Characterization of specific differences in protein phosphorylation of the plasma membrane and the endoplasmic reticulum of mouse fibroblasts

Endogenous phosphorylation was studied with highly purified fractions of the plasma membrane and the endoplasmic reticulum of SV40-transformed mouse fibroblasts using [γ-³²P]ATP and [γ-³²P]GTP as precursors. With ATP maximum overall incorporation of ³²P into both membrane fractions occured at pH 7.8 in the presence of 10 mM MgCl₂ after incubation for 1 min. GTP could be utilized only by the plasma membrane fraction showing maximum incorporation of ³²P at pH 7.8 and 10 mM MgCl₂ after incubation for 3 min.The pattern of phosphoproteins of the plasma membrane is represented by more than 15 proteins whereas the endoplasmic reticulum essentially contained only one phosphorylated component of 35 000 molecular weight. The comparison of ATP- and GTP-specific phophorlation of the plasma membrane revealed GTP to be a less efficient precursor yielding a similar phosphoprotein pattern with one significant difference: the GTP-specific main component exhibited a molecular wieght of about 100 000 and the ATP-specific main component a molecular weight of 110 000.The relative distribution of individual phosphoproteins in the pattern of the plasma membrane was dependent on pH but not on MgCl₂ concentration or time of incubation. Increasing concentrations of plasma membrane protein altered the patterns of phosphoproteins dramatically: At high protein concentrations the ATP-specific main component (M_r = 110 000) was no more phosphorylated whereas with GTP the main component M_r = 100 000 was essentially the sole phosphorylated protein.     

Characterization and Intraorganellar Distribution of Protein Kinases in Amyloplasts Isolated from Cultured Cells of Sycamore (Acer pseudoplatanus L.)

Incubation of amyloplasts isolated from cultured cells of sycamore (Acer pseudoplatanus L.) with [γ-³²P]ATP resulted in the rapid phosphorylation (half-time of 40 seconds at 25 degrees Celcius) of organellar polypeptides. The preferred substrate for amyloplast protein kinases was Mg²⁺. ATP, and recovery of only [³²P]serine after partial acid hydrolysis indicated the predominance of protein serine kinases in the organelle. These activities were located in the envelope and stromal fractions of the plastid, which showed different specificities toward exogenous protein substrates and distinct patterns of phosphorylation of endogenous polypeptides. A 66-kilodalton polypeptide, inaccessible to an exogenously added protease, was one of the major phosphorylated products found in intact amyloplasts at low [γ-³²P] adenosine triphosphate concentrations. This polypeptide represented the major phosphoprotein observed with the isolated envelope fraction. The patterns of polypeptide phosphorylation found in intact amyloplasts and chloroplasts from cultured cell lines of sycamore were clearly distinguishable. The overall results indicate the presence of protein phosphorylation systems unique to this reserve plastid present in nonphotosynthetic tissues.     

Human osteogenic sarcoma cells exhibit enhanced protein phosphorylation

Protein phosphorylation was compared in normal human cells and human osteogenic sarcoma cells. The phosphorylation of endogenous cellular protein substrates was measured by two independent methods, incubation of homogenized cells with [γ-³²P]ATP or labeling of intact cells with Na₂H³²PO₄. Phosphorylated proteins were identified by SDS-polyacrylamide gel electrophoresis and autoradiography. The stained protein bands of all four osteosarcoma cell lines were nearly identical to those of the normal cells. However, each of the osteosarcoma cell lines showed autoradiographic evidence of enhanced phosphorylation in many different protein bands which was neither cyclic AMP-dependent nor a function of cellular growth rate or density. When normal and tumor cell homogenates were mixed prior to incubation with [γ-³²P]ATP, the resulting phosphoprotein patterns resembled those obtained with the tumor cells alone. In addition, a surgically derived osteogenic sarcoma was cultured and an established line obtained; another portion of the fresh tumor was immediately homogenized and used in a phosphorylation assay. The same enhanced phosphorylation pattern was obtained with the homogenized fresh tumor as with the cell line established from it. These results suggest thathuman osteogenic sarcoma cells are able to perform a significantly increased amount of phosphorylation of endegenous cellular protein substrates when compared to normal human cells.     

