Characterization of a protease that acts specifically on the 22-kDa protein in thylakoid membranes from green spores of the fern Osmunda japonica

A proteolytic enzyme responsible for the breakdown of a 22-kDa protein, whose abundance decreases in thylakoid membranes during germination of green spores of the fern Osmunda japonica , was partially purified from the thylakoid membranes of quiescent spores by a combination of ammonium sulfate fractionation, ion-exchange chromatography on DEAE-Toyopearl 650 S and size-fractionation HPLC on G3000SW. The enzyme was found to be a cysteine endoproteinase, as judged by its dependency on sulfhydryl reagents and inhibition by E-64 and iodoacetate, and by the appearance of distinct proteolytic products that were not further degraded during prolonged reaction time. Highest protease activity was observed around pH 9.7, the activity being partially suppressed by cations. The K_m of the 22-kDa protein as a substrate in the proteolysis was 67 μg ml⁻¹, equivalent to 3 μ M . The enzyme, with a native molecular mass of about 43 kDa, showed high specificity against the 22-kDa protein as a substrate. The isolated protease could not degrade the 22-kDa protein associated with fresh thylakoid membranes but digested the protein in the presence of 0.05% Triton X-100.     

Isolation and partial characterization of membrane-associated cyclophilin and a related 22-kDa glycoprotein.

The presence of membrane-associated proteins which stereospecifically bind cyclosporin A and react with anti-cyclophilin antibodies has been documented in rat tissues. Extraction of membranes with 6 M urea or 0.5% Chaps releases cyclosporin-binding activity that is 5-12% of that found in cytosol. Cyclosporin-A-binding proteins are present in most subcellular organelles of liver, but microsomes contain the greatest activity. These proteins can be purified by adsorption onto a cyclosporin-A affinity column and elution with cyclosporin A. Two major fractions are resolved on SDS/PAGE: an 18-kDa fraction is comprised of two isoforms that are similar if not identical to the two major cytosolic isoforms of cyclophilin. In addition, in microsomes an approximately equal quantity of a 22-kDa glycoprotein was detected. Based on partial sequencing (five peptides, 89 amino acids) this protein is similar but not identical to human cyclophilin B. This 22-kDa isoform is poorly recognized by affinity-purified anti-cyclophilin antibodies and comprises several predominant isoforms (pI approximately 9.3-9.6). Selective binding of membrane 22-kDa cyclophilin to peanut lectin suggests the oligosaccharides contain a terminal galactosyl-N-galactosamine residue.     

Isolation and characterization of the 10-kDa and 22-kDa polypeptides of higher plant photosystem 2

Two polypeptides of 10 kDa and 22 kDa, shown to be components of the higher plant photosystem 2, were purified and examined. A NaCl/Triton X-100 treatment was designed, which released these two polypeptides from the thylakoid membrane, in concert with the extrinsic 16-kDa and 23-kDa proteins, concomitant with a loss in oxygen-evolution activity. After this treatment the oxygen-evolving activity of the photosystem 2 membranes devoid of the 10-kDa and the 22-kDa polypeptides could be restored with CaCl2, but not by readdition of the purified 23-kDa protein. This deficiency was caused by an inability of the 23-kDa protein to rebind to the photosystem 2 membranes. In analogy, the oxygen-evolution activity of a highly purified photosystem 2 core preparation, devoid of the 10-kDa and 22-kDa polypeptides, was stimulated by CaCl2, but not by the 23-kDa protein. We, therefore, suggest that the 10-kDa or the 22-kDa polypeptides provide a binding-site for the extrinsic 23-kDa protein to the thylakoid membrane. The 10-kDa and 22-kDa polypeptides were isolated through ion-exchange chromatography in the presence of detergents. They both displayed hydrophobic properties, verified by their low proportion of polar amino acid residues and their partition to the hydrophobic phase during Triton X-114 fractionation. The purified polypeptides did not contain metallic cofactors or substances with absorption in the visible region of the spectrum.     

