Purification, Properties, and Phosphorylation by Protein Kinase C of Two Phosphoinositidase C Isozymes from Rat Brain

Two forms of phosphoinositidase C have been purified from the soluble fraction of rat brain. The purification scheme included gel filtration followed by chromatography on cellulose phosphate, phenyl-Sepharose, and Mono Q. Gradient sodium dodecyl sulphate-polyacrylamide gel electrophoresis gave apparent molecular masses of 151 kDa and 147 kDa. Western blotting with monoclonal antibodies showed that the isozymes corresponded to PLC-beta-1 and PLC-gamma of bovine brain. With both enzymes phosphatidylinositol 4,5-bisphosphate was a better substrate than phosphatidylinositol at neutral pH and low calcium ion concentrations. Both enzymes produced a proportion of inositol 1:2-cyclic phosphates from each substrate, particularly at acid pH. Some GTPase activity was seen in the early stages of purification, but was separated from PLC-beta-1 and PLC-gamma on Mono Q. Purified rat brain protein kinase C phosphorylated PLC-gamma but not PLC-beta-1. Incubation with the kinase increased the activity of both enzymes however, possibly by phosphorylation of another protein in the preparations.     

Phosphoprotein Component of Vaccinia Virions

The recent discovery of a protein kinase activity in vaccinia virions led us to search for a viral protein which is phosphorylated in vivo. Vaccinia virus was radioactively labeled by infecting cells in the presence of (32)P(1). A phosphoprotein was isolated from purified delipidated virions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The phosphoprotein appeared to be a specific viral component induced after infection. More than 60% of the phosphoprotein was associated with viral cores. The electrophoretic mobility of the protein suggested that it has a molecular weight of 11,000 to 12,000. Phosphoserine was liberated by acid hydrolysis and identified by electrophoresis with known standards. Tryptic digests of the purified phosphoprotein were analyzed by two-dimensional electrophoresis and chromatography on thin-layer cellulose plates, and a single major phosphopeptide was resolved. The high selectivity of phosphorylation suggested that the process has a specific function.     

Purification and properties of a multifunctional calcium/calmodulin-dependent protein kinase from rat pancreas

A calcium/calmodulin-dependent protein kinase (Ca/calmodulin protein kinase) was purified from rat pancreas using hydrophobic chromatography followed by gel filtration and affinity chromatography. Ca/calmodulin protein kinase from pancreas resembled previously described multifunctional Ca/calmodulin protein kinases from other tissues with respect to substrate specificity, autophosphorylation on serine and threonine residues, and catalytic and hydrodynamic properties. While Ca/calmodulin protein kinase from other tissues contains subunits of 53-60 kDa with variable proportions of a smaller 50-52 kDa subunit, pancreatic Ca/calmodulin protein kinase was found to contain a single component of 51 kDa. Experiments mixing brain Ca/calmodulin protein kinase with pancreatic homogenate suggest that the absence of a larger subunit in the pancreatic Ca/calmodulin protein kinase is not due to proteolytic degradation during enzyme preparation. Ca/calmodulin protein kinase binding to 125I-labeled calmodulin in solution was demonstrated using the photoaffinity cross-linker, N-hydroxysuccinimidyl-4-azidobenzoate. 125I-labeled calmodulin binding to Ca/calmodulin protein kinase was also demonstrated using filters containing Ca/calmodulin protein kinase transferred from polyacrylamide gels after two-dimensional gel electrophoresis. Finally, the ribosomal substrate for Ca/calmodulin protein kinase was identified as the ribosomal protein, S6. The purification procedure presented in this study promises to be useful in characterizing Ca/calmodulin protein kinase in other tissues and in clarifying the role of these enzymes in cellular function.     

Phosphorylation of an acidic mol. wt. 80 000 cellular protein in a cell-free system and intact Swiss 3T3 cells: a specific marker of protein kinase C activity.

