Detection and characterization of GTP-binding proteins in the chloroplast envelope of spinach (Spinacia oleracea)

Proteins binding guanosine triphosphate (GTP) have emerged as important regulators in several cellular processes in plants. To investigate any role of such proteins in chloroplast functions, we subjected envelope, stroma and thylakoid fractions isolated from spinach chloroplasts to two different GTP-binding assays. With both methods, we detected GTP-specific binding only in the envelope fraction. Two chloroplast envelope proteins with the apparent molecular weights of 30.5 and 33.5 kDa, respectively, bound [α-³²P]GTP after SDS-PAGE followed by electroblotting onto a PVDF-membrane and renaturation. Both proteins were intrinsic proteins located in the outer chloroplast envelope. Also, when the fractions were incubated with [α-³²P]GTP, followed by periodate oxidation and borohydride reduction to cross-link GTP to proteins, two proteins in the envelope fraction, of apparent molecular weights of 28 and 39 kDa, appeared to specifically bind GTP. When agents that stimulate heterotrimeric G-proteins, cholera toxin or the mastoparan analogue mas7, were added to isolated chloroplast envelope, the binding of radiolabelled GTP to the 39 kDa protein, a protein of the inner chloroplast envelope, was stimulated, whereas GTP-binding of the 28 kDa protein, a protein of the outer envelope, was unchanged. Mas7 also stimulated synthesis of monogalactosyl diacylglycerol in isolated chloroplast envelope. The occurrence and regulation of GTP-binding proteins in the chloroplast envelope suggests that GTP-binding proteins could be involved in communication with the extraplastidic compartment during chloroplast biogenesis and development.     

Chloroplast biogenesis. Cell-free transfer of envelope monogalactosylglycerides to thylakoids.

An ATP- and temperature-dependent transfer of monogalactosylglycerides from the chloroplast envelope to the chloroplast thylakoids was reconstituted in a cell-free system prepared from isolated chloroplasts of garden pea (Pisum sativum) or spinach (Spinacia oleracea). Isolated envelope membranes, in which the label was present exclusively in monogalactosylglycerides, were prepared radiolabeled in vitro with [14C]galactose from UDP-[14C]galactose to label galactolipids as the donor. ATP-dependent transfer of radioactivity from donor to unlabeled acceptor thylakoids, immobilized on nitrocellulose strips, was observed. In some experiments linear transfer for longer than 30 min of incubation was facilitated by the addition of stroma proteins but in other experiments stroma was without effect or inhibitory suggesting no absolute requirements for a soluble protein carrier. Transfer was donor specific. No membrane fraction tested (plasma membrane, tonoplast, endoplasmic reticulum, nuclei, Golgi apparatus, mitochondria or thylakoids) (isolated from tissue radiolabeled in vivo with [14C]acetate) other than chloroplast envelopes demonstrated any significant ability to transfer labeled membrane lipids to immobilized thylakoids. Acceptor specificity, while not absolute, showed a 3-10-fold greater ATP-dependent transfer of labeled galactolipids from chloroplast envelopes to immobilized thylakoids than to other leaf membranes. The results provide independent confirmation of the potential for transfer of galactolipids between chloroplast envelopes and thylakoids suggested previously from ultrastructural studies and of the known location of thylakoid galactolipid biosynthetic activities in the chloroplast envelope.     

Protein Methylation in Cerebellar Synaptosomes

Abstract: Synaptosomes from five regions of adult rat brain were isolated, analyzed for methyl acceptor proteins, and probed for methyltransferases by photoaffinity labeling. Methylated proteins of 17 and 35 kDa were observed in all regions, but cerebellar synaptosomes were enriched in a 21–26-kDa family of methyl acceptor proteins and contained a unique major methylated protein of 52 kDa and a protein of 50 kDa, which was methylated only in the presence of EGTA. When cerebellar and liver subcellular fractions were compared, the cytosolic fractions of each tissue contained methylated proteins of 17 and 35 kDa; liver membrane fractions contained few methylated proteins, whereas cerebellar microsomes had robust methylation of the 21–26-kDa group. Differential centrifugation of lysed cerebellar synaptosomes localized the 17- and 35-kDa methyl acceptor proteins to the synaptoplasm, the 21–26-kDa family to the synaptic membranes, and the 52-kDa to synaptic vesicles. The 21–26-kDa family was identified as GTP-binding proteins by [α-³²P]GTP overlay assay; these proteins contained a putative methylated carboxyl cysteine, based on the presence of volatile methyl esters and the inhibition of methylation by acetylfarnesylcysteine. The 52-kDa methylated protein also contained volatile methyl esters, but did not bind [α-³²P]GTP. When synaptosomes were screened for putative methyltransferases by S -adenosyl-L-[ methyl -³H]methionine photoaffinity labeling, a protein of 24 kDa was detected only in cerebellum, and this labeled protein was localized to synaptic membranes.     