Partial purification of a binding protein for polychlorinated biphenyls from rat lung cytosol: physicochemical and immunochemical characterization

A binding protein for certain methyl sulfone metabolites of polychlorinated biphenyls (PCB) was partially purified from lung cytosol of untreated female rats. The protein has an Mr of 13,000 and a pI of 5.3 in the absence of reducing agents. In the presence of dithioerythreitol or beta-mercaptoethanol, the protein is split into subunits with a more basic pI. The 13-kDa protein was electroeluted from SDS-polyacrylamide gels, and an antiserum against the protein was raised in rabbit. The immunoglobulin fraction was shown to contain monospecific antibodies against the 13-kDa protein as determined by Western immunoblots. The antibodies retained partially purified binding protein labeled with radioactive ligand when subjected to protein A-Sepharose chromatography and caused a shift in the elution of the labeled protein from Sephadex G-75 and a shift in its sedimentation behavior on sucrose gradients. Due to striking similarities in physicochemical characteristics of the 13-kDa protein and a protein purified from rabbit lung and uterus, uteroglobin, the anti 13-kDa protein antibodies were tested for cross-reactivity. As judged by Western immunoblots, the anti 13-kDa protein antibodies did not cross-react with uteroglobin and the two proteins, although similar, do not seem to be identical. The 13-kDa protein is proposed to be responsible for the accumulation of certain methylsulfonyl-PCBs in lung tissue of rats. Monospecific antibodies against the 13-kDa protein should constitute immunochemical probes of great value in attempts to elucidate the physiological role of the protein as well as its possible role in PCB-induced respiratory toxicity.     

Characteristics of 30-, 63-, and 89-kilodalton proteins whose secretion from mouse fibroblasts is altered by beta-interferon

Quiescent mouse BALB/c-3T3 cells were treated with beta-interferon to induce the secretion of proteins of 30 and 89 kDa and with a platelet-derived growth factor preparation to induce the secretion of a 63-kDa protein. To label the secreted proteins the cultures were supplemented with [35S]methionine after addition of the inducer. The proteins in the culture fluid were fractionated resulting in a radioactively pure 63-kDa protein and 30- and 89-kDa protein preparations with residual minor radioactive impurities. The secreted 89-kDa protein shared at least one characteristic with some interferon-induced cell-associated enzymes: it bound double-stranded RNA tightly. The 63-kDa protein was undetectable in the culture fluid from resting BALB/c-3T3 cells and was barely or not at all detectable in the culture fluids from growing BALB/c-3T3 and NIH 3T3 cells, respectively. The protein was, however, among the three major constitutively secreted proteins in the case of growing Kirsten murine sarcoma virus-transformed NIH 3T3 cells. Treatment with 1000 units/ml beta-interferon decreased the accumulation of the 63-kDa protein in the culture fluid of quiescent BALB/c-3T3 cells which had been treated with a platelet-derived growth factor preparation by over 80% and that in the culture fluid of Kirsten murine sarcoma virus-transformed NIH 3T3 cells by about 50%. This decrease was not a consequence of an inhibition of cell growth.     

Biosynthesis of rat insulin-like growth factor II. I. Immunochemical demonstration of a approximately 20-kilodalton biosynthetic precursor of rat insulin-like growth factor II in metabolically labeled BRL-3A rat liver cells

BRL-3A rat liver cells synthesize mature 7484-dalton rat insulin-like growth factor II (rIGF-II) as a approximately 22-kDa precursor, presumably prepro-rIGF-II. In the present study, we have biosynthetically labeled intact BRL-3A cells with [35S]cysteine and immunoprecipitated cell lysates and media with antisera to rIGF-II. A approximately 20-kDa protein was identified in immunoprecipitates of cell lysates having properties consistent with pro-rIGF-II. The approximately 20-kDa protein is precipitated by immune sera but not by nonimmune serum. Its immunoprecipitation is specifically inhibited by unlabeled rIGF-II but not by insulin. It is not precipitated from labeled lysates of a subclone of BRL-3A cells (BRL-3A2) that does not synthesize rIGF-II. The approximately 20-kDa protein is rapidly labeled intracellularly (10 min) but is not detected in BRL-3A media. In pulse-chase experiments, radioactivity in the approximately 20-kDa protein disappears during the chase and appears, at later times, in specifically immunoprecipitated approximately 19-, approximately 10-, approximately 8-, and approximately 7-kDa proteins in media and, to a limited extent, intracellularly. A protein with electrophoretic mobility identical to that of the approximately 20-kDa protein observed in cell lysates is immunoprecipitated from 35S-proteins whose synthesis is directed by BRL-3A RNA in a reticulocyte lysate cell-free translation system supplemented with microsomal membranes, and presumably arises by cotranslational removal of the signal peptide from approximately 22-kDa prepro-rIGF-II. Processing of the approximately 20-kDa protein in intact BRL-3A cells to intermediate and mature rIGF-II species appears to occur at the time of secretion and/or shortly thereafter, with the different forms appearing at approximately the same time.     