Comparison of the Protein Kinase and Acid Phosphatase Activities of Five Species of Leishmania1

Promastigotes from log phase and stationary phase cultures of Leishmania donovani, L. braziliensis panamensis, L. tropica, L. major, and L. mexicana amazonensis were analyzed for their content of protein kinase and acid phosphatase activities. Cell surface, historic-specific protein kinase activity was 1.3- to 2.8-fold higher in stationary phase cells of all species except for L. tropica in which the activities of stationary and log phase cells were equal; L. mexicana amazonensis had the highest histone-specific protein kinase activity and L. donovani the lowest. When viable, motile promastigotes of all five species were incubated for 10 min with [γ-³²P]ATP and Mg²⁺ (10 mM) in the absence of exogenous histone acceptor; about one dozen proteins were phosphorylated in each case. Both log phase and stationary phase promastigotes of all five species extensively phosphorylated a 50-kDa protein that had the mobility of tubulin. Incubation of pure calf brain tubulin with [γ-³²P]ATP and purified L. donovani protein kinase resulted in extensive phosphorylation of the former. Highly infective metacyclic forms (PNA^-) of L. major, isolated from a stationary culture using the peanut agglutinin (PNA), contained eight times more histone-specific protein kinase activity than noninfective log phase cells (PNA⁺). The PNA^- and PNA⁺ forms of L. major both phosphorylated a 50-kDa protein when incubated with [γ-³²P]ATP and magnesium or manganese ions (10 mM); the 50-kDa protein was precipitated by anti-tubulin rabbit antibodies. Extracts of all five species contained large amounts of acid phosphatase activity. With the exception of L. braziliensis panamensis for which late log phase organisms contained 12-fold more tartrate-resistant acid phosphatase activity than did early log phase cells, stationary and log phase parasites contained approximately the same amount of acid phosphatase activity.     

Blue-light-induced phosphorylation of a plasma-membrane protein from phototropically sensitive tips of maize coleoptiles

Tips of maize coleoptiles, which function as esential light sensors for the phototropic growth reaction, exhibit a rapid blue-light-induced phosphorylation of a plasma-membrane-associated 100-kDa protein. Characteristics of this reaction are as follows: (i) The functional unit involved in the light-dependent phosphorylation consists of a photoreceptor, a protein kinase and the 100-kDa protein. This complex is only localized in the plasma membrane of tips but not in other parts of the seedling, (ii) The photoreceptor is a cryptochrome-like compound, (iii) The pH optimum of the light-dependent phosphorylation on isolated plasma membranes is around pH 7.8 whereas the light-independent phosphorylation of other membrane proteins occurs at lower values (pH 6.2). (iv) The light-induced in-vitro phosphorylation of the 100-kDa protein is strongly inhibited by the protein-kinase inhibitor staurosporine (IC₅₀=4 nM). (v) The ³²P-moiety of a ³²P-[100 kDa]-protein complex generated after a light pulse with the aid of a membrane-associated protein kinase in the presence of [γ-³²P]ATP cannot be removed by a 100-fold higher level of (unlabelled) ATP. This fact indicates that protein and phosphate are covalently connected and that the complex is not a short-lived intermediate. (vi) The 100-kDa protein is not identical to the plasma-membrane H⁺-ATPase, as shown by immunostaining on Western blots. (vii) Irradiation-dependent in vivo phosphorylation of the 100-kDa protein in tips is already saturated by a light pulse of 5 s. In contrast, the de-phosphorylation of the protein in the dark is a slow reaction lasting about 30 min. It is suggested that the blue-light-triggered phosphorylated status of the 100-kDa protein is an early step in phototropism of the coleoptile, affecting the transport of auxin primarily in the irradiated flank.     

