Specific protein phosphorylation during cyclic AMP-mediated morphological reversion of transformed cells

Treatment of transformed Chinese hamster ovary cells with dibutyryl cAMP or other agents that elevate cAMP results in the acquisition of growth and morphology characteristic of normal fibroblasts. The role of specific protein phosphorylation in this process of morphological reversion has been examined using metabolic labelling of Chinese hamster ovary (CHO) cells with ³²P-orthophosphate in the presence or absence of N⁶O^2′-dibutyryladenosine 3′:5′-cyclic monophosphoric acid (Bt₂cAMP). Analysis of labelled cultures by SDS gel electrophoresis and radioautography demonstrate dramatic changes in the phosphorylation of only 2 cellular proteins during reverse transformation. A 55,000 dalton protein (pp55) was phosphorylated and a 20,000 dalton protein (pp20) was dephosphorylated. The time course of these events was consistent with the kinetics of morphological reversion. The lower molecular weight species, pp20, was dephosphorylated within 15–30 minutes, prior to all morphological changes except membrane tranquilization. The higher molecular weight protein, pp55, was maximally phosphorylated over 1–2 hours following addition of Bt₂cAMP, paralleling early stages in the establishment of fibroblastic form. The phosphorylated forms of pp20 and pp55 were both extracted from cellular cytoskeletons by 0.5% Triton X-100, but analysis of ³⁵S-methioninelabelled cultures suggested that unphosphorylated pp 20 may be bound to the cytoskeleton. Since pp20 was found to comigrate with the 20,000 dalton myosin light chain, it is possible that dephosphorylation of CHO cell myosin induced by cAMP may alter its interaction with actin microfilaments and modulate the assembly of stress fibers during morphological reversion.     

Phosphorylation upon cold stress in rice (Oryza sativa L.) seedlings

The response of plants to cold stress is not well understood at the biochemical level, although it has been studied extensively at the ecological level. To investigate whether protein phosphorylation may play an important role in cold stress, we exposed rice seedlings to low temperatures, prepared protein extracts from the leaves and incubated these in the presence of [γ-³²P]ATP. The proteins were then separated by two-dimensional polyacrylamide gel electrophoresis. While several proteins were found to be phosphorylated upon cold stress one protein, pp35, which has an isoelectric point of 8.0, was more phosphorylated than the others. The pp35 protein was found to be phosphorylated when rice seedlings were incubated for 6 h at 5°C before the leaf protein extract was prepared and radioactive labeling was performed. The pp35 was, however, significantly more phosphorylated in cold-tolerant rice varieties. Antibodies were raised against purified pp35 in adult rabbits. Using this pp35 antibody, which can recognize the RuBisCO large-chain subunit (LSU), and from amino acid sequencing of pp35, we were able to identify and confirm the pp35 protein as the fragment of RuBisCO LSU (EC 4.1.1.39). Phosphorylation of the RuBisCO LSU may be important in cold tolerance. Received: 7 July 1998 / Accepted: 19 December 1998     

RNA tumor virus phosphoproteins: phosphorylation of precursor and processed polypeptides.

Monospecific antisera made against the 30,000 molecular weight major internal polypeptide (p30) and the 12,000 molecular weight phosphorylated polypeptide (pp12) of a wild mouse type C oncovirus were used to immunoprecipitate precursor polypeptides from extracts of isotopically labeled cells infected with the oncovirus. Analysis of the immunoprecipitates by SDS-polyacrylamide gel electrophoresis led to the detection of several precursor polypeptide (Pr) species containing the determinants of both p30 and pp12. These species, namely Pr>100, Pr100, Pr77, Pr62, and Pr50, were all found to be phosphorylated in pulse experiments. The polypeptide pp12 was, however, the major phosphorylated species immunoprecipitated by anti-pp12 sera in pulse and chase experiments. These data and a relatively high degree of phosphorylation in the processed pp12 suggested that phosphorylation of oncovirus protein is initiated at the polyprotein level and the phosphoprotein moiety is further phosphorylated subsequent to processing.     

