Effect of Zinc on Calmodulin-Stimulated Protein Kinase II and Protein Phosphorylation in Rat Cerebral Cortex

The effect of increasing concentrations of Zn2+ (1 microM-5 mM) on protein phosphorylation was investigated in cytosol (S3) and crude synaptic plasma membrane (P2-M) fractions from rat cerebral cortex and purified calmodulin-stimulated protein kinase II (CMK II). Zn2+ was found to be a potent inhibitor of both protein kinase and protein phosphatase activities, with highly specific effects on CMK II. Only one phosphoprotein band (40 kDa in P2-M phosphorylated under basal conditions) was unaffected by addition of Zn2+. The vast majority of phosphoprotein bands in both basal and calcium/calmodulin-stimulated conditions showed a dose-dependent inhibition of phosphorylation, which varied with individual phosphoproteins. Two basal phosphoprotein bands (58 and 66 kDa in S3) showed a significant stimulation of phosphorylation at 100 microM Zn2+ with decreased stimulation at higher concentrations, which was absent by 5 mM Zn2+. A few Ca2+/calmodulin-stimulated phosphoproteins in P2-M and S3 showed biphasic behavior; inhibition at less than 100 microM Zn2+ and stimulation by millimolar concentrations of Zn2+ in the presence or absence of added Ca2+/calmodulin. The two major phosphoproteins in this group were identified as the alpha and beta subunits of CMK II. Using purified enzyme, Zn2+ was shown to have two direct effects on CMK II: an inhibition of Ca2+/calmodulin-stimulated autophosphorylation and substrate phosphorylation activity at low concentrations and the creation of a new Zn(2+)-stimulated, Ca2+/calmodulin-independent activity at concentrations of greater than 100 microM that produces a redistribution of activity biased toward autophosphorylation and an alpha subunit with an altered mobility on sodium dodecyl sulfate-containing gels.     

Ribosomal proteins of Tetrahymena thermophila. Correlation of one- and two-dimensional electrophoretic migration patterns and characterization of additional small and large subunit proteins

Further analysis of the protein complement of the cytoplasmic ribosome of the protozoon Tetrahymena thermophila has led to the identification and characterization of seven additional proteins, three in the small and four in the large subunit of this ribosome. Several of these proteins are poorly soluble or insoluble in the absence of high concentrations of urea and are not seen in the electrophoretic distribution patterns of ribosomal proteins in two-dimensional polyacrylamide gels unless 6 M urea is added to electrode buffers in contact with protein samples (first dimension) and first-dimension gels (second dimension). The migration patterns of the 40S and 60S subunits of the T. thermophila ribosome in one-dimensional polyacrylamide SDS gels and in two-dimensional gels prepared by means of the basic-acidic system of Kaltschmidt and Wittmann**, and the basic-SDS system of Zinker and Warner*** have been correlated.     

Protein phosphorylation in intact bovine epididymal spermatozoa: identification of the type II regulatory subunit of cyclic adenosine 3',5'-monophosphate-dependent protein kinase as an endogenous phosphoprotein

An obstacle to the study of protein phosphorylation in mammalian spermatozoa has been the inability to incorporate sufficient amounts of 32Pi into cellular adenosine triphosphate (ATP) (Babcock et al., 1975). We report conditions under which 32Pi is effectively incorporated into the ATP of intact bovine spermatozoa. In the presence of a bicarbonate-buffered medium containing glucose, spermatozoa incorporated 32P into intracellular ATP in a time-dependent manner; after 2 h of incubation, the specific activity of [gamma-32P]ATP (2.3 X 10(4) cpm/nmol ATP) was estimated to be 50-65% of the specific activity of the intracellular phosphate pool. In the absence of glucose or other added substrates, the specific activity of [gamma-32P]ATP was 10-25% that of the specific activity observed in the presence of glucose. Washed spermatozoa incubated in carrier-free 32Pi for 2 h at 37 degrees C, and solubilized in a solution containing final concentrations of 6.8 M urea, 6% NP4O, and 5% beta-mercaptoethanol contained in excess of 40 32Pi-labeled proteins as assessed by two-dimensional polyacrylamide gel electrophoresis. Major phosphoproteins had approximate molecular weights of 93,000, 40,000, and 22,000. A different two-dimensional gel pattern was observed when cells were extracted with a solution containing 38.5 mM 2[N-cyclohexylamino] ethanesulfonic acid (CHES), pH 9.5/1.5% sodium dodecyl sulphate (SDS) at 100 degrees C. In contrast to the urea/Nonidet P-40 (NP40)/beta-mercaptoethanol extract, a 56,000 Mr phosphoprotein represented a major component while the 40,000 Mr and several of the 22,000 Mr polypeptides were markedly reduced in radioactive intensity. The 56,000 Mr species present in the CHES/SDS extract comigrated with the purified, phosphorylated regulatory subunit (RII) of cyclic adenosine 3',5'-monophosphate-dependent protein kinase from bovine heart. Antibodies to RII immunoprecipitated a 56,000 Mr, 32P-labeled polypeptide from the CHES/SDS extract that comigrated with purified, [32P] RII after two-dimensional electrophoresis. RII, then, appears to represent one of the endogenous phosphoproteins of intact bovine epididymal spermatozoa.     