Hypoxia Induces Synthesis of a Novel 22-kDa Protein in Neonatal Rat Oligodendrocytes

Neonatal (3-day-old) rat oligodendrocytes grown in monolayer culture and exposed to increasingly hypoxic culture conditions showed a dramatic reduction in myelin basic protein synthesis but no significant inhibition of Tran35S-label incorporation into oligodendrocyte proteins in general or into structural proteins such as actin. However, there was a dramatic increase in synthesis of a novel 22-kDa protein. Reoxygenation of cultures reversed the synthesis of the 22-kDa protein, and thiol and calpain protease inhibitors (EP-459 and leupeptin) did not prevent synthesis of the protein, suggesting that it did not result from proteolysis. The 22-kDa protein (which we have called hypoxin) was coimmunoprecipitated by a polyclonal antibody to actin but did not react with the anti-actin antibody on western blots. The synthesis of hypoxin accounted for up to 50% of the Tran35S-label incorporated into immunoprecipitated protein, suggesting that it plays a major role in the cell's response to hypoxia. Subcellular fractionation revealed that the 22-kDa protein was largely associated with the cytosolic/cytoskeletal compartment. However, it is unlikely to be one of the cytoskeleton-associated Rho or Rac low-molecular-mass (20-24 kDa) GTP-binding proteins because it did not bind [alpha-32P]GTP on western blots. Oligodendrocytes did not synthesize a 22-kDa protein in response to heat shock but did synthesize the typical 70- and 90-kDa heat-shock proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of CCAAT/enhancer binding proteins alpha and beta in the porcine ovary and regulation in primary cultures of granulosa cells

CCAAT/enhancer binding proteins alpha and beta (CEBPA/ CEBPB) were evaluated in the porcine ovary during the estrous cycle. CEBPB mRNA was present in antral follicles and was significantly increased in healthy corpora lutea (CL), whereas CEBPA mRNA was constitutively expressed in these structures. Both isoforms of CEBPA (42 and 30 kDa) exhibited greater expression in preovulatory follicles, and the 42-kDa isoform increased in CL, whereas the 30-kDa isoform decreased. All major isoforms of CEBPB (38, 34, and 20 kDa) were expressed, with the 34- and 20-kDa isoforms being more abundant in preovulatory follicles and further increased in CL. The effects of FSH and cAMP analogue on the distribution of CEBP isoforms were evaluated in primary cultures of porcine granulosa cells. FSH and 8-Br-cAMP had little stimulatory effect on isoform distribution, but cAMP treatment for 24 h tended to decrease the 30-kDa form of CEBPA and the 34-kDa form of CEBPB. The 34-kDa form of CEBPB was decreased by the protein kinase A inhibitor H89 at 4 h (with FSH treatment), and by both protein kinase A and phosphatidylinositol 3-kinase inhibitors at 24 h of treatment. In transfected granulosa cells, FSH and cAMP analogue stimulated a CEBP consensus sequence-reporter construct that was blocked by H89. These data implicate protein kinase A as the major regulator of CEBPB isoform distribution and CEBP-mediated transactivation in granulosa cells. The differential expression of specific CEBPA/B isoforms observed in maturing follicles and CL may contribute to changes in follicular cell differentiation and increasing steroidogenic capacity.     