Relationship between the subunit structure of insulin receptor and its competence to bind insulin and undergo phosphorylation

Insulin receptor partially purified from human placenta by chromatography on immobilized wheat germ agglutinin was subjected to affinity cross linking to determine the relationship between the subunit structure of the multiple forms of the insulin receptor and their competence to bind insulin and undergo autophosphorylation. It was demonstrated that, whereas the 340-kDa intact receptor undergoes autophosphorylation, the 290- and 320-kDa insulin binding forms of the receptor do not. Phosphorylation at tyrosyl residues in the intact receptor was verified using a new facile method for determination of phosphorylated amino acids. The competence of the phosphorylated 340-kDa protein to bind insulin was demonstrated using a double-probe labeling protocol wherein receptor phosphorylated with [γ-³²P]ATP was cross-linked with disuccinimidyl suberate (DSS) in the presence of N^?B29-biotinylinsulin. The observation that succinylavidin, by virtue of its interaction with biotinyl residues, decreased the electrophoretic mobility of receptor radiochemically labeled with ³²P indicated that the phosphorylated 340-kDa protein was competent to bind insulin. This result is compelling evidence that the 340-kDa phosphorylated species is insulin receptor itself, rather than a closely associated contaminant. Treatment of the receptor with the crosslinking agent DSS produced (after reduction and denaturation) α-dimer, β-dimer, and a smaller amount of tetramer. This observation is consistent with a symmetrical, tetrameric, α₂β₂structure for insulin receptor from human placenta, and excludes previously proposed alternative structures containing one α and One β Chain.     

Echistatin inhibits pp72 and pp125 phosphorylation in fibrinogen-adherent platelets

The adhesion of ADP-stimulated platelets to immobilized fibrinogen induces the tyrosine phosphorylation of multiple proteins which include pp72^syk and pp125^FAK. The phosphorylation of these two proteins increases as function of time of platelet adhesion to fibrinogen; however, pp72^syk results strongly phosphorylated already after 15 min. whereas pp125^FAK reaches high levels of phosphorylation after 1 h of platelet adhesion. Phosphorylation of both proteins is only slightly detectable when platelets are held in suspension or when platelets are allowed to adhere to bovine serum albumin, a non-specific substrate. Echistatin, an Arg-Gly-Asp (RGD)-containing snake-venom protein, affects protein tyrosine phosphorylation promoted by platelet adhesion to fibrinogen, by causing an approximately 44% and 39% decrease of pp72^syk and pp125^FAK phosphorylation, respectively. The interaction of echistatin with fibrinogen receptor glycoprotein Ilb-Illa on platelet surface might be responsible for the block of integrin-mediated signaling cascade, including pp72^syk and pp125^FAK inactivation.     

Enzymatic characteristics of pp60v-src isolated from vanadium-treated transformed cells

The transforming protein of Rous sarcoma virus (RSV) typically appears as a single phosphorylated polypeptide designated pp60^v-src, pp60^v-src possesses a protein kinase activity specific for tyrosine residues on select protein substrates. Treatment of RSV-transformed cells with vanadium ions resulted in the appearance of an electrophoretic variant of pp60^v-src and was paralleled by a significant increase in the src kinase specific activity in purified enzyme preparations. Both the normal (standard) src kinase and the src kinase preparations obtained from vanadium-treated cells exhibited similar optimal activity profiles for MgCl₂, KCl, and pH. Furthermore, their site specificities of phosphorylation of the substrates casein and vinculin were the same. The reaction kinetic profile of the standard src kinase showed a nonlinear pattern, while the vanadium enzyme exhibited conventional linear Michaelis-Menten kinetics. These results are discussed with respect to the possible functional regulation of pp60^v-src activity by a vanadium-sensitive protein phosphatase activity.     

A rapid enzymatic procedure for production of 1-beta-D-(3H)arabinofuranosyl cytosine 5'-triphosphate.

A rapid method of sequentially phosphorylating picomole quantities of [³H]-araC to [³H]araCTP is described (ara = 1-β-d-arabinofuranosyl). The procedure utilizes a system of phosphorylating enzymes isolated from rat spleen and requires a single incubation step. The [³H]araCTP product is isolated by ion-exchange chromatography and analyzed by PEI-cellulose thin-layer chromatography. At low concentrations of [³H]araC as much as 80% can be phosphorylated to the triphosphate, and the produet may be obtained in radiochemical purity greater than 97%.     