Activation of the endogenous Ca2+-activated phospholipid-dependent protein kinase (protein kinase C) by Ca2+, phosphatidylserine (PS) and phorbol dibutyrate (PBt2) in detergent-solubilized extracts of Swiss 3T3 cells resulted in a very rapid increase (detectable within seconds) in the phosphorylation of an 80 000 mol. wt. protein (termed 80 K). Neither cyclic AMP nor Ca2+ had any effect on 80 K phosphorylation. The 80 K phosphoproteins generated after activation of protein kinase C, both in cell-free conditions and in intact fibroblasts, are identical as judged by one and two-dimensional polyacrylamide slab gel electrophoresis and peptide mapping. Prolonged treatment of cells with phorbol esters causes a selective decrease in protein kinase C activity and prevents the stimulation of 80 K phosphorylation in intact fibroblasts. We now show that extracts from PBt2-treated cultures fail to stimulate 80 K phosphorylation after the addition of the protein kinase C activators. This effect was due to the lack of protein kinase C activity since the addition of exogenous protein kinase C from mouse brain stimulated 80 K phosphorylation in both control and PBt2-treated preparations. The 80 K phosphoprotein generated by activation of endogenous and exogenous protein kinase C yielded similar phosphopeptide fragments after peptide mapping by limited proteolysis. We conclude that the detection of changes in the phosphorylation of 80 K provides a useful approach to ascertain which extracellular ligands activate protein kinase C in intact cells.     

Dephosphin, a 96,000 Da substrate of protein kinase C in synaptosomal cytosol, is phosphorylated in intact synaptosomes

A 96,000 dalton phosphoprotein, called dephosphin, is phosphorylated in intact synaptosomes from rat brain and is rapidly dephosphorylated upon depolarisation-dependent calcium entry. A 96,000 dalton phosphoprotein is also a substrate of protein kinase C in synaptosomal cytosol, and the aim of the study was to determine whether the two proteins may be the same. Dephosphin in intact synaptosomes and the 96,000 dalton protein kinase C substrate comigrated on polyacrylamide gels. Both phosphoproteins had identical phosphopeptide maps after digestion with V8 protease. Both phosphoproteins ran on isoelectric focussing gels with a pI of 6.3-6.7 and focussed as a series of 5-6 spots. Both proteins were phosphorylated exclusively on serine. Both proteins could be resolved into a doublet on longer polyacrylamide gels. The two subunits were of 96 and 93 kDa in both phosphorylation conditions and had dissimilar phosphopeptide maps. However, phosphopeptide maps of either the 96 or 93 kDa subunits were identical in intact synaptosomes compared with synaptosomal cytosol. These results show that a phosphoprotein phosphorylated in intact synaptosomes and a 96,000 dalton protein kinase C substrate from rat brain synaptosomal cytosol are the same, and raise the possibility that protein kinase C is the protein kinase responsible for dephosphin phosphorylation in intact synaptosomes.     

Vasopressin stimulates the phosphorylation of an 83,000M r protein in the superior cervical ganglion

1. 32P-Labeled proteins from the superior cervical ganglion of the rat were separated by two-dimensional gel electrophoresis and visualized by autoradiography. 2. The most heavily labeled phosphoprotein in the ganglion had a relative molecular weight of 83,000 and a pI of 4.5. Phosphorylation of this protein was increased by phorbol 12,13-dibutyrate, an activator of the Ca2+/phospholipid-dependent protein kinase, protein kinase C. This protein appears to be similar or identical to a specific protein kinase C substrate that has been described in other tissues (Blackshear, P. J., et al., J. Biol. Chem. 261:1459-1469, 1986). 3. Phosphorylation of this protein was also increased by treatment of the ganglion with phospholipase C (Bacillus cereus) but was not increased by 8-bromo-cyclic AMP or by nicotinic agonists. Vasopressin increased the hydrolysis of inositol-containing phospholipids in the ganglion and also increased the labeling of the 83,000 Mr protein. Thus, vasopressin appears to activate protein kinase C in the ganglion. 4. Muscarine, which also increased phospholipid metabolism in the ganglion, did not increase the phosphorylation of the 83,000 Mr protein. Muscarine and vasopressin stimulate phospholipid metabolism in different structures within the ganglion (Horwitz, J., et al., J. Pharmacol. Exp. Ther. 237:312-317, 1986). Muscarine may increase phospholipid metabolism in structures that do not contain significant amounts of the 83,000 Mr protein.     

Self-phosphorylation of cyclic guanosine 3':5'-monophosphate-dependent protein kinase from bovine lung. Effect of cyclic adenosine 3':5'-monophosphate, cyclic guanosine 3':5'-monophosphate and histone.

Incubation of purified cyclic guanosine 3':5'-monophospate-dependent protein kinase with [gamma-32P]ATP and Mg2+ led to formation of one 32P-labeled protein, Mr = 75,000, which corresponded to the single protein band detected after polyacrylamide gel electrophoresis in sodium dodecyl sulfate. When electrophoresis was performed without detergent, the labeled protein coincided with the position of cGMP-dependent protein kinase activity. Phosphorylation was enhanced severalfold by either histone or cAMP and was inhibited by the addition of cGMP. Low concentrations of cGMP blocked the stimulatory effects of cAMP or histone (or both). Since neither cAMP-dependent protein kinase nor cGMP-dependent phosphoprotein phosphatase activities were detected in the purified enzyme, we concluded that the cGMP-dependent protein kinase is a substrate for its own phosphotransferase activity and that other protein substrates (histone) and cyclic nucleotides modulate the process of self-phosphorylation.     