Interaction of GTP with proteins during the cell cycle of Streptomyces coelicolor

Abstract Six putative GTP binding proteins were detected by ultraviolet light in the presence of [α-³²P]GTP during the developmental cycle of Streptomyces coelicolor . Four out of six were true GTP binding proteins. Immunological reactions carried out with antiserum which recognizes the α-common subunit of G regulatory proteins identified two bands of 67 kDa and 30 kDa. Studies with [γ-³²P]GTP showed significant changes in protein phosphorylation during the cell cycle. The results show that at least three different systems of GTP protein interaction are present in S. coelicolor .     

Proteins that interact with GTP in Streptomyces griseus and its possible implication in morphogenesis

Abstract By cross-linking with [α-³²P]GTP or [γ-³²P]GTP with or without UV treatment, several proteins of Streptomyces griseus were shown to interact with GTP in specific ways. After gel electrophoresis, 19 bands of radioactivity were found; 12 bands were assigned as GTP-binding proteins and 6 bands as phosphorylated proteins. One band was assumed to be a guanylylated protein. The profile of radioactive bands was similar between cells prepared from liquid or solid culture, but markedly different between growth phases. A mutant (strain M-1) defective in aerial mycelium formation, which was originally found as a decoyinine-resistant isolate, was found to have a different profile of phosphorylated proteins.     

Multiple GTP-binding proteins in sea urchin sperm: Evidence for Gs and small G-proteins

Sea urchin sperm plasma membranes isolated from heads and flagella were used to examine the presence of Gs (stimulatory guanine nucleotide-binding regulatory protein) and small G-proteins. Flagellar plasma membranes incubated with [³²P]NAD and cholera toxin (CTX) displayed radiolabeling in a protein of 48 kDa, which was reactive by immunoblotting with a specific antibody against mammalian Gs. CTX-catalyzed [³²P]ADP-ribosylation in conjunction with immunoprecipitation with anti-Gs, followed by electrophoresis and autoradiography, revealed one band of 48 kDa. Head plasma membranes, in contrast, did not show substrates for ADP-ribosylation by CTX. In flagellar and head plasma membranes pertussis toxin (PTX) ADP-ribosylated the same protein described previously in membranes from whole sperm; the extent of ADP-ribosylation by PTX was higher in flagellar than in head membranes. Small G-proteins were investigated by [³²P]GTP-blotting. Both head and flagellar plasma membranes showed three radiolabeled bands of 28, 25 and 24 kDa. Unlabeled GTP and GDP, but not other nucleotides, interfered with the [α-³²P]GTP-binding in a concentration-dependent manner. A monoclonal antibody against human Ras p21 recognized a single protein of 21 kDa only in flagellar membranes. Thus, sea urchin sperm contain a membrane protein that shares characteristics with mammalian Gs and four small G-proteins, including Ras . Gs, Gi and Ras are enriched in flagellar membranes while the other small G-proteins do not display a preferential distribution along the sea urchin sperm plasma membrane. The role of these G-proteins in sea urchin sperm is presently under investigation.     