Action of sweet-potato beta-amylase on phosphodextrin of potato starch

The specificity of sweet-potato beta-amylase in the vicinity of the phosphate ester groups was studied by determining the structures of the phosphorylated oligosaccharides (alpha-phosphodextrin and beta-limit-alpha-phosphodextrin) formed by its action on potato starch. The beta-limit-alpha-phosphodextrin was separated by chromatography on Dowex-1 (HCOO^?) resin into three fractions that were distinguishable by the d.p. and by the ratio of d-glucose 6-phosphate residues to total organic phosphate. Each fraction contained linear molecules having one phosphate ester group that was not located at the reducing or non-reducing terminals. The smallest phosphodextrin was 6²-phosphorylmaltotriose. It was deduced that beta-amylase hydrolysed (1→4)-α-d linkages from the non-reducing end until one or two d-glucosyl residues remained attached to the phosphorylated residue, depending on whether there was originally an odd or even number of glucosyl residues on the non-reducing side of the phosphorylated residue.     

Insulin-stimulated tyrosine phosphorylation of a 43 kDa protein in rat liver membranes

The insulin receptor (IR) tyrosine kinase is essential for the regulation of different cellular functions by insulin. This may occur by a direct phosphorylation of membrane and/or cytoplasmic proteins by the IR tyrosine kinase. Hence it is important to identify putative physiological substrates for the IR tyrosine kinase. In this study we found that the glycoprotein fraction from rat liver membranes contain a 43 kDa protein (pp43) which, like the -subunit of IR, is phosphorylated in an insulin-dependent manner. A 25-fold enhancement of ³²P incorporation into pp43 by insulin was found under optimal conditions. Half-maximal phosphorylation of pp43 and the -subunit of IR were attained at 66 nM and 60 nM insulin, respectively. Mn²⁺ (K_a = 1.0 mM) was much better than Mg²⁺ (K_a = 6.3 mM) in supporting pp43 phosphorylation. Insulin-stimulated phosphorylation of pp43 (t_1/2 = 3.6 min) proceeded at a much slower rate compared to that of the -subunit of IR (t_1/2 = 1.2 min). Phosphoamino acid analysis of pp43 revealed that both tyrosine and serine are phosphorylated in the ratio 4 : 1. Tyrosine, but not serine, phosphorylation was increased 12-fold by insulin. Phosphorylation of pp43 occurred on 4 major tryptic peptides. Comparison to the tryptic phosphopeptides from IR -subunit suggest that pp43 was not derived from IR -subunit by proteolysis. Our results suggest that pp43 may be an endogenous substrate for the IR tyrosine kinase.     

Separation of phosphohydroxyamino acids by high-performance liquid chromatography

A high-performance liquid chromatography system has been developed which resolves O-phosphoserine, O-phosphothreonine, and O⁴-phosphotyrosine. Separation is performed on an anion-exchange resin eluted with phosphate buffer of low pH. Detection of the amino acid derivatives is accomplished using O-phthalaldehyde in an in-stream fluorometric system. This highly sensitive method has been applied to the detection of phosphohydroxyamino acids in bovine myelin basic protein phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase and in whole bovine myelin phosphorylated by endogenous kinases.     

Dephosphorylation of 40S ribosomal subunits by phosphoprotein phosphatase activity from rabbit reticulocytes.

Phosphoprotein phosphatase activities which remove phosphoryl groups from ribosomal protein have been partially purified from rabbit reticulocytes by chromatography on DEAE-cellulose. Two major peaks of phosphoprotein phosphatase activity were observed when 40S ribosomal subunits, phosphorylated $\text{in vitro}$ with cyclic AMP-regulated protein kinases and (γ-³²P)ATP, were used as substrate. The phosphatase activity eluting at 0.14 M KCl was characterized further using ribosomal subunits phosphorylated $\text{in situ}$ by incubation of intact reticulocytes with radioactive inorganic phosphate. Phosphate covalently bound to 40S ribosomal subunits and 80S ribosomes was removed by the phosphatase activity. The enzyme was not active with phosphorylated proteins associated with 60S ribosomal subunits.     