A pantothenate derivative is covalently bound to mitochondrial proteins in Neurospora crassa

The presence of protein-bound pantothenate in Neurospora crassa was investigated by labelling a pantothenate auxotroph (pan-2) with [14C]pantothenate and examining mycelial homogenates on dodecyl sulfate/polyacrylamide gels. Five peaks of radioactivity were found, with apparent molecular masses of 200, 140, 22, 19, and 9 kDa. The 200-kDa peak was identified as fatty acid synthetase, based on its absence in a fatty acid synthetase mutant. The 22-kDa and 19-kDa peaks co-purified with mitochondrial markers on sucrose gradients. When purified mitochondria were fractionated, the 19-kDa protein was associated with the inner membrane and the 22-kDa protein was enriched in the soluble mitochondrial fraction. The label was quantitatively recovered from the mitochondrial proteins as 4'-phosphopantetheine after mild alkaline hydrolysis. Although the function of this post-translational modification of mitochondrial proteins is not known, several possibilities are discussed: the 4'-phosphopantetheine may act as a carrier group in an enzymatic reaction, or it may perform a regulatory function as part of an enzyme complex.     

Identification and partial purification of a band 3-like protein from rabbit renal brush border membranes

A monoclonal antibody against the membrane domain of human erythrocyte band 3 was tested for its ability to bind to rabbit renal brush border membranes. A single brush border protein with a molecular mass of 43 kDa was recognized by the band 3 antibody. Using DNase I coupled to an agarose-bead support this 43-kDa protein was partially purified by removing actin and a number of actin-bound proteins from the brush border membranes. The partially purified 43 kDa-band was eluted from sodium dodecyl sulfate-polyacrylamide gels and used to make a highly sensitive and specific guinea pig antiserum. This antiserum, but not serum from control guinea pigs, cross-reacts with purified band 3 from human, rabbit, and bovine erythrocytes confirming the immunologic similarity among these proteins. The 43-kDa protein can be stained by the periodic acid-Schiff base method and binds wheat germ agglutinin and concanavalin A, demonstrating that it is a glycoprotein. Furthermore, in the absence of dithiothreitol, the immunoreactive brush border protein migrates with a molecular mass of 86 kDa on an sodium dodecyl sulfate-polyacrylamide gel suggesting that under nonreducing conditions it exists as a dimer. The 43-kDa protein could be solubilized in octyl glucoside and was further purified using gel filtration chromatography. The amino acid composition of the 43-kDa brush border protein was obtained, and its similarity with erythrocyte band 3 is discussed.     

Signal transduction mechanism leading to enhanced proliferation of primary cultured adult rat hepatocytes treated with royal jelly 57-kDa protein

A 57-kDa protein in royal jelly (RJ) was previously shown to stimulate hepatocyte DNA synthesis and prolongs the proliferation of hepatocytes as well as increasing albumin production [Kamakura, M., Suenobu, N., and Fukushima, M. (2001) Biochem. Biophys. Res. Commun. 282, 865-874]. In this study, I investigated the signal transduction mechanisms involved in the induction of hepatocyte DNA synthesis and the promotion of cell survival by this 57-kDa protein in primary cultures of adult rat hepatocytes. Hepatocyte DNA synthesis induced by the 57-kDa protein was not influenced by several alpha- and beta-adrenoceptor antagonists, but was dose-dependently abolished by an inhibitor of a tyrosine-specific protein kinase, genistein. A phospholipase C inhibitor (U-73122) and a protein kinase C (PKC) inhibitor (sphingosine) inhibited 57-kDa protein-stimulated he-patocyte DNA synthesis, whereas a protein kinase A inhibitor (H-89) did not. The 57-kDa protein also activated PKC in rat hepatocytes. Various inhibitors of intracellular signal transduction elements (PD98059, p21 ras farnesyltransferase inhibitor, wortmannin and rapamycin) also blocked hepatocyte DNA synthesis induced by the 57-kDa protein. Furthermore, the 57-kDa protein activated mitogen-activated protein (MAP) kinase in rat hepatocytes. The activation of MAP kinase by the 57-kDa protein was inhibited by PD98059 and sphingosine. The 57-kDa protein also activated protein kinase B, which is a key regulator of cell survival. These results suggest that, like growth factors, the 57-kDa protein activates several important intracellular signaling factors involved in the stimulation of hepatocyte DNA synthesis and the protection of cells from apoptosis.     