Characterization of insulin-stimulated microtubule-associated protein kinase. Rapid isolation and stabilization of a novel serine/threonine kinase from 3T3-L1 cells

A protein kinase, termed microtubule-associated protein (MAP) kinase, which phosphorylates microtubule-associated protein 2 (MAP-2) in vitro and is stimulated 1.5-3-fold in extracts from insulin-treated 3T3-L1 cells has been identified (Ray, L.B., and Sturgill, T.W. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 1502-1506). Here, we describe chromatographic properties of MAP kinase and provide biochemical characterization of the partially purified enzyme. Isolation of the enzyme is facilitated by its unusually high affinity for hydrophobic interaction chromatography matrices. The molecular weight of the partially purified enzyme was determined to be 35,000 by gel filtration chromatography and 37,000 by glycerol gradient centrifugation. MAP kinase activity of chromatographic fractions correlated precisely with the presence of a 40-kDa phosphoprotein detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. MAP kinase has a Km of 7 microM for ATP and does not utilize GTP. Acetyl-CoA carboxylase, ATP citrate-lyase, casein, histones, phosvitin, protamine, and ribosomal protein S6 were all poor substrates relative to MAP-2. The enzyme is inhibited by fluoride and beta-glycerol phosphate but not by heparin. These properties of MAP kinase distinguish it from protein kinases previously described in the literature.     

A Protein Modulator Stimulates C Kinase-Dependent Phosphorylation of a 90K Substrate in Synaptic Membranes

We have identified and partially purified an acidic, heat-stable, noncalmodulin protein from bovine brain cytosol that stimulates Ca2+-dependent phosphorylation of an Mr 90K substrate in crude rat brain synaptic membranes. We show that this modulator of phosphorylation (MOP) enhances Ca2+- and phospholipid-dependent protein kinase (C kinase) phosphorylation of this 90K substrate. The 90K substrate is a higher Mr form of an 87K substrate that is a major C kinase substrate in rat brain. The Ca2+-dependent phosphorylation of both substrates is inhibited by the Ca2+-binding proteins S-100 and calmodulin. Both substrates yield phosphopeptide fragments of Mr 9K and 13K after limited proteolysis with V8 protease. Two-dimensional polyacrylamide gel electrophoresis reveals that they have similar acidic isoelectric points (pI 5.0). MOP enhances Ca2+-dependent phosphorylation of the 90K substrate whereas the phosphorylation of 87K is diminished. This reciprocal relationship suggests that the mobility of the 87K substrate in sodium dodecyl sulfate-polyacrylamide gels is decreased to 90K with increasing phosphorylation. MOP may be a novel protein modulator of C kinase-mediated phosphorylation in the nervous system.     

Protein inhibitors of phosphorylase phosphatase and cyclic AMP-dependent protein kinase from rabbit skeletal muscle

Summary A heat- and acid-stable proten inhibitor of phosphorylase phosphatase is present in a highly purified preparation of protein inhibitor of cyclic AMP-dependent protein kinase from rabbit skeletal muscle. Although these two inhibitors have strikingly similar properties to each other, such as sensitivity to trypsin and behavior on gel permeation chromatography, they can be separated by polyacrylamide disc gel electrophoresis. This indicates that the phosphatase-inhibitory and kinase-inhibitory activities reside with different protein species. The inhibition of both the enzymes is not altered by incubating the inhibitor preparation with a general phosphoprotein phosphatase, with phosvitin kinase, or with cyclic AMP-dependent protein kinase. Inhibition of phosphorylase phosphatase is of a non-competitive type supporting the idea that the phosphatase inhibitor is not an alternative substrate for the enzyme. Inhibition of phosphatase activity is selective in that it does not occur when phosphorylated histone or phosphorylated protamine are used as substrates.     