Changes in the pattern of protein phosphorylation during meiosis reinitiation in starfish oocytes

Endogenous protein phosphorylation in oocytes of Marthasterias glacialis was examined by incubating living oocytes with [³²P]phosphate and cortical and endoplasmic fractions with [γ-³²P]ATP. Individual phosphorylated proteins were detected by autoradiography after bidimensional and monodimensional electrophoresis, using SDS-polyacrylamide gradient gel slabs. An increased phosphorylation of several protein species was observed as early as 5 min following in vivo hormonal stimulation by 1-methyladenine. No dephosphorylation nor any change in protein staining of the gels was observed. In vitro phosphorylation patterns were consistent with those observed in vivo. They did not change upon in vitro 1-methyladenine addition and remained unaffected when the incubations were carried out in the presence of cAMP or beef heart protein kinase inhibitor. Cortical phosphorylation was inhibited by calcium ions. The results suggest that the hormone promotes alterations in the availability of phosphorylation sites in some proteins already present in the control oocytes which, as well as the corresponding activated cAMP-independent protein kinases, may play a significant role during formation of the maturation promoting factor.     

On the role of protein phosphorylation in the ATP-dependent permeabilization of transformed cells

Incubation of transformed mouse fibroblasts with external ATP in alkaline medium low in divalent cations causes an increase in the permeability of the plasma membrane to nucleotides and other small molecules. Previous suggestions that the phosphorylation of a 44,000 dalton membrane protein is involved in this permeabilization process have been pursued. Fractionation of cells that had been incubated with [γ-³²P] ATP revealed that the labeled 44K phosphoprotein was found in both the membrane and mitochondrial fractions. Incubation of fractions isolated from unlabeled cells with [γ-³²P] ATP resulted in substantial formation of ³²P-44K in the mitochondrial fraction and less incorporation in the membrane fraction. The 44,000 dalton protein was identified as the α-subunit of mitochondrial pyruvate dehydrogenase by partial proteolytic mapping and immunological cross-reactivity with antibodies prepared against bovine pyruvate dehydrogenase. The phosphorylation of this protein in whole cells by externally added ATP is suppressed by inclusion in the incubation medium of carboxyatractyloside (CAT) and EDTA. These substances have no effect on ATP-dependent permeabilization, indicating that the phosphorylation of pyruvate dehydrogenase is not involved in this process.     

In vitro phosphorylation of maize leaf phosphoenolpyruvate carboxylase

Autoradiography of total soluble maize (Zea mays) leaf proteins incubated with ³²P-labeled adenylates and separated by denaturing electrophoresis revealed that many polypeptides were phosphorylated in vitro by endogenous protein kinase(s). The most intense band was at 94 to 100 kilodaltons and was observed when using either [γ-³²P]ATP or [β-³²P]ADP as the phosphate donor. This band was comprised of the subunits of both pyruvate, Pi dikinase (PPDK) and phosphoenolpyruvate carboxylase (PEPCase). PPDK activity was previously shown to be dark/light-regulated via a novel ADP-dependent phosphorylation/Pi-dependent dephosphorylation of a threonyl residue. The identity of the acid-stable 94 to 100 kilodalton band phosphorylated by ATP was established unequivocally as PEPCase by two-dimensional gel electrophoresis and immunoblotting. The phosphorylated amino acid was a serine residue, as determined by two-dimensional thin-layer electrophoresis. While the in vitro phosphorylation of PEPCase from illuminated maize leaves by an endogenous protein kinase resulted in a partial inactivation (~25%) of the enzyme when assayed at pH 7 and subsaturating levels of PEP, effector modulation by l-malate and glucose-6-phosphate was relatively unaffected. Changes in the aggregation state of maize PEPCase (homotetrameric native structure) were studied by nondenaturing electrophoresis and immunoblotting. Enzyme from leaves of illuminated plants dissociated upon dilution, whereas the protein from darkened tissue did not dissociate, thus indicating a physical difference between the enzyme from light- versus dark-adapted maize plants.     

Rapid protein kinase assay using phosphocellulose-paper absorption.

Protein kinase (EC 2.7.1.37) catalyzes the phosphorylation of serine and threonine residues of a number of proteins. Histone is widely used as an acceptor substrate in measuring the activity of this enzyme isolated from a variety of sources. We have devised a rapid procedure for resolving phosphohistone from ATP and its metabolites based on the specific absorption of phosphorylated histone onto phosphocellulose paper. Using [γ-³²P]ATP as the phosphoryl donor, aliquots of the protein kinase assay mixture are applied to phosphocellulose-paper disks that are then immersed in water which elutes [γ-³²P]ATP and metabolites. After brief organic solvent extraction and drying, bound radioactivity is measured by liquid scintillation spectrometry.     