Sensitization of G Protein-Coupled Benzodiazepine Receptors in the Striatum of 6-Hydroxydopamine-Lesioned Rats

Abstract: The nonselective benzodiazepine (BZ) agonist diazepam is a potent inhibitor of adenylyl cyclase (AC) activity in the rat striatum. To examine this inhibitory action of diazepam further, its effects were examined in 6-hydroxydopamine-lesioned animals, which reportedly exhibit sensitization of the striatal AC pathway. As previously observed, inhibition of AC activity by diazepam was biphasic, with the first phase being receptor-mediated, whereas the second phase involves a direct action on the enzyme itself. In the presence of NaCl (120 m M ), a marked sensitization to the receptor-mediated inhibitory effect of diazepam on AC activity was observed in striatal membranes of lesioned animals. EC₅₀ values were 10.4 ± 1.1 and 4.8 ± 0.9 n M ( p < 0.05) for intact and lesioned striata, respectively. An examination of [³H]diazepam binding revealed a significant increase in the density of binding sites in denervated striata, with no change in affinity. A time-dependent increase in [α-³²P]GTP labeling of two distinct striatal proteins with apparent molecular masses of 40 and 45 kDa, suggestive of the α subunits of G_i and G_s, respectively, was observed. There was a significant increase in basal [α-³²P]GTP binding to both proteins in lesioned striata. In addition, diazepam stimulated [α-³²P]GTP binding to the 40-kDa protein, especially in lesioned striata. These data indicate that the sensitization of the receptor-mediated inhibitory effect of diazepam on AC activity in denervated striata may involve up-regulation of BZ receptors as well as enhanced functional coupling of these receptors to inhibitory G proteins.     

Effects of drought on [1-14C]-oleic and [1-14C]-linoleic acid desaturation in cotton leaves

The effects of water stress on [1-¹⁴C]-oleic and [1-¹⁴C]-linoleic acid desaturations were studied in leaves of two varieties of cotton ( Gossypium hirsutum L.), one drought-sensitive (Reba) and the other more resistant (Mocosinho). After 24 h incorporation, [1-¹⁴C]-oleate led to the appearance of linoleate in phospholipids and, additionally, of linolenate in galactolipids. [1-¹⁴C]-Linoleate was desaturated to linolenate only in galactolipid fractions. Water stress markedly inhibited the incorporation of the precursors into the leaf lipids. The two desaturation steps were affected, particularly the transformation of linoleate to linolenate in monogalactosyldiacylglycerol in the drought-sensitive variety of cotton. The metabolic implications of the inhibition of the biosynthesis of C₁₈-polyunsaturated fatty acids are discussed.     

Galactosyltransferases involved in galactolipid biosynthesis are located in the outer membrane of pea chloroplast envelopes

The galactosylation steps in the biosynthesis of galactolipids involve two different enzymes; a UDP-Gal:diacylglycerol galactosyltransferase and a galactolipid:galactolipid galactosyltransferase. Previous localization studies have shown that in spinach these enzymes are located in the chloroplast envelope. Our results with peas (Pisum sativum var Laxton's Progress No. 9) confirm these results and extend the localization by providing evidence that the galactosyltransferases are in the outer membrane of the envelope. The specific activity of UDP-Gal:diacylglycerol galactosyltransferase in outer membrane preparations was 6 to 10 times greater than that exhibited by inner membrane preparations. In addition, using quantitative sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it was possible to show that the UDP-Gal:diacylglycerol galactosyltransferase activity associated with inner membrane preparations could be accounted for by outer membrane contamination. It is concluded from these results that this enzyme is located predominantly, if not exclusively, in the outer membrane of the envelope. An analysis of the galactolipid products synthesized by the highly purified outer membrane showed that the galactolipid:galactolipid galactosyltransferase is also present, suggesting that this enzyme is also an outer membrane enzyme. The implication of these results is that the final assembly of galactolipids is carried out on the outer membrane of the chloroplast envelope.     

Phosphorylation of phospholipase B in the plasma membrane of Saccharomyces cerevisiae

Abstract Plasma membrane vesicles from Saccharomyces cerevisiae were incubated with [γ-³²P]ATP. Several phosphorylated protein bands were separated by LiDS polyacrylamide gel electrophoresis. One of these bands with an apparent M _r of 145 000 was identified by immunoprecipitation as a membrane-bound phospholipase.     