Protein phosphorylation in human peripheral nerve: Altered phosphorylation of a 25-kDa glycoprotein in leprosy

Protein phosphorylation in a low speed supernatant of human peripheral nerve (tibial and sural) homogenate was investigated. The major phosphorylated proteins had molecular mass in the range of 70, 55, 45, and 25 kDa. Mg²⁺ or Mn²⁺ was essential for maximum phosphorylation although Zn²⁺, Co²⁺, and Ca²⁺, could partially support phosphorylation. External protein substrates casein and histone were also phosphorylated. The protein phosphatase inhibitor orthovanadate enhanced the phosphorylation of the 45 and 25 kDa proteins significantly. Concanavalin A-Sepharose chromatography of the phosphorylated peripheral nerve proteins showed that the 25 kDa protein was a glycoprotein. Protein phosphorylation of peripheral nerves from leprosy affected individuals was compared with normals. The phosphorylation of 25 kDa protein was decreased in most of the patients with leprosy.     

Properties of phosphorylated NADP+-specific glutamate dehydrogenase fromEscherichia coli

The NADP⁺-specific glutamate dehydrogenase (GDH) fromEscherichia coli strain D₅H₃G₇, an enzyme that catalyzes the interconversion of -ketoglutarate andl-glutamate, has been shown to be phosphorylated in vitro in an ATP-dependent enzymatic reaction. The phosphorylated protein is extremely acid labile and is unstable at high pH. Treatment of GDH with diethyl pyrocarbonate (DEP), a histidine-modifying reagent, blocked the incorporation of³²P from [-³²P]ATP. GDH catalytic activity was also inhibited by DEP treatment. Hydroxylamine, a reagent hydrolyzing phosphoramidates, catalyzed the removal of phosphate from phosphorylated GDH, suggesting that GDH may be phosphorylated at a histidine residue(s). A total enzymatic hydrolysis of phosphorylated GDH, which was electroeluted from a native polyacrylamide gel, was analyzed by a Dowex 1-8X anion exchange chromatography. The presence of³²P-labeled 3-phosphohistidine, characterized and identified from this hydrolysate, demonstrates that a histidine residue(s) is the site of phosphorylation.     

Apparent activation of the MgATP-dependent protein phosphatase by pp60v-src identification of an activity like that of glycogen synthase kinase 3 in immunoaffinity purified pp60v-src preparations

Immunoaffinity purified pp60^v-src was found to activate the MgATP-dependent protein phosphatase in the presence of MgATP. Although preliminary evidence suggested that phosphorylation of the inhibitor-2 subunit on tyrosine residues was responsible for the activation, preincubation of the pp60^v-src preparation at 41°C resulted in a rapid loss of its protein kinase activities towards both casein and inhibitor-2 while its ability to activate the protein phosphatase complex was relatively insensitive to this treatment. This result demonstrated that pp60^v-src was not responsible for activation of the MgATP-dependent protein phosphatase. A protein kinase activity which phosphorylated glycogen synthase on serine residues was detected in the pp60^v-src preparation. The protein kinase was active in the presence of inhibitors of phosphorylase kinase, glycogen synthase kinase 5/casein kinase II, and cAMP-dependent protein kinase. It is, therefore, likely that activation of the MgATP-dependent protein phosphatase resulted from the presence of a glycogen synthase kinase 3 like activity in the pp60^v-src preparation. Our results illustrate the importance of applying multiple criteria to link the phosphorylation of a protein with an observed change in its activity.     

LOCALIZATION OF SH-PTP1 TO SYNAPTIC VESICLES: A POSSIBLE ROLE IN NEUROTRANSMISSION

The tyrosine kinase pp60^src is known to phosphorylate synaptophysin and in doing so may regulate neurotransmitter release. The tyrosyl phosphorylated state of synaptophysin is dependent on pp60^src kinase and the unknown protein tyrosine phosphate phosphohydrolase (PTPase, EC 3.1.3.48). Here we report the protein tyrosine phosphate phosphohydrolase SH-PTP1, to associate with synaptic vesicles and interact with synaptophysin. These studies identify SH-PTP1 as a new member of the secretory machinery at the nerve terminal and suggest its involvement in neurotransmission.     

The mobile ferrous iron pool in Escherichia coli is bound to a phosphorylated sugar derivative

Based on in vivo Mössbauer spectroscopy it has previously been demonstrated that the intracellular iron pool of Escherichia coli, grown in iron deficient media supplemented with siderophores as the sole iron source, is dominated by a single Fe²⁺ and a single Fe³⁺ species. We have isolated the ferrous ion species and have purified it employing native column PAGE, chromatography and ultrafiltration. The purified compound displays an M _app of 2.2 kDa and an extremely low isoelectric point (pI) of 1.05. It is shown that this ferrous ion binding compound is neither a protein nor a nucleotide, rather it is composed mainly of phosphorylated sugar derivatives. This compound binds approximately 40% of the cytoplasmic iron. Therefore it is proposed that this oligomeric ferrous carbohydrate phosphate represents the long sought after mobile, low molecular mass iron pool.     