Phosphorylation of the GABAa/Benzodiazepine Receptor α Subunit by a Receptor-Associated Protein Kinase

Abstract: Partially purified preparations of GABA_a/benzodiazepine receptor from rat brain were found to contain high levels of a protein kinase activity that phosphorylated a small number of proteins in the receptor preparations, including a 50-kilodalton (kD) phosphoprotein that comigrated on two-dimensional electrophoresis with purified, immunolabeled, and photolabeled receptor α subunit. Further evidence that the comigrating 50-kD phosphoprotein was, in fact, the receptor α subunit was obtained by peptide mapping analysis: the 50-kD phosphoprotein yielded one-dimensional peptide maps identical to those obtained from iodinated, purified α subunit. Phosphoamino acid analysis revealed that the receptor α subunit is phosphorylated on serine residues by the protein kinase activity present in receptor preparations. Preliminary characterization of the receptor-associated protein kinase activity suggested that it may be a second messenger-independent protein kinase. Protein kinase activity was unaltered by cyclic AMP, cyclic GMP, calcium plus calmodulin, calcium plus phosphatidylserine, and various inhibitors of these protein kinases. Examination of the substrate specificity of the receptor-associated protein kinase indicated that the enzyme preferred basic proteins as substrates. Endogenous phosphorylation experiments indicated that the receptor α subunit may also be phosphorylated in crude membranes by a protein kinase activity present in those membranes. As with phosphorylation of the receptor in purified preparations, its phosphorylation in crude membranes also appeared to be unaffected by activators and inhibitors of second messenger-dependent protein kinases. These findings raise the possibility that the phosphorylation of the α subunit of the GABA_a/ benzodiazepine receptor by a receptor-associated protein kinase plays a role in modulating the physiological activity of the receptor in vivo.     

Purification and characterization of hormone-regulated isoforms of the regulatory subunit of type II cAMP-dependent protein kinase from rat ovaries

The regulatory subunit (R-II) of cAMP-dependent protein kinase type II is induced in rat ovarian granulosa cells by the synergistic actions of estradiol and follicle-stimulating hormone. The R-II from rat ovaries was compared with R-II from rat heart, rat brain, bovine heart, and bovine brain using immunological methods, 8-N3[32P]cAMP photoaffinity labeling and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Three isoforms of R-II were identified in rat ovarian cell extract (R-II54 Mr = 54,000, R-II52 Mr = 52,000, R-II51 Mr = 51,000), two isoforms of R-II in rat brain cell extract (Mr = 54,000, Mr = 52,000), and one isoform of R-II in rat heart cell extract (Mr = 54,000). Rat ovarian R-II54, heart R-II, and brain R-II (Mr = 54,000) were recognized by antiserum against rat heart R-II, whereas rat ovarian R-II52/R-II51 and rat brain R-II (Mr = 52,000) were not. In contrast, an antiserum raised against bovine heart R-II recognized all three isoforms of ovarian R-II as well as the lower molecular weight form of rat brain R-II. Ovarian types R-II52 and R-II51 but not R-II54 were increased selectively in granulosa cells by estradiol and follicle-stimulating hormone. In addition: 1) ovarian R-II52/51 subunits were purified to homogeneity and shown to recombine with C subunit from bovine heart to form a cAMP-dependent protein kinase; 2) pure R-II52/51 were not interconvertible to a higher molecular weight form by C subunit-dependent phosphorylation; 3) pure rat heart R-II (Mr = 54,000) and ovarian R-II52/51 exhibited distinct differences based on one- and two-dimensional peptide mapping; and 4) by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis pure R-II52/51 were resolved as three (rather than two) isoelectric variants which were clearly different from pure rat heart R-II54. Thus, the hormone-regulated form of R-II in rat ovarian granulosa cells appears to represent a gene product distinct from R-II54 in rat heart.     

Protein kinase C located in rat liver nuclei. Partial purification and biochemical and immunochemical characterization

In the rat liver homogenate, maximal protein kinase C activity was found at two calcium concentrations (1.75 and 3.5 mM). Subcellular fractionation of the liver homogenate revealed that the protein kinase C activity requiring 1.75 mM calcium was present only in the cytosolic and particulate subcellular fractions. The protein kinase C activity requiring 3.5 mM calcium concentration was mainly located in the rat liver nuclei preparation. About 19% of the liver homogenate protein kinase C activity requiring 3.5 mM calcium was present in the nuclei. Goat anti-rat brain protein kinase C antibodies revealed a single immunoreactive band at 80-82 kDa in the rat liver nuclear, particulate, or cytosolic fractions. Based on the ratio of plasma membrane marker enzyme activity determined in the nuclear preparation, the purity of the isolated nuclei was ascertained. Rat liver nuclear protein kinase C activity has been partially purified. The purification steps sequentially employed were Triton X-100 extraction of isolated nuclei, DEAE-cellulose chromatography, Phenyl-Superose, and Mono Q (fast protein liquid) chromatography. The final purification step revealed, by silver nitrate staining on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, two protein bands at 80 and 66 kDa, respectively. These findings provide definitive data regarding the nuclear location of protein kinase C. The nuclear location of protein kinase C may lead to an understanding of the molecular pathway involved in signal transduction from the plasma membrane to the nucleus.     