Phosphorylation of ribosomal proteins in rat cerebral cortex in vitro.

Incubation of cerebral cortical tissue from immature rats in the presence of [32P]orthophosphate resulted in similar rates of incorporation of radioactivity into the proteins of free and membrane-bound ribosomes. Incorporation of label into ribosomal proteins of both species continued actively for at least 3 hours. Since recovery of membrane-bound ribosomes from rat cerebral cortex is quite low, further analyses of the radioactive phosphoproteins were restricted to the free ribosome population. A significant fraction of the radioactivity which was precipitated with trichloroacetic acid was not removed by heating in trichloroacetic acid at 90 degrees or extracted with organic solvents and therefore was presumed to be covalently bound to protein. The radioactive phosphoryl groups present in the ribosomal proteins were mainly in ester linkages since they were readily removed by exposure to 1 N NaOH, relatively unaltered by 1N HCl, and unaffected by hydroxylamine. This conclusion was supported by the isolation of labeled o-phosphoserine and o-phosphothreonine residues from hydrolysates of ribosomal proteins. A significant fraction of the labeled phosphoproteins in the purified ribosomes appeared to be bound tightly to the ribosome structure since only 40% of the radioactivity could be removed by extraction of these ribosomes with 1 M KCl. Phosphorylation of proteins of cerebral monoribosomes was more rapid than the same process in polyribosomes from the same source. Eight radioactive phosphoprotein bands could be detected by electrophoresis of proteins obtained from unfractionated cerebral ribosomes on unidimensional polyacrylamide gels containing sodium dodecyl sulfate. The protein nature of these materials was confirmed by pronase digestion. Proteins of subribosomal particles isolated from the total free ribosomal population were labeled differentially. When dissociation was carried out in the presence of EDTA, the small subunit contained four radioactive phosphoprotein bands, whereas the large subunit contained five. Three of the radioactive phosphoprotein components of the small subunit were removed when dissociation of cerebral ribosomes which were previously washed with high salt media was carried out in the presence of puromycin and high salt. However, only the largest labeled phosphoprotein band of the large subunit was removed by this procedure. This component exhibited the same electrophoretic mobility as one of the radioactive phosphoprotein bands which was removed from the small subunit by high salt treatment..     

Fat cell protein phosphorylation. Identification of phosphoprotein-2 as ATP-citrate lyase.

We have purified to apparent homogeneity a phosphoprotein from rat adipose tissue which is rapidly phosphorylated in vitro by ATP. The native phosphoprotein has an approximate sedimentation coefficient of 14.8 S. On sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis, the protein dissociated into identical subunits of Mr = 128,000. A phosphoprotein with similar properties was also isolated from liver. Purified phosphoproteins from fat cells and liver had ATP-citrate lyase activity and co-migrated on sodium dodecyl sulfate gels with fat cell phosphoprotein-2, the phosphorylation of which is increased by incubating fat cells with insulin. The phosphoamino acid residue of the cells with insulin. The phosphoamino acid residue of the phosphoprotein was identified as tau-phosphohistidine. These evidences suggest that fat cell phosphoprotein-2 is ATP-citrate lyase.     

