substrate, and a low level of activity of one that phosphorylates exogenous Kemptide in a cAMP-dependent manner. To begin to elucidate its role in neutrophil function, we have characterized several properties of the releasable ecto-protein kinase activity on human neutrophils. This enzyme activity was inhibited by impermeant stilbene disulfonic acids, which are known to alter neutrophil function, as well as by impermeant sulfhydryl reactive agents. Enzyme activity was detectable at physiologic concentrations of Mg2+, but was higher in the presence of Mn2+. Protein kinase activity was strongly inhibited by heparin, whereas trifluoperazine, cAMP, and cGMP had little effect on kinase activity. Protein kinase activity was selectively removed from the cell surface by incubation with the ecto-kinase substrates casein and phosvitin, but the enzyme was not released by phosphatidylinositol-specific phospholipase C. Repeated exposure of neutrophils to substrate depleted ecto-protein kinase activity from the cell surface, but activity was rapidly restored by incubation in buffer lacking substrate. The released protein kinase had a Km for ATP of approximately 0.5 microM and a pH maximum between 7.0 and 7.5. At least four ecto-protein kinase substrates were detected in serum; vitronectin was identified as one of these substrates by immunoprecipitation studies. Although the exact role of ecto-protein kinase activity in neutrophil function remains undefined, the identification of vitronectin as a serum substrate suggests that it interacts with a physiologically important substrate.     

A comparison of the intrinsic protein kinase activities of membrane preparations from various tissues.

The ability of membrane preparations from different tissues to catalyse the phosphorylation of their endogenous protein (intrinsic protein kinase activity) was determined. It was found that membrane fragments prepared from a large variety of tissues contain this activity although the actual level varies quite widely. Preparations from vas deferens and brain have nearly ten times more activity than preparations from heart, kidney, or erythrocytes. Plasma membranes from skeletal muscle have no detectable activity. The intrinsic protein kinase activity of membrane fragments from most tissues is stimulated by cyclic AMP although the phosphorylation of proteins in preparations of kidney microsomes or heart plasma membranes, is not affected. cyclic GMP (10 micronM) has no effect on the intrinsic protein kinase activity of any membrane preparation examined. A specific inhibitor of soluble, cyclic AMP-stimulated, protein kinase has no effect on the intrinsic protein kinase activity of any of the membrane preparations examined. This suggests that the intrinsic protein kinase activity of membrane preparations may be due to the presence of a specific protein kinase. It is suggested that an examination of the distribution of membrane-bound intrinsic protein kinase activity among different tissues may be helpful in determining the function of the reaction.     

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.     

Synaptic membrane proteins as substrates for cyclic AMP-stimulated protein phosphorylation in various regions of rat brain.

Synaptosomal plasma membranes from mammalian brain contain protein kinase activity which phosphorylates endogenous membrane proteins and is stimulated by cyclic AMP. Using polyacrylamide gel electrophoresis it was shown that at least ten proteins in the synaptosomal plasma membrane fraction could be phosphorylated by endogenous cyclic AMP-stimulated protein kinase activity. The number of proteins whose phosphorylation was stimulated by cyclic AMP was strongly influenced by the pH and Mg2+ concentration used in the phosphorylation reaction. A complex pattern of cyclic AMP-stimulated protein phosphorylation was obtained only with synaptosomal plasma membranes and a crude microsomal fraction. Mitochondrial and myelin fractions exhibited no cyclic AMP-stimulated protein kinase activity. Investigation of the distribution of substrates for cyclic AMP-stimulated phosphorylation among various brain regions failed to reveal any regional differences.     

Correlation of Ca2+-and calmodulin-dependent protein kinase activity with secretion of insulin from islets of Langerhans.

A Ca2+-activated and calmodulin-dependent protein kinase activity which phosphorylates predominantly two endogenous proteins of 57kDa and 54kDa was found in a microsomal fraction from islet cells. Half-maximal activation of the protein kinase occurs at approx. 1.9 microM-Ca2+ and 4 micrograms of calmodulin/ml (250 nM) for phosphorylation of both protein substrates. Similar phosphoprotein bands (57kDa and 54kDa) were identified in intact islets that had been labelled with [32P]Pi. Islets prelabelled with [32P]Pi and incubated with 28 mM-glucose secreted significantly more insulin and had greater incorporation of radioactivity into the 54 kDa protein than did islets incubated under basal conditions in the presence of 5 mM-glucose. Thus the potential importance of the phosphorylation of these proteins in the regulation of insulin secretion is indicated both by activation of the protein kinase activity by physiological concentrations of free Ca2+ and by correlation of the phosphorylation of the substrates with insulin secretion in intact islets. Experiments undertaken to identify the endogenous substrates indicated that this calmodulin-dependent protein kinase may phosphorylate the alpha- and beta-subunits of tubulin. These findings suggest that Ca2+-stimulated phosphorylation of islet-cell tubulin via a membrane-bound calmodulin-dependent protein kinase may represent a critical step in the initiation of insulin secretion from the islets of Langerhans.     