Tyrosine phosphorylation of a 22-kDa protein is correlated with transformation by Rous sarcoma virus

Recent studies from this laboratory have identified novel cytoskeletal proteins that are phosphorylated on tyrosine in vivo in Rous sarcoma virus-transformed chick fibroblasts (Glenney, J. R., Jr., and Zokas, L. (1989) J. Cell Biol. 108, 2401-2408). In the present report, the phosphorylation of these proteins was examined in cells expressing the nonmyristylated mutants of src that are not transformed. A good correlation was found between transformation and the tyrosine phosphorylation of a 22-kDa protein. Tyrosine phosphorylation of the 22-kDa protein was reduced more than 95% in cells expressing the nonmyristylated mutants of src. Size fractionation revealed that the 22-kDa phosphoprotein in transformed chick fibroblasts is found in a Mr 150,000 complex. Monoclonal antibodies were used to screen various chicken tissues where the 22-kDa protein was found at high levels in muscle and lung with low levels in epithelial cells and brain. The 22-kDa protein becomes an excellent candidate for a mediator of transformation by the tyrosine kinase class of oncogenes.     

Extraordinarily stable disulfide-linked homodimer of human growth hormone

Although a 22-kDa human growth hormone (hGH) is the predicted protein product of the hGH-N gene, a pleiotropic collection of uncharacterized molecular weight and charge isoforms is also produced. Using chromatography and preparative SDS-PAGE under reducing conditions we isolated an unusually stable mercaptoethanol-resistant (MER) 45-kDa hGH. A 5-h incubation at 100 degrees C in the presence of 2-mercaptoethanol was required to convert approximately 90% of MER-45-kDa hGH into a 22-kDa hGH. Other reductants were not as effective in splitting MER-45-kDa hGH. After fracturing MER-45-kDa hGH, the 22-kDa hGH fragments would spontaneously reassociate if the reductant was removed; however, alkylation of cysteine residues prevented their reassociation. Identical amino acid sequences for the first six N-terminal residues were obtained for MER-45-kDa hGH and its 22-kDa hGH cleavage product. Structural identity of MER-45-kDa hGH and 22-kDa hGH was demonstrated by MALDI-TOF mass spectrometry of tryptic digests. MER-45-kDa hGH did not break up upon incubation with EDTA and EGTA. The significance of this work to our understanding of the structure of hGH isoforms is that it demonstrates that MER-45-kDa hGH is not a single chain polypeptide but is instead a homodimer of 22-kDa hGH monomers. The MER-45-kDa hGH dimer is held together by interchain disulfide bonds and not by divalent metal cation bridges. Additionally, MER-45-kDa hGH's interchain disulfide links are exceptionally resistant to reducing agents and thus confer extreme stability to the homodimer.     

Phosphorylation of the Core Protein of Hepatitis B Virus by a 46-Kilodalton Serine Kinase

Core protein is the major component of the core particle (nucleocapsid) of human hepatitis B virus. Core particles and core proteins are involved in a number of important functions in the replication cycle of the virus, including RNA packaging, DNA synthesis, and recognition of viral envelope proteins. Core protein is a phosphoprotein with most, if not all, of the phosphorylation on C-terminal serine residues. In this study, we identified a serine kinase activity from the ribosome-associated protein fraction of cytoplasm that could specifically bind and phosphorylate the C-terminal portion of recombinant core protein. This kinase is referred to as core-associated kinase (CAK). CAK could be inhibited by the kinase inhibitors heparin and manganese ions but not by spermidine, DRB, H89, or H7, indicating that CAK is distinct from protein kinase A and protein kinase C. CAK could be partially purified by heparin-Sepharose CL-6B and phosphocellulose P11 columns. By using a far-Western assay, three specific proteins, of 46, 35, and 13 kDa, were shown to interact with the C-terminal part of the core protein. These three proteins were present only in the eluted fractions that contains the CAK activity. An in-gel kinase assay showed that a 46-kDa kinase in the same fraction could bind and phosphorylate the C-terminal part of the recombinant core protein. These results indicate that this 46-kDa kinase is most probably CAK. A similar 46-kDa kinase, which exhibits the same profile of sensitivity to kinase inhibitors as that of CAK, is present in both purified intracellular core particles and extracellular 42-nm virions, suggesting that CAK is a candidate for the core particle-associated kinase.     