PHOSPHORYLATION OF NUCLEAR PROTEINS FROM RABBIT CEREBRUM, CEREBELLUM AND LIVER IN VITRO

Abstract— Phosphorylation of nuclear protein was investigated with isolated nuclei from rabbit cerebral cortex, cerebellum and liver by using [γ-³²P]ATP. The results were compared with the previously reported findings on phosphorylation with tissue slices and [³²P]phosphate. Cerebral cortex showed a very high level of phosphorylation, while liver showed the lowest, the difference being several fold in magnitude. With each tissue source, the extent of phosphorylation was maximum at incubation period for 2–3 min with steady decline afterwards. When nuclear proteins were further fractionated into 0.14 m -NaCl-soluble, 0.25 n -HCl-soluble (mainly histone) and acidic phenol-soluble proteins, NaCl-soluble protein showed the highest phosphorylation while HCl-soluble the lowest. The ratio among these tissue sources studied and the ratio among various protein fractions in each tissue source were strikingly similar to what had been shown with tissue slices. Further separation of acidic phenol-soluble protein with polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed retention of the characteristic difference of the pattern of phosphorylation between liver and the CNS tissue as having been observed with tissue slices, although phosphorylation of proteins with molecular weights of less than 40,000 was much reduced with the isolated nuclei. Although other methods with extracted protein kinase or chromatic protein fractions might be more desirable under ordinary situations, the system for nuclear protein phosphorylation with isolated nuclei and [γ-³²P]ATP may be useful under certain experimental conditions provided the incubation condition is carefully selected.     

A 30-kDa Protein in the Surface Complex and Flagella of Euglena has Protein Kinase Activity

ABSTRACT A protein kinase (PK) was partially purified from NaCl extracts of the cell surface complex of Euglena using DEAE-cellulose chromatography. Tubulins extracted either from flagella or from the cell surface complexes of Euglena were readily phosphorylated when incubated with [γ-³²P]-ATP and the PK. Protein kinase activity was augmented with 5 mM Mn²⁺ or Mg² and was inhibited or had greatly reduced activity with 5 mM Ca²⁺, Co^2-, Cu²⁺ or Zn²⁺. Incorporation was much lower when [γ-³²P]-GTP was the phosphate donor. Serine and threonine were the major radiolabeled phosphoamino acids in tubulins; label was also found in phosphotyrosine. Alpha-tubulin solubilized from flagella was a relatively poor substrate for the PK, but a Euglena α-tubulin cDNA overexpressed as a Trx-fusion protein incorporated [γ-³²P]-ATP into serine and threonine when incubated with cell surface extracts. Alpha- and β-tubulins from cell surface complexes were equally good substrates for the PK. No incorporation was observed in intact microtubules either from the cell surface complex or from isolated flagella. In-gel assays identified a polypeptide of about 30 kDa that phosphorylated tubulins in extracts of both flagella and the cell surface complexes, and dephosphorylated casein was a competitive substrate for the partially purified kinase. In vivo incubation with [³²P]-orthophosphate produced numerous radiolabeled bands in acrylamide gels of NaCl extracts of the cell surface complex, but none of these bands could be positively related to tubulins extracted from surface complex microtubules.     

Galactolipid Synthesis in Vicia faba Leaves: III. Site(s) of Galactosyl Transferase Activity

Leaves of Vicia faba were fed ¹⁴CO₂ in light for periods of up to 6 hours. At intervals, leaf samples were homogenized and separated into fractions which contained “broken” and “intact” chloroplasts, and three other high speed centrifugal fractions containing other cell membranes and chloroplast envelopes. Analyses of the radioactive labeling of galactose from the galactolipids in these fractions and in purified chloroplast envelopes indicated that the major site of galactosyl transferase enzyme activity was in the chloroplast envelope. The data suggest that in time much of the radioactive galactolipid was transferred from the envelope to the thylakoid-containing fractions. The major site of galactolipid synthesis appears to be in the envelope but there is some evidence of another site in the thylakoids.     

Lipid biosynthesis by chloroplasts from olive tree leaves

In vitro incubation of isolated chloroplasts from young olive tree leaves ( Olea europaea L. cv. Marteño) in acetate-1-¹⁴C showed a high labelling of saturated fatty acids (palmitic + stearic) and, above all, of the monounsatured ones (oleic); the low biosynthetic rate of α-linolenic acid being noteworthy. These fatty acids are mainly found as free ones, or incorporated in mono-and diglyceride molecules. Phosphoand galactolipids, the most abundant acyl-lipid components of chloroplast lamellae, showed low incorporation rates. The fatty acid synthesis by isolated chloroplasts depends on exogenous CoA, ATP, NADPH and, especially, on added ACP (acyl carrier protein) preparation from Escherichia coli , whereas it was strongly inhibited by Triton X-100.
In vivo experiments with acetate-1-¹⁴C infiltration into young excised leaves showed a high labelling of chloroplast phospholipids, but a low ¹⁴C incorporation into galactolipids, a remarkable feature because these latter are main components of chloroplast lamellae. The high biosynthetic rate of α-linolenic acid is noteworthy and appears mainly linked to monogalactosyldiglycerides. Also the low incorporation of saturated fatty acids to neutral lipids is remarkable. The low in vitro synthesis of α-linolenic acid in comparison with that of the in vivo conditions, suggests the existence of a cooperation between chloroplasts and other parts of the cell to carry out the synthesis of this compound.     