Partial purification and properties of a cyclic 3',5'-AMP-independent protein kinase from rat liver.

1. A cyclic 3',5'-AMP-independent protein kinase (ATP : protein phosphotransferase, EC 2.7.1.37) from rat liver cytosol was partially purified and characterized. Purification by (NH4)2SO4 precipitation, DEAE-cellulose, Bio Gel A-0.5 m and cellulose phosphate chromatography increased the specific activity about 700-fold. 2. An endogenous protein substrate was closely associated with the protein kinase and was not separable from this enzyme up to the cellulose phosphate stage. After phosphorylation, chromatography with Bio Gel A-0.5 m partially separated this endogenous phosphoprotein from the enzyme activity; this dissociation had no apparent effect on kinase activity with casein or phosvitin as substrates, or on the apparent molecular weight of the enzyme (approx. 158,000). 3. This protein kinase with casein, phosvitin, or the endogenous substrate was totally insensitive to the thiol reagents, p-hydroxymercuribenzoate, 5,5'-dithiobis(2-nitrobenzoic acid), iodoacetamide, and N-ethylmaleimide. The enzyme was also unaffected by cyclic 3',5'-AMP, heat-stable protein kinase inhibitor, and the regulatory subunit of a cyclic 3',5'-AMP-dependent protein kinase.     

Phosphorylation of endogenous and exogenous peptides by rat heart sarcolemma

Rat heart plasma membranes contain a calcium-dependent protein kinase which phosphorylates endogenous protein substrates as well as added histones. The major endogenous protein phosphorylated is of 17 kDa on SDS-polyacrylamide gel electrophoresis. Proteins of 85 kDa and 60 kDa were also phosphorylated. Treatment of a rat heart homogenate with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate increased the recovery of kinase activity in the sarcolemmal membranes by up to 10-fold. The activity in such membranes was no longer calcium dependent. Although several histones were effective substrates for the enzyme, myosin light chain and phosvitin were not phosphorylated. These membranes contain a very active ATP hydrolysing activity which necessitated very brief incubation times to avoid loss of substrate. The membranes also contain cyclic AMP dependent protein kinase activity which is not active unless cyclic AMP is added to the incubations. The calcium dependent endogenous kinase, which is not inhibited by the heat stable inhibitor protein of cyclic AMP-dependent kinase, or by trifluoperazine, has several properties in common with protein kinase C. Preincubation of the sarcolemmal membranes with a high concentration of insulin caused inhibition of the phosphorylation of the endogenous 17 kDa and 85 kDa bands. There was no effect on the phosphorylation of the 60 kDa peptide. This effect of insulin was specific for the hormone and required preincubation of the hormone with the membranes for 20 min.     

Protein substrate specificity of a calmodulin-dependent protein kinase isolated from bovine heart

The protein substrate specificity of a calmodulin-dependent protein kinase activity from the cytosolic fraction of bovine heart was examined. Prior to the experiments, the kinase activity was purified more than 50-fold with a recovery of greater than 10% of the homogenate activity. Two endogenous protein substrates of molecular weight 57,000 and 73,000 were phosphorylated in these kinase preparations. The kinase preparation was also able to phosphorylate exogenous synapsin, phospholamban, glycogen synthase, MAP-2, myelin basic proteins and κ-casein, but not tubulin, pyruvate kinase, the regulatory subunit of cAMP protein kinase II, myosin light chain or phosphorylase b. High levels of calmodulin were required for activation of the kinase activity toward the 57,000 and 73,000 molecular weight endogenous substrates (K_0.5 = 93 +/- 5 nM), glycogen synthase (K_0.5 = 127 +/- 10 nM), and κ-casein (K_0.5 = 321 +/- 107 nM). The kinase possessed a high affinity for glycogen synthase (half maximal activity at 0.9 +/- 0.4 μM) but a low affinity for κ-casein (21 +/- 2 μM). Sucrose density gradient centrifugation separated the calmodulin-dependent protein kinase activity into two fractions with apparent molecular weights of approximately 900,000 and 100,000. Both fractions phosphorylated the endogenous 57,000 molecular weight substrate and glycogen synthase similarly. These results indicate that cardiac calmodulin-dependent protein kinase previously observed to phosphorylate endogenous protein substrate possesses a wide range of substrate specificity.     

Plasma membrane cyclic AMP-dependent protein phosphorylation system in L6 myoblasts.