Nuclear phosphoprotein kinase activities in normal and neoplastic tissues.

Nuclear phosphoprotein kinases from normal rat liver and transplantable neoplasms were fractionated and compared. A phosphoprotein kinase fraction activated by Mn2+ was found to be present only in the neoplasms. This nuclear protein kinase phosphorylated nuclear proteins represented by one major and several minor bands as determined by polyacrylamide gel electrophoresis (M approximately 50,000).     

Neuromodulin (GAP43): a neuronal protein kinase C substrate is also present in 0-2A glial cell lineage. Characterization of neuromodulin in secondary cultures of oligodendrocytes and comparison with the neuronal antigen

Neuromodulin (also called GAP43, G50, F1, pp46), a neural-specific calmodulin binding protein, is a major protein kinase C substrate found in developing and regenerating neurons. Here, we report the immunocytochemical characterization of neuromodulin in cultured 0-2A bipotential glial precursor cells obtained from newborn rat brain. Neuromodulin is also present in oligodendrocytes and type 2 astrocytes (stellate-shaped astrocytes), which are both derived from the bipotential glial 0-2A progenitor cells, but is absent of type 1 astrocytes (flat protoplasmic astrocytes). These results support the hypothesis of a common cell lineage for neurons and bipotential 0-2A progenitor cells and suggest that neuromodulin plays a more general role in plasticity during development of the central nervous system. The expression of neuromodulin in secondary cultures of newborn rat oligodendrocytes and its absence in type 1 astrocytes was confirmed by Northern blot analysis of isolated total RNA from these different types of cells using a cDNA probe for the neuromodulin mRNA and by Western blot analysis of the cell extracts using polyclonal antibodies against neuromodulin. The properties of the neuromodulin protein in cultured oligodendrocytes and neuronal cells have been compared. Although neuromodulin in oligodendrocytes is soluble in 2.5% perchloric acid like the neuronal counterpart it migrates essentially as a single protein spot on two-dimensional gel electrophoresis whereas the neuronal antigen can be resolved into at least three distinct protein spots. To obtain precise alignments of the different neuromodulin spots from these two cell types, oligodendrocyte and neuronal cell extracts were mixed together and run on the same two-dimensional gel electrophoresis system. Oligodendroglial neuromodulin migrates with a pI identical to the basic forms of the neuronal protein in isoelectric focusing gel. However, the glial neuromodulin shows a slightly lower mobility in the second dimensional lithium dodecyl sulfate-PAGE than its neuronal counterpart. As measured by 32Pi incorporation, neuromodulin phosphorylation in oligodendrocytes is dramatically increased after short-term phorbol ester treatments, which activate protein kinase C, and is totally inhibited by long-term phorbol ester treatments, which downregulates protein kinase C, thus confirming its probable specific in vivo phosphorylation by protein kinase C. In primary cultures of neuronal cells, two of the three neuromodulin spots were observed to be phosphorylated with an apparent preferential phosphorylation of the more acid forms.     

Vasopressin rapidly stimulates protein kinase C in quiescent Swiss 3T3 cells

Addition of vasopressin to quiescent cultures of Swiss 3T3 cells caused a rapid increase in the phosphorylation of an acidic molecular weight 80,000 cellular protein (termed 80K). The effect was concentration- and time-dependent; enhancement in 80K phosphorylation could be detected as early as 30 sec after the addition of the hormone. Recently, a rapid increase in the phosphorylation of an 80K cellular protein following treatment with phorbol esters or diacylglycerol has been shown to reflect the activation of protein kinase C in intact Swiss 3T3 cells. Here we show that the 80K phosphoproteins generated in response to vasopressin and phorbol 12,13-dibutyrate (PBt2) were identical as judged by one- and two-dimensional polyacrylamide gel electrophoresis (PAGE) and peptide mapping following partial proteolysis with Staphylococcus aureus V8 protease. In addition, prolonged pretreatment of 3T3 cells with PBt2 which leads to the disappearance of protein kinase C activity blocked the ability of vasopressin to stimulate the phosphorylation of 80K. The effect of vasopressin on 80K phosphorylation and mitogenesis was selectively blocked by the vasopressin antagonist (Pmp1-O-Me-Tyr2-Arg8) vasopressin suggesting that these responses are mediated by its specific receptor in these cells. The removal of vasopressin leads to dephosphorylation (within minutes) of the 80K phosphoprotein. We conclude that vasopressin rapidly stimulates protein kinase C activity in intact 3T3 cells.     