ATP-induced Secretion in PC12 Cells and Photoaffinity Labeling of Receptors

Abstract— Secretion of catecholamines by rat PC12 cells is strongly stimulated by extracellular ATP via a P₂-type pur-inergic receptor. ATP-induced norepinephrine release was inhibited 80% when extracellular Ca²⁺ was absent. Only four nucleotides, ATP, ATPγS, benzoylbenzoyl ATP (BzATP), and 2-methylthio-ATP, gave substantial stimulation of norepinephrine release from PC12 cells. ATP-induced secretion was inhibited by Mg²⁺, and this inhibition was overcome by the addition of excess ATP suggesting that ATP^4-was the active ligand. ATP-induced secretion of catecholamine release was enhanced by treatment of cells with pertussis toxin or 12- O -tetradecanoylphorbol 13-acetate. The stimulatory effects of 12- O -tetradecanoyl-phorbol 13-acetate and pertussis toxin on norepinephrine release were additive. After brief exposure of intact cells to the photoaffinity analog, [α-³²P]BzATP, two major proteins of 44 and 50 kDa and a minor protein of 97 kDa were labeled. An excess of ATP-γS and BzATP but not GTP blocked labeling of the proteins by [³²P]BzATP. Labeling of the 50-kDa protein was more sensitive to competition by 2-methylthio-ATP than the other labeled proteins, suggesting that the 50-kDa protein represents the P₂ receptor responsible for ATP-stimulated secretion in these cells.     

Small GTP-binding Proteins Associated with Secretory Vesicles of Paramecium

GTP-binding proteins act as molecular switches in a variety of membrane-associated processes, including secretion. One group of GTP-binding proteins, 20–30 kDa, is related to the product of the ras proto-oncogene. In Saccharomyces cerevisiae, ras -like GTP-binding proteins regulate vesicular traffic in secretion. The ciliate protist Paramecium tetraurelia contains secretory vesicles (trichocysts) whose protein contents are released by regulated exocytosis. Using [α-³²P]GTP and an on-blot assay for GTP-binding, we detected at least seven GTP-binding proteins of low molecular mass (22–31 kDa) in extracts of Paramecium tetraurelia. Subcellular fractions contained characteristic subsets of these seven; cilia were enriched for the smallest (22 kDa). The pattern of GTP-binding proteins was altered in two mutants defective in the formation or discharge of trichocysts. Trichocysts isolated with their surrounding membranes intact contained two minor GTP-binding proteins (23.5 and 29 kDa) and one major GTP-binding protein (23 kDa) that were absent from demembranated trichocysts. This differential localization of GTP-binding proteins suggests functional specialization of specific GTP-binding proteins in ciliary motility and exocytosis.     

Incorporation of label from root-applied N6[8-14C]furfiuryIadenine into the guanine nucleotide fraction of pea bud ribonucleic acid

The pattern of incorporation of label into the nucleotides of axillary bud ribonucleic acid was investigated in Pisum sativum L. cv. Meteor following the application of N ⁶[8-^I4C]furfuryladenine or of [8-¹⁴C]adenine to the root system of decapitated plants and to cultured excised buds. When N ⁶[8-¹⁴C]furifaryladenine was applied to the root system label was confined to the guanine nucleotide moiety of the axillary bud ribonucleic acid; label from [8-¹⁴C]adenine was incorporated preferentially into adenine nucleotide in the molar ratio adenine nucleotide/guanine nucleotide = 3.23. When isolated buds were incubated in media containing [8-¹⁴C]adenine or N ⁶[8-¹⁴C]furfuryladenine, label was incorporated into both purine moieties of the ribonucleic acid. However, the relative incorporation into the guanine nucleotide fraction was considerably greater for N ⁶[8-^I4C]furfuryladenine (adenine nucleotide/guanine nucleotide = 2.23) than for [8-¹⁴C]adenine (ratio = 4.67).
It was concluded that the pattern of metabolism of adenine to guanine and its incorporation into the guanine nucleotide moiety of pea axillary bud ribonucleic acid, is influenced by the presence of a substitution in the N ⁶ position of the adenine base.     