Developmental changes in the cyclic AMP-dependent phosphorylation and dephosphorylation of a protein endogenous to murine brain and liver.

Brain and liver cytosol extracts from mice of different ages were incubated with (γ-³²P)ATP. The phosphorylated substrates were separated by gel electrophoresis and examined by autoradiography. The amount of P³² that could be incorporated into a 49,000 M.W. protein (called protein 49) postnatally increased in brain but decreased in liver. Cyclic AMP stimulated both the phosphorylation and dephosphorylation of liver protein 49 to a greater extent in adults than in neonates. Brain protein 49 phosphorylation was more sensitive to cyclic AMP in neonates than in adults.     

Nuclear protein phosphorylation in isolated nuclei from HeLa cells. Evidence that 32P incorporation from [γ-32P]GTP is catalyzed by nuclear kinase II

A nuclear system for studying nuclear protein phosphorylation is characterized, using as phosphate donor either low levels of [γ-³²P]GTP, low levels of [γ-³²P]ATP, or low levels of labeled ATP plus excess unlabeled GTP. Since nuclear casein kinase II is the only described nuclear protein kinase to use GTP with high affinity, low levels of GTP should specifically assay this enzyme. ATP should measure all kinases, and ATP plus unlabeled GTP should measure all kinases except nuclear casein kinase II (ATP-specific kinases). The results are consistent with these predictions. In contrast with the ATP-specific activity, endogenous phosphorylation with GTP was enhanced by 100 mM NaCl, inhibited by heparin and quercetin, stimulated by polyamines, and did not use exogenous histone as substrate. The GTP- and ATP-specific kinases phosphorylated different subsets of about 20 endogenous polypeptides each. Addition of purified casein kinase II enhanced the GTP-supported phosphorylation of the identical proteins that were phosphorylated by endogenous kinase. These results support the hypothesis that activity measured with GTP is catalyzed by nuclear casein kinase II, though other minor kinases which can use GTP are not ruled out. Preliminary observations with this system suggest that the major nuclear kinases exist in an inhibited state in nuclei, and that the effects of polyamines on nuclear casein kinase II activity are substrate specific. This nuclear system is used to determine if the C-proteins of hnRNP particles, previously shown to be substrates for nuclear casein kinase II in isolated particles, is phosphorylated by GTP in intact nuclei. The results demonstrate that the C-proteins are effectively phosphorylated by GTP, but in addition they are phosphorylated by ATP-specific kinase activity.     

Evidence for the utilization of extracellular [γ-32P]ATP for the phosphorylation of intracellular proteins in the squid giant axon

Proteins in the squid giant axon were labeled with ³²P by in vitro incubation of isolated axoplasm with radioactive [γ-³²P]adenosine triphosphate (ATP) and separated by polyacrylamide sodium dodecyl sulfate gel electrophoresis. The two major phosphorylated regions on the gel had molecular weights of 400 000 and 200 000. These two peaks appear to be neurofilament proteins of squid axoplasm. The same set of proteins was phosphorylated in the axoplasm regardless of whether the [γ-³²P]ATP was applied in situ intracellularly or extracelarly. These results suggest that ATP in the extracellular space is, by some ATP-translocation mechanism, utilized in the process of intracellular phosphorylation. Measurements of the apparent influx of ATP across the squid axon membrane yielded results consistent with the view that ATP in the extracellular fluid could be transported into the axoplasm.     

Endogenous Protein Phosphorylation in Rat Brain Mitochondria: Occurrence of a Novel ATP-Dependent Form of the Autophosphorylated Enzyme Succinyl-CoA Synthetase