Evidence for a beta-Aspartyl Phosphate Residue in the Phosphorylated Intermediate of the Red Beet Plasma Membrane ATPase

A borohydride reduction method was used to identify the phosphorylated amino acid in the phospho-enzyme of the red beet (Beta vulgaris L.) plasma membrane ATPase. Plasma membrane fractions were phosphorylated with unlabeled ATP in the presence of MgSO₄ at pH 6.5 and then treated with sodium [³H]borohydride. The borohydride-treated samples were subjected to hydrolysis in 6 normal HCl at 110°C for 22 hours and then analyzed by high voltage paper electrophoresis and thin layer chromatography. This analysis demonstrated the formation of labeled homoserine as the major reduction product when phosphorylated membrane samples were treated with sodium [³H]borohydride. This suggests that the phosphoryl group in the plasma membrane ATPase of red beet storage tissue is attached to the β-carboxyl side chain of an aspartic acid residue in the active site of the enzyme.     

Phosphorylated α(1→4)Glucans as Substrate for Potato Starch-Branching Enzyme I

The possible involvement of potato (Solanum tuberosum L.) starch-branching enzyme I (PSBE-I) in the in vivo synthesis of phosphorylated amylopectin was investigated in in vitro experiments with isolated PSBE-I using ³³P-labeled phosphorylated and ³H end-labeled nonphosphorylated α(1→4)glucans as the substrates. From these radiolabeled substrates PSBE-I was shown to catalyze the formation of dual-labeled (³H/³³P) phosphorylated branched polysaccharides with an average degree of polymerization of 80 to 85. The relatively high molecular mass indicated that the product was the result of multiple chain-transfer reactions. The presence of α(1→6) branch points was documented by isoamylase treatment and anion-exchange chromatography. Although the initial steps of the in vivo mechanism responsible for phosphorylation of potato starch remains elusive, the present study demonstrates that the enzyme machinery available in potato has the ability to incorporate phosphorylated α(1→4)glucans into neutral polysaccharides in an interchain catalytic reaction. Potato mini tubers synthesized phosphorylated starch from exogenously supplied ³³PO₄³⁻ and [U-¹⁴C]Glc at rates 4 times higher than those previously obtained using tubers from fully grown potato plants. This system was more reproducible compared with soil-grown tubers and was therefore used for preparation of ³³P-labeled phosphorylated α(1→4)glucan chains.     

Identification of an adipocyte protein that binds to calmodulin in the absence of Ca2+ and is phosphorylated in response to insulin and tumor-promoting phorbol esters

The present experiments were performed to identify calmodulin-binding proteins phosphorylated in response to insulin. Homogenates were prepared from 32Pi-labeled rat adipocytes. After centrifugation, the supernatants (+/- Ca2+) were applied to calmodulin-Sepharose columns. The bound proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and phosphoproteins were visualized by autoradiography. Several proteins bound to the affinity resin in the presence of Ca2+, two bound +/- Ca2+, but only one protein, Mr = 170,000 (denoted pp170), bound in the absence of Ca2+. Binding of pp170 was inhibited by adding calmodulin (micromolar) or Ca2+ (nanomolar) to extracts prior to affinity chromatography. Physiological concentrations of insulin rapidly and reversibly increased (by as much as 4-fold) 32P-labeled pp170. Phorbol 12-myristate 13-acetate (PMA) increased (up to 3-fold) phosphorylation of pp170; but 4 alpha-phorbol 12,13-didecanoate was without effect. Phosphorylation of pp170 in response to insulin and PMA occurred predominantly on serine residues; no phosphotyrosine was detected. Protein kinase C inhibitors attenuated PMA-stimulated phosphorylation of pp170, but had no effect on insulin-stimulated phosphorylation. Peptide mapping indicated that pp170 was phosphorylated on multiple sites and that insulin stimulated the phosphorylation of at least one site not phosphorylated in response to PMA. The results indicate that insulin and PMA stimulate the phosphorylation of pp170 via different pathways, the latter presumably via protein kinase C.