Human brain creatine kinase binding to immobilized cibacron blue F3GA: characterization and use in purification of the enzyme.

Creatine kinase (ATP: creatine N-phosphotransferase, EC 2.7.3.2) from adult human brain grey matter was purified by cibacron blue F3GA-Sepharose affinity chromatography. By gel electrophoresis of the purified enzyme under non-denaturing conditions a single protein band was observed. The dye-bound enzyme was eluted using its substrate, ATP. Reversibility of the binding of purified creatine kinase to blue Sepharose by ATP in a concentration-dependent manner indicated that the cibacron blue molecule which structurally mimics nucleotides occupied the substrate binding site of the enzyme. Also the marked dependence of enzyme binding to blue Sepharose on Mg2+ concentration suggested that Mg2+ ion is capable of combining with the dye moiety to form a site-specific binding complex that is similar to the physiological substrate of creatine kinase, namely Mg(2+)-ATP or Mg(2+)-ADP.     

A protein kinase C inhibitory activity is present in rat brain homogenate

The partial purification and characterization of (a) factor(s) from rat brain which inhibit(s) the activity of calcium and phospholipid-dependent protein kinase from the same tissue is described. This factor, present in 100 000 X g rat brain homogenate supernatant, is inactivated upon treatment by trypsin and pepsin and is therefore assumed to be a protein. It was partially purified by ion-exchange chromatography on DEAE-cellulose, ammonium sulfate precipitation and gel filtration. This inhibitor is not stable to heating at 70 degrees C for 10 min, however partial renaturation of the inhibitory activity can be observed after incubation of the denatured inhibitor for 24 h at 4 degrees C. It is precipitable by 10% trichloroacetic acid and by 2 M ammonium sulfate. It exhibits a Stokes radius of 20 A by gel exclusion chromatography, corresponding to a molecular mass of 20 kDa assuming a globular shape. Kinetic analysis of the inhibition of calcium-phospholipid-dependent histone kinase activity indicates that the inhibitor is competitive with respect to the protein substrate. No change was observed in the kinetic values of the kinase for ATP, Ca2+ and phospholipids.     

Detection and identification of a tumor-associated heteroorganic antigen of Zajdela hepatoma in non-histone proteins of chromatin

By polyacrylamide gel electrophoresis, a phosphoprotein with mol. weight of 42 kDa was detected in non-histone proteins (NHP) of chromatin of Zajdela ascitic hepatoma cells eluted from phosphocellulose with 0.4-0.5 M NaCl. A protein of the same mol. weight is present in narrow fractions of rat kidney chromatin, but is absent in rat liver. It is suggested that the revealed protein corresponds to the tumor-associated heteroorganic NHP antigen detected earlier in NHP chromatin of rat tumor cells. By MALDI mass spectrometry, this phosphoprotein was identified as ERK2/mitogen-activated protein kinase.     

Purification of two distinct proteins of approximate Mr 80,000 from human epithelial cells and identification as proper substrates for protein kinase C.

A Mr-80,000 acidic phosphoprotein ('80K protein') is a specific substrate for protein kinase C. We attempted to purify the 80K protein from a human squamous-cell carcinoma cell line, Ca9-22, by the sequential use of heat treatment, (NH4)2SO4 precipitation, Mono Q column chromatography, proRPC column chromatography and gel filtration. The 80K protein was assayed by phosphorylation in vitro by using partially purified human type III protein kinase C, and was fractionated into two distinct molecular species with slightly different Mr values, designated 80K-L and 80K-H proteins. Phosphorylation occurred mainly at serine residues of these proteins. Two-dimensional phosphopeptide maps after trypsin digestion and kinetic profiles of phosphorylation were different from each other. Ca2(+)- and phospholipid-dependency of the phosphorylation in vitro confirmed that both 80K-L and 80K-H proteins are true substrates for three subtypes of protein kinase C. The 80K-L protein was a preferential substrate for type III protein kinase C, and the 80K-H protein was phosphorylated more effectively by type I and type II protein kinase C. The possible roles of these two distinct 80K proteins in signal transduction are discussed.