Abstract: When rat brain mitochondria are incubated with [γ-³²P]ATP, there is a rapid (10 s) phosphorylation of proteins designated E, and F of M.W. 42,000 and 32,000, respectively. Although [γ-³²P]ATP was the preferred substrate for protein F, a small amount of labeling did occur with [γ-³²P]GTP. Phosphorylation of E₁ was absolutely ATP-dependent. On the other hand, a 32,000 M.W. protein from rat liver mitoplasts (mitochondria devoid of an outer membrane) was highly phosphorylated when [γ-³²P]GTP was used but not at all phosphorylated within short time periods with [γ-³²P]ATP. Both the ATP-labeled brain phosphoprotein F and GTP-labeled liver protein migrated to identical positions on high-resolution two-dimensional polyacrylamide gels, and both contained acid-labile phosphoryl groups. Furthermore, both phosphoproteins were identified as the autophosphorylated subunit of succinyl-CoA synthetase (SCS, EC 6.2.1.4) by using antibody directed against purified GTP-dependent porcine SCS. However, immunotitration experiments with anti-porcine SCS revealed that ATP- and GTP-labeled protein F in brain differed in their interactions with antibody, suggesting that in rat brain mitochondria two different forms of the enzyme exist that are immunologically distinct and differ in substrate specificity. When mitochondrial preparations enriched in particular brain cell or subcellular types were examined, an unequal distribution of E₁ and the two forms of protein F were observed. A brain subfraction containing neuronal cell body and glial mitochondria (CM) was found to contain E₁ and approximately equal amounts of the ATP- and GTP-dependent forms of protein F. Light synaptic mitochondria(SM₁) contained ATP-dependent protein F almost exclusively and were depleted in E₁. Dense synaptic mitochondria (SM₂) are rich in the ATP form of SCS but also contain low amounts of the GTP enzyme.     

Phosphorylation of membrane proteins from Plasmodium berghei-infected red cells.

Membrane from Plasmodium berghei-infected mouse red cells has a different pattern of phosphorylation by (γ-³²P)ATP from normal membrane. A phosphorylated membrane protein of apparent molecular weight 42,000, absent in membrane from normal cells, can be detected in membrane from infected cells. The new phosphorylated protein can be extracted by 0.1 mM EDTA but not by triton X-100, indicating that it may be red cell actin.     

Partial characterization of protein kinase-catalyzed phosphorylation of low molecular weight proteins in purified preparations of pigeon heart sarcolemma and sarcoplasmic reticulum

Pigeon heart microsomes contain three minor size protein kinase substrates of minimal molecular weights of 22 000, 15 000, and 11 500, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When the microsomes were partially loaded with calcium oxalate and subjected to rate zonal and isopynic centrifugations in sucrose density gradient columns, the 22 000 and the 15 000 dalton proteins settled in the heaviest fraction, which was composed mainly of vesicles of sarcoplasmic reticular membranes; the 11 500 dalton protein was concentrated in the lightest fractions, which consisted chiefly of vesicles of sarcolemmal origin. During incubation of the membrane fractions with Mg[γ-³²P]ATP significant amounts of ³²P were incorporated into all these proteins. Incorporation of ³²P into the 15 000 dalton protein was moderately and ³²P incorporation into the 22 000 dalton protein was markedly enhanced in the presence of exogenous soluble cyclic AMP-dependent protein kinase and cyclic AMP. The phosphorylation of the three proteins was virtually unaffected by CA²⁺ concentrations up to 0.1 mM and by ethyleneglycol-bis(β-aminoethylether)-N,N′-tetraacetic acid in the absence of added Ca²⁺.Phosphorylation of the 22 000 and the 11 500 dalton proteins occurred mainly at serine residues. In the 15 000 dalton protein threonine residues were the main site of endogenous phosphorylation. Nearly equal amounts of [³²P]-phosphate were incorporated into threonine and serine residues of this protein when phosphorylation was supported by exogenous cyclic AMP-dependent protein kinase and cyclic AMP.The 15 000 dalton protein could be removed from its membrane attachment by extraction with an acidic chloroform/methanol mixture. This step opens the way for the purification of this membrane-bound protein kinase substrate.     

Characteristics of Membrane Protein Phosphorylation in Plasmodium berghei-Infected Mouse Erythrocytes1

ABSTRACT. Membrane protein phosphorylation in Plasmodium berghei-infected erythrocytes was studied by incubating intact cells with (³²P)orthophosphate and incubating isolated membrane with (γ-³²P)ATP. Phosphorylated proteins were detected by autoradiography after sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis or isoelectric focusing followed by gel electrophoresis. New phosphorylated proteins were found in membrane from infected erythrocytes, including a protein with electrophoretic mobility identical to band 5, with M, 43,000. The molar ratio of phosphate to protein ranged between 0.1 and 0.5. Isoelectric focusing-SDS polyacrylamide gel electrophoresis, peptide mapping, extractability properties, and reduction of susceptibility to DNase I inhibition suggested that this protein is phosphorylated actin. In contrast, spectrin phosphorylation in infected erythrocytes was mostly unchanged.     