Identification and characterization of viral structural proteins of infectious salmon anemia virus

Infectious salmon anemia virus (ISAV) is an unclassified Orthomyxovirus that has been shown to contain a segmented genome with eight single-stranded RNA species coding for 10 viral proteins. Four major structural proteins were characterized in the present study: two glycosylated proteins with estimated molecular masses of 42 and 50 kDa, one 66-kDa phosphoprotein, and one 22-kDa protein. Examination of lysed virions revealed the two glycoproteins and the 22-kDa protein in the soluble fraction, while the 66-kDa phosphoprotein and a minor part of the 22-kDa protein were found in the pelleted fraction. Immunofluorescence staining of infected cells demonstrated that the 22-kDa protein was a late protein accumulating in the nucleus. We conclude that the 66-kDa protein is the nucleoprotein, the 22-kDa protein is the matrix protein, and the 42- and 50-kDa proteins are the surface proteins. Radioimmunoprecipitation analysis of the 42-kDa glycoprotein, which was previously shown to represent the ISAV hemagglutinin, indicated that this protein exists at least as dimers. Further, by labeling of purified ISAV with [1,3-(3)H]diisopropyl fluorophosphate, it was also demonstrated that the viral esterase is located with the hemagglutinin. This finding was confirmed by demonstration of acetylesterase activity in affinity-purified hemagglutinin preparations. Finally, the active-site serine residue could be tentatively identified at position 32 within the amino acid sequence of the hemagglutinin of ISAV strain Glesvaer/2/90. It is proposed that the ISAV vp66 protein be termed nucleoprotein, the gp42 protein be termed HE protein, and the vp22 protein be termed matrix protein.     

A protein partially expressed on the surface of HepG2 cells that binds lipoproteins specifically is nucleolin

Nucleolin, a major nucleolar protein of rapidly growing eukaryotic cells, has been thought to be predominantly if not exclusively located in the nucleolus. Recent data however [Borer, R.A., Lehner, C.F., Eppenberger, H.M., & Nigg, N.A. (1989) Cell 56, 379-390] suggest that the protein shuttles constantly between the nucleus and cytoplasm. Ligand blotting studies of whole cell extracts of HepG2 cells identified, in addition to the LDL receptor, another LDL binding protein of Mr 109,000. The 109-kDa protein was partially purified by HPLC and, like the LDL receptor, bound apoB- and apoE-containing lipoproteins but not HDL. However, unlike the LDL receptor, the 109-kDa protein bound lipoproteins in the presence of EDTA and reducing agents, had a lower affinity for lipoproteins than the LDL receptor, and did not react with two antibodies raised against the LDL receptor. The protein sequences of three separate peptides derived from the partially purified 109-kDa species were determined and were identical except for one residue to three separate regions of the published sequence of nucleolin. On immunoblot analysis the 109-kDa protein reacted with a nucleolin-specific antibody, and purified nucleolin reacted both with anti-109-kDa antibody and with LDL. When intact HepG2 cells were treated with Pronase before harvest, there was a 46% decrease in 109-kDa protein while recovery of actin, an intracellular protein, was unaffected. When intact HepG2 cells were surface iodinated and the proteins subjected to HPLC fractionation, the 109-kDa protein was found to be iodinated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Serine/threonine-specific protein kinase activity associated with viral pp60src protein