Plasma membranes can be isolated without disruption of cells by the plasma membrane vesiculation technique (Scott, R.E. (1976) Science 194, 743-745). A major advantage of this technique is that it avoids contamination of plasma membranes with intracellular membrane components. Using this method, we prepared plasma membranes from L6 myoblasts grown in tissue culture and studied the characteristics of the protein phosphorylation system. We found that these plasma membrane preparations contain protein kinase which is tightly bound to the membrane and cannot be removed by washing in EDTA or in high ionic strength salt solutions. This protein kinase activity can catalyze the phosphorylation of several exogenous substrates with decreasing efficiency as acceptors of phosphate: calf thymus histones f2b, protamine and caseine. Cyclic AMP causes a dose-dependent stimulation of protein kinase activity; the highest stimulation (4-fold) is achieved at concentration 10(-5) M cyclic AMP. Cyclic AMP-dependent stimulation can be completely inhibited by heat-stable protein kinase inhibitor isolated from rabbit skeletal muscle. On the other hand, cyclic GMP does not affect the activity of protein kinase. Plasma membrane-bound protein kinase also catalyzes the phosphorylation of endogenous membrane protein substrates and this is also stimulated by addition of cyclic AMP. Analysis of plasma membrane proteins by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis showed that specific polypeptides are phosphorylated by cyclic AMP-independent and by cyclic AMP-dependent protein kinase systems. The results of these studies demonstrate the presence of endogenous cyclic AMP-dependent and -independent protein phosphorylating systems (enzyme activity and substrates) in purified plasma membrane preparations. These data provide a basis for further investigations on the role of plasma membrane phosphorylation as a regulator of membrane functions including those that may control cellular differentiation.     

Cyclic nucleotide-independent protein kinase from pea shoots

Protein kinase has been isolated from 6-day old etiolated pea shoots. Crude homogenates contain endogenous protein substrates for the kinase. Casein or phosvitin, but not histone, can serve as substrates for assay. DEAE-cellulose columns distinguish several forms of protein kinase activity. Cyclic nucleotides do not modify the activity of these protein kinases $\text{in vitro}$.     

Phosphoproteins of the Adrenal Chromaffin Granule Membrane

A fraction of chromaffin granule membranes contained a number of substrates for endogenous protein kinase activity as well as endogenous phosphatase activity. The major 32P-labelled polypeptide of molecular weight 43,000 appeared to be the alpha-subunit of pyruvate dehydrogenase of residual mitochondria. Several polypeptides showed cyclic AMP stimulation of phosphorylation of which the major polypeptide of molecular weight 59,000 shows half-maximal phosphorylation with 0.49 microM cyclic AMP. The phosphorylation of several other polypeptides is inhibited at high cyclic AMP concentrations. From studies with immunoprecipitation and two-dimensional electrophoresis it was found that alpha- and beta-tubulin and actin were absent from the granule membranes. However 32P labelling of a proportion of the copies of dopamine-beta-hydroxylase was demonstrated. The majority of the substrates for endogenous protein kinase activity are probably on the cytoplasmic side of the granule membrane.     

Measuring MAP kinase activity in immune complex assays

I present an overview of published methods for measuring mitogen activated protein (MAP) kinase activity on endogenous associated substrates, exogenously added substrates as well as determination of activation loop phosphorylation as a read-out of kinase activity in vivo. Detailed procedures for these assays are given for two MAP kinases (MAPKs) Fus3 and Kss1 and compared with other published protocols, including the protocols for Hog1 and Mpk1 MAPKs. Measuring kinase activity in immune complex assays can serve as an approach for identification of potential substrates of protein kinases as well as for detecting other kinase-associated proteins.     

CD66 monoclonal antibodies recognize a phosphotyrosine-containing protein bearing a carcinoembryonic antigen cross-reacting antigen on the surface of human neutrophils.

The CD66 Ag is a neutrophil-specific "activation Ag" in that it is detected in low density on resting cells but its surface expression is up-regulated by stimulation (with the chemotactic peptide FMLP, the calcium ionophore A23187, and 12-O-tetradeconoyl-phorbol-13-acetate). Phosphorylation is an important mechanism of regulation of protein function. Although most studies of protein phosphorylation have focused on intracellular reactions, recent studies have provided evidence for the existence of ectoprotein kinase activity on the surface of several types of cells including human neutrophils. The role of ectoprotein kinase activity in cell function is unknown and little is known about the endogenous substrates of this enzyme system. The identification and characterization of physiologic substrates of ectoprotein kinase activity should aid the understanding of the role of this enzyme activity in cell function. Immunoprecipitation and subsequent gel electrophoresis of proteins from neutrophils labeled with [gamma-32P]ATP revealed that CD66 mAb specifically recognize a approximately 180-kDa phosphoprotein on the surface of human neutrophils. This protein was one of the major endogenous substrates for human neutrophil ectoprotein kinase activity. Phosphoamino acid analysis of the 180-kDa protein revealed that it contained predominantly phosphotyrosine. Preclearing studies demonstrated that this protein was also recognized by CD15 mAb, and by polyclonal anticarcinoembryonic Ag antiserum. In addition, the CD66 mAb reacted with purified carcinoembryonic Ag, biliary glycoprotein, and "nonspecific cross-reacting Ag." Thus, the neutrophil protein recognized by CD66 mAb appears to be a approximately 180-kDa form of the classical "nonspecific cross-reacting Ag" on human neutrophils.