Cross-linking study on protein neighborhoods at the subunit interface of rat liver ribosomes with 2-iminothiolane

Rat liver 80 S ribosomes were cross-linked with 2-iminothiolane. Proteins extracted from the cross-linked 80 S ribosomes were separated into 25 fractions by chromatography on carboxy methylcellulose. Each protein fraction was analyzed by diagonal polyacrylamide-sodium dodecyl sulfate gel electrophoresis. Eight pairs characteristic of 80 S ribosomes were detected which did not appear when isolated 40 S and 60 S subunits were cross-linked, and the cross-linked proteins were analyzed in similar manners. The cross-linked components were radioiodinated and then analyzed by two-dimensional gel electrophoresis, followed by autoradiography. Eight kinds of cross-links between 60 S subunit proteins and 40 S subunit proteins were identified as follows: SA30 (acidic protein with Mr 30,000)-LA33 (acidic protein with Mr 33,000), S2-LA33, S2-L11, S3a-L11, S4-L5, S25-L5, S4-L24 and S6-L24.     

Depolarization-Dependent Protein Phosphorylation in Rat Cortical Synaptosomes: Characterization of Active Protein Kinases by Phosphopeptide Analysis of Substrates

Depolarization of synaptosomes is known to cause a calcium-dependent increase in the phosphorylation of a number of proteins. It was the aim of this study to determine which protein kinases are activated on depolarization by analyzing the incorporation of 32Pi into synaptosomal phosphoproteins and phosphopeptides. The following well-characterized phosphoproteins were chosen for study: phosphoprotein "87K," synapsin Ia and Ib, phosphoproteins IIIa and IIIb, the catalytic subunits of calmodulin kinase II, and the B-50 protein. Each was initially identified as a phosphoprotein in lysed synaptosomes after incubation with [gamma-32P]ATP. Mobility on two-dimensional polyacrylamide gels and phosphorylation by specific protein kinases were the primary criteria used for identification. A technique was developed that allowed simultaneous analysis of the phosphopeptides derived from all of these proteins. Phosphopeptides were characterized in lysed synaptosomes after activating cyclic AMP-, calmodulin-, and phospholipid-stimulated protein kinases in the presence of [gamma-32P]ATP. Phosphoproteins labelled in intact synaptosomes after incubation with 32Pi were then compared with those seen after ATP-labelling of lysed synaptosomes. As expected from previous work, phosphoprotein "87K," and synapsin Ia and Ib were labelled, but for the first time, phosphoproteins IIIa, IIIb, and the B-50 protein were identified as being labelled in intact synaptosomes; the calmodulin kinase II subunits were hardly phosphorylated. From a comparison of the phosphopeptide profiles it was found that cyclic AMP-, calmodulin-, and phospholipid-stimulated protein kinases are all active in intact synaptosomes and their activity is dependent on extrasynaptosomal calcium. The activation of cyclic AMP-stimulated protein kinases in intact synaptosomes was confirmed by the addition of dibutyryl cyclic AMP and theophylline which specifically increased the labelling of phosphopeptides in synapsin Ia and Ib and in phosphoproteins IIIa and IIIb. On depolarization of intact synaptosomes, a number of phosphopeptides showed increased labelling and the pattern suggested that cyclic AMP-, calmodulin-, and phospholipid-stimulated protein kinases were all activated. No new peptides were phosphorylated, suggesting that depolarization simply increased the activity of already active protein kinases and that there was no depolarization-specific increase in protein phosphorylation.     

Influence of glucagon and cyclic adenosine 3':5'-monophosphate on the phosphorylation of rat liver ribosomal protein S6.

The administration of glucagon, adenosine 3':5'-monophosphate, or N6,O2'-dibutyryl adenosine 3':5'-monophosphate caused an increase in the phosphorylation of rat liver ribosomes. The increase (approximately 3-fold) was in the protein of the small ribosomal subunit. The proteins were separated by two-dimensional polyacrylamide gel electrophoresis and radioautographs were made of the gels. The effect of the hormone and of the nucleotides was entirely due to an increase in the phosphorylation of the 40 S ribosomal subunit protein S6.     