The Galactolipid, Phospholipid, and Fatty Acid Composition of the Chloroplast Envelope Membranes of Vicia faba. L

The galactolipid, phospholipid, and fatty acid composition of chloroplast envelope membrane fractions isolated from leaves of Vicia faba L. has been determined. The major lipids in this fraction are: monogalactosyldiglyceride, 29%; digalactosyldiglyceride, 32%; phosphatidylcholine, 30%; and phosphatidylglycerol 9%. The lipid composition of the chloroplast envelope membranes is qualitatively similar to that of the lamellar membranes isolated from the same plastids, but the proportion of each lipid present is very different. The total galactolipid to total phospholipid ratio was 1.6: 1 in the envelope and 11.1: 1 in the lamellae. The monogalactosyldiglyceride-digalactosyl-diglyceride ratio was 0.9: 1 in the envelope and 2.4: 1 in the lamellae. Both membranes lack phosphatidylethanolamine.     

Metabolism of Thiamine Triphosphate in Rat Brain: Correlation with Chloride Permeability

Abstract: Our results show that a net synthesis of thiamine triphosphate (TTP) can be demonstrated in vitro using rat brain extracts. The total homogenate was preincubated with thiamine or its diphosphate derivative (TDP), centrifuged, and washed twice. With TDP (1 m M ) as substrate, a 10-fold increase in TTP content was observed in this fraction (nuclear fraction, membrane vesicles). A smaller, but significant, increase was observed in the P₂ fraction (mitochondrial/synaptosomal fraction). In view of the low TTP content of our fractions, it was carefully assessed that authentic TTP was being formed. Incorporation of radioactivity from [β-³²P]TDP and [γ-³²P]ATP in TTP suggests that these two compounds are its precursors. Furthermore, TTP synthesis was inhibited by ADP and relatively low concentrations of Zn²⁺. These results suggest that TTP synthesis is catalyzed by an ATP:TDP transphosphorylase rather than by the cytoplasmic adenylate kinase that may be present in the vesicles. After osmotic lysis of the vesicles at alkaline pH, TTP was recovered in protein-bound form. Concomitantly, a soluble thiamine triphosphatase, with alkaline pH optimum, was also released from the vesicles. No net synthesis could be obtained in the cytosolic fraction or in detergent-solubilized systems. Like TTP synthesis, chloride permeability of the vesicles was increased when the homogenate had been incubated with thiamine and particularly with TDP. Our results suggest a regulatory role of TTP on chloride permeability, but the target remains to be characterized.     

Characterization of the protein kinase activity in beet root plasma membranes

Two protein kinase activities were found in plasma membrane-enriched preparations from red beet ( Beta vulgarix L.). The kinases in these preparations produced the phosphorylation of several membrane polypeptides. These kinases also phosphorylated histone III-S and casein. The activities of two different kinases could be distinguished: one was half-maximally stimulated by 1 μ M free Ca²⁺ phosphorylated histone III-S better than casein, showed half-maximal activity at an ATP concentration of 0.071 m M . had an optimum pH of 7, and was poorly inhibited by GTP, CTP or UTP. Another, much lower, kinase activity that phosphorylated casein was also observed; it was Ca²⁺ independent, showed half-maximal activity at ATP concentrations of 0.017 and 0.287 m M , exhibited a broad pH optimum about pH 7 and was inhibited by GTP, CTP, UTP or GDP to a greater extent than the calcium-stimulated activity. When plasma membrane proteins were solubilized with lysophosphatidyicholine and treated with [γ-³²P]ATP at several dilutions, a 125-kDa polypeptide was autophosphorylated in the absence of Ca²⁺, while 77-, 71- and 65-kDa polypeptides were autophosphorylated in its presence. Autophosphorylation in gels after electrophoresis showed a Ca²⁺-stimulated phosphoprotein band at 64 kDa.