Three different types of experiments are presented in this paper, the results of which converge to indicate that the viral src protein associates with and modulates the activity and/or the specificity of a serine/threonine protein kinase. Firstly, a 60-kDa protein from extracts of FR3T3 rat fibroblasts transformed by wild-type Rous sarcoma virus (SRD-FR3T3) is shown to be immunoprecipitated with a monoclonal antibody (mAb) raised against bacterially produced pp60v-src, the mAb327 [Lipsich, L. A., Lewis, A. J. & Brugge, J. S. (1983) J. Virol. 48, 352-360] and to be phosphorylated in vitro at serine/threonine/tyrosine residues, in the ratio 25:53:22. Under the same experimental conditions, the pp60c-src protein immunoprecipitated with mAb327 from extracts of NIH c-src overexpresser cells is phosphorylated exclusively on tyrosine residues. Secondly, the results of immunoprecipitation experiments using a tumor-bearing rabbit (TBR) serum and reported in an earlier work [David-Pfeuty, T. & Hovanessian, A. (1984) Eur. J. Biochem. 140, 325-342], together with those reported here, suggest that the TBR-immunoprecipitated pp60v-src coprecipitates with a cellular protein related to the 60-kDa subunit of the Ca2+/calmodulin protein kinase II from brain. Finally, partially purified preparations of pp60v-src, but not of pp60c-src, are shown to contain a Ca2+/calmodulin-dependent protein kinase activity that phosphorylates a 52-kDa protein substrate.     

The identification and characterization of an epidermal growth factor-stimulated phosphorylation of a specific low molecular weight GTP-binding protein in a reconstituted phospholipid vesicle system

The abilities of different GTP-binding proteins to serve as phosphosubstrates for the epidermal growth factor (EGF) receptor/tyrosine kinase have been examined in reconstituted phospholipid vesicle systems. During the course of these studies we discovered that a low molecular mass, high affinity GTP-binding protein from bovine brain (designated as the 22-kDa protein) served as an excellent phosphosubstrate for the tyrosine-agarose-purified human placental EGF receptor. The EGF-stimulated phosphorylation of the purified 22-kDa protein occurs on tyrosine residues, with stoichiometries approaching 2 mol of 32Pi incorporated/mol of [35S]guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S)-binding sites. The EGF-stimulated phosphorylation of the brain 22-kDa protein requires its reconstitution into phospholipid vesicles. No phosphorylation of this GTP-binding protein is detected if it is simply mixed with the purified EGF receptor in detergent solution or if detergent is added back to lipid vesicles containing the EGF receptor and the 22-kDa protein. The EGF-stimulated phosphorylation of this GTP-binding protein is also markedly attenuated by guanine nucleotides, i.e. GTP, GTP gamma S, or GDP, suggesting that maximal phosphorylation occurs when the GTP-binding protein is in a guanine nucleotide-depleted state. Purified preparations of the 22-kDa phosphosubstrate do not cross-react with antibodies against the ras proteins. However, they do cross-react against two different peptide antibodies generated against specific sequences of the human platelet (and placental) GTP-binding protein originally designated Gp (Evans, T., Brown, M. L., Fraser, E. D., and Northrup, J. K. (1986) J. Biol. Chem. 261, 7052-7059) and more recently named G25K (Polakis, P. G., Synderman, R., and Evans, T. (1989) Biochem. Biophys. Res. Commun. 160, 25-32). When highly purified preparations of the human platelet Gp (G25K) protein are reconstituted with the purified EGF receptor into phospholipid vesicles, an EGF-stimulated phosphorylation of the platelet GTP-binding protein occurs with a stoichiometry approaching 2 mol of 32Pi incorporated/mol of [35S]GTP gamma S-binding sites. As is the case for the brain 22-kDa protein, the EGF-stimulated phosphorylation of the platelet GTP-binding protein is attenuated by guanine nucleotides. Overall, these results suggest that the brain 22-kDa phosphosubstrate for the EGF receptor is very similar, if not identical, to the Gp (G25K) protein. Although guanine nucleotide binding to the brain 22-kDa protein or to the platelet. GTP-binding protein inhibits phosphorylation, the phosphorylated GTP-binding proteins appear to bind [35S]GTP gamma S slightly better than their nonphosphorylated counterparts.