On substrate specificity of the donor site of the Escherichia coli ribosomal peptidyl transferase center: Synthesis of dipeptides from 3′-terminal fragments of aminoacyl-tRNA

The insulin receptor of human placenta even after extensive purification is phosphorylated in the presence of [γ-³²P]ATP and NaF, and is dephosphorylated again on incubation in NaF-free medium. Insulin stimulates phosphate incorporation into the M_r95 000 subunit probably by activation of the phosphorylation step. Our data suggest that the insulin receptor contains both kinase and phosphatase activities that may control the phosphorylation state of the receptor.     

Determination of phosphorylase kinase activity in crude homogenates by affinity chromatography on 5′-AMP Sepharose

A sensitive method for measuring phosphorylase kinase activity by the incorporation of ³²P from [γ-³²]ATP into phosphorylase in the presence of other phosphorylation reactions is described. The kinase reaction is carried out in a crude homogenate. After stopping the reaction, a portion of the reaction mixture is withdrawn for assay of phosphorylase conversion and the rest is applied on a 5′-AMP Sepharose column. Phosphorylase in both forms is retained on the column while other phosphorylated proteins and [γ-³²P]ATP are washed out. The phosphorylase is then eluted by 10 mm AMP and the radioactivity incorporated is counted.     

Identification of the 64 kilodalton chloroplast stromal phosphoprotein as phosphoglucomutase

Phosphorylation of the 64 kilodalton stromal phosphoprotein by incubation of pea (Pisum sativum) chloroplast extracts with [γ-³²P]ATP decreased in the presence of Glc-6-P and Glc-1,6-P₂, but was stimulated by glucose. Two-dimensional gel electrophoresis following incubation of intact chloroplasts and stromal extracts with [γ-³²P]ATP, or incubation of stromal extracts and partially purified phosphoglucomutase (EC 2.7.5.1) with [³²P]Glc-1-P showed that the identical 64 kilodalton polypeptide was labeled. A 62 kilodalton polypeptide was phosphorylated by incubation of tobacco (Nicotiana sylvestris) stromal extracts with either [γ-³²P]ATP or [³²P]Glc-1-P. In contrast, an analogous polypeptide was not phosphorylated in extracts from a tobacco mutant deficient in plastid phosphoglucomutase activity. The results indicate that the 64 (or 62) kilodalton chloroplast stromal phosphoprotein is phosphoglucomutase.     

Protein kinases in brown adipose tissue of developing rats I. GTP as nucleotide substrate for histone phosphorylation

100 000 × g soluble extracts from interscapular brown adipose tissue catalyzed the transfer of the terminal phosphoryl group from GTP to histone. Maximal velocity was achieved only with both cyclic AMP and ATP present. The cyclic AMP dose-response curve was the same as for the ATP-utilizing enzyme, with maximum stimulation at 0.5 μM. ATP (1–100 μM) increased the rate of histone phosphorylation with GTP as the radioactive substrate. Higher concentrations had a dilution effect similar to that of GTP on the ATP-utilizing enzyme. Similar effects were observed with ADP and AMP. The apparent K_m values for histone were the same with both GTP and ATP as nucleotide substrates. The effects of pH, purified beef muscle kinase inhibitor and of NaCl were also the same. Maximum velocities of histone phosphorylation from ATP and those from GTP were almost the same in brown fat of all age groups tested. Separated on histone-Sepharose, the GTP-utilizing activity was absolutely dependent on the re-addition of the ATP-utilizing enzyme (a linear relationship with a slope of approx. 0.95). An extremely active nucleotide phosphotransferase activity was found in the same subcellular fraction. The rate of equilibration of the γ-³² P between GTP and ATP could account for all the histone phosphorylation with [γ-³² P] GTP. It is concluded that, in spite of the presence of nucleotide phosphotransferase and ATP-protein kinase activities, a direct transfer from GTP to a protein substrate cannot be excluded. Also, histone may not be the natural protein acceptor for GTP-linked phosphorylation.