Large-scale identification of novel mitosis-specific phosphoproteins

Systematic identification of phosphoproteins is essential for understanding cellular signalling pathways since phosphorylation plays important roles in cellular regulation. Monoclonal antibody MPM-2 recognizes a discrete set of mitosis-specific phosphoproteins and constitutes a specific tool to investigate the significance of phosphorylation in cell cycle. However, due to the difficulties in identifying antigens revealed on immunoblot membrane, only minority of MPM-2 antigens have been identified. Here we originated proteomics approaches for large-scale identification of MPM-2 phosphoproteins. Mitotic extracts were run on several two-dimensional gel electrophoresis (2D) in parallel, and stained by Coomassie Blue. Each individual spot on one of the gels was excised, and proteins in it were further resolved by regular SDS-electrophoresis and blotted on membrane for MPM-2 stain. Counterparts of the positive proteins were selected on another parallel 2D gel and identified by mass-spectrometry. Using this strategy, 100 spots were excised from Coomassie-stained 2D gel and screened by 1D immunoblots for MPM-2 reactivity, and 22 proteins containing potential MPM-2 epitope were identified in addition to a known MPM-2 antigen, laminin-binding protein. These results were further validated by immunofluorescence, co-immunoprecipitation and in vitro phosphorylation assay. The identification of an unprecedented number of potential MPM-2 phosphoprotein antigens gives new insight into the range of proteins involved in the regulation of the early stages of cell division. Meanwhile, this strategy could be used wherever unknown antigens are explored, especially for antibodies that can recognize more than one antigen.     

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

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

Endogenous, cyclic 3'-5' AMP-dependent phosphorylation of human red cell pyruvate kinase.

Ribosomal proteins of $\text{Physarum}\text{polycephalum}$ were labelled $\text{in}\text{vivo}$ with ³²PO₄. Three acid phosphoproteins were observed in the large subunit, while two basic ones were present in the small subunit. Ribosomal phosphoprotein S3 accounted for 70% of the total radioactivity and may be equivalent to S6 from rat liver.     

Ribosomal proteins from rabbit reticulocytes: number and molecular weights of proteins from ribosomal subunits.

The ribosomal proteins from 40 S and 60 S subunits of rabbit reticulocytes were separated by two-dimensional polyacrylamide gel electrophoresis. The protein spots stained with Coomassie brilliant blue were cut out and the proteins were extracted. The material extracted from each spot was mixed with proteins of known molecular weight and then analyzed by electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate. Both the total number and the molecular weights of each of the proteins were determined by these procedures. Thirty-two proteins were identified in the 40 S subunits; their molecular weights ranged from 8000 to 39,000 (average mol. wt = 25,000). Thirty-nine proteins were identified in the 60 S subunit; their molecular weights ranged from 9000 to 58,000 (average mol. wt = 31,000). The sum of the molecular weights of the individual proteins from each subunit is in agreement with previous estimations, derived from physico-chemical measurements of the total protein in mammalian ribosomal subunits. The molecular weight distribution obtained for the isolated proteins was nearly identical to that derived from spectrophotometric analysis of polyacrylamide-sodium dodecyl sulfate gels of the total protein mixtures from each subunit stained with Coomassie brilliant blue. The results are consistent with the hypothesis that reticulocyte ribosomes contain one copy of most of their protein constituents.     

The increased phosphorylation of ribosomal protein S6 in Arbacia punctulata is not a universal event in the activation of sea urchin eggs

Eggs of the sea urchins Arbacia punctulata (Ap), Lytechinus pictus (Lp), and Strongylocentrotus purpuratus (Sp) were labeled to equilibrium with 32PO3-4. Approximately 65-70% of the label in extractable adenine nucleotides comigrates chromatographically with ATP. Autoradiograms of one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) slab gels show that each species possesses a distinct complement of phosphate-exchangeable phosphoproteins. No changes in the phosphoprotein composition are detected in Lp and Sp eggs as a result of fertilization or development for 2.5 hr (with the possible exception of a 43,000 Mr protein in Lp). In Ap, increases in the phosphorylation of bands at Mr's 30,000, 55,000, and 105,000 are seen during the first 10 min postinsemination. The 30,000 Mr band in Ap eggs has previously been identified as ribosomal protein S6 and the hypothesis presented that its increased phosphorylation may be an important step in the activation of protein synthesis at fertilization (D. G. Ballinger and T. Hunt, 1981, Dev. Biol. 87, 277-285). In Lp and Sp eggs S6 (identified by two-dimensional PAGE) is heavily phosphorylated in the unfertilized state and the extent of labeling does not increase after fertilization. If the increased phosphorylation of S6 seen in Ap is indeed related to translational activation, then these results suggest that different sea urchin species may rely on different mechanisms for the activation of protein synthesis.     

Characterization of the major phosphoprotein and its kinase on the surface of the rat adipocyte

Intact rat fat cells exposed to 12.5 microM [gamma-32P]ATP incorporate label into specific proteins within minutes. By solubilizing the reaction mixture with SDS which by passes the subcellular fractionation steps, the labeled proteins can be identified in autoradiographs of SDS-PAGE gels. The most prominently labeled protein has an Mr of 42,000. Localization of this component to the cell surface can be made on the basis of inhibition of phosphorylation by addition of a protein derived from the rat brain with protein kinase inhibitory property, susceptibility of the phosphorylated protein to tryptic digestion, whereas the unphosphorylated protein is unaffected by digestion with trypsin (15 min), inhibition of phosphorylation of this protein after brief exposure to melittin, and the consistent observation that more label is associated with the 42,000 Mr band in homogenates and permeabilized cells than in comparable numbers of intact cells exposed to the same amount of label. A 42,000 Mr phosphoprotein is also present in mitochondria which is most likely the alpha subunit of pyruvate dehydrogenase. To rule out the possibility that the cell surface protein might be a mitochondrial contaminant from broken cells, 32Pi-labeled and [gamma-32P]ATP-labeled cells were solubilized with Triton and chromatographed on a rabbit anti-pyruvate dehydrogenase antibody-Sepharose 4B column. A single labeled peak was detected upon elution of the bound fraction only in the 32Pi-labeled sample, and not in the [gamma-32P]ATP-labeled sample. Subcellular fractionation studies of intact cells labeled with [gamma-32P]ATP showed differences in the recovery of phosphoproteins of 42,000 Mr depending on whether a continuous sucrose gradient (27.6-54.1%, g/ml) or a discontinuous sucrose gradient (16, 35 and 48%, g/ml) was used. Phosphoproteins of 42,000 Mr were located in the mitochondrial and membrane fractions collected by discontinuous sucrose gradient separation, whereas a phosphoprotein of 42,000 Mr was found primarily in the mitochondrial fraction after continuous sucrose gradient separation. By 5'-nucleotidase activity measurements, the latter approach appears to result in the isolation of a heavy fragment of the plasma membrane with the mitochondrial light fraction which is 42,000 in Mr and labeled. Finally, comparison of the autoradiographs of two-dimensional (2D) gels (isoelectric focusing followed by 10% SDS-PAGE) show different isoelectric points for 42,000 Mr components in [gamma-32P]ATP- and 32Pi-labeled cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Phosphoprotein profiling by PA-GeLC-MS/MS

A significant consequence of protein phosphorylation is to alter protein-protein interactions, leading to dynamic regulation of the components of protein complexes that direct many core biological processes. Recent proteomic studies have populated databases with extensive compilations of cellular phosphoproteins and phosphorylation sites and a similarly deep coverage of the subunit compositions and interactions in multiprotein complexes. However, considerably less data are available on the dynamics of phosphorylation, composition of multiprotein complexes or that define their interdependence. We describe a method to identify candidate phosphoprotein complexes by combining phosphoprotein affinity chromatography, separation by size, denaturing gel electrophoresis, protein identification by tandem mass spectrometry, and informatics analysis. Toward developing phosphoproteome profiling, we have isolated native phosphoproteins using a phosphoprotein affinity matrix, Pro-Q Diamond resin (Molecular Probes-Invitrogen). This resin quantitatively retains phosphoproteins and associated proteins from cell extracts. Pro-Q Diamond purification of a yeast whole cell extract followed by 1-D PAGE separation, proteolysis and ESI LC-MS/MS, a method we term PA-GeLC-MS/MS, yielded 108 proteins, a majority of which were known phosphoproteins. To identify proteins that were purified as parts of phosphoprotein complexes, the Pro-Q eluate was separated into two fractions by size, <100 kDa and >100 kDa, before analysis by PAGE and ESI LC-MS/MS and the component proteins queried against databases to identify protein-protein interactions. The <100 kDa fraction was enriched in phosphoproteins indicating the presence of monomeric phosphoproteins. The >100 kDa fraction contained 171 proteins of 20-80 kDa, nearly all of which participate in known protein-protein interactions. Of these 171, few are known phosphoproteins, consistent with their purification by participation in protein complexes. By comparing the results of our phosphoprotein profiling with the informational databases on phosphoproteomics, protein-protein interactions and protein complexes, we have developed an approach to examining the correlation between protein interactions and protein phosphorylation.     

Dephosphorylation of S6 and expression of the heat shock response in Drosophila melanogaster.

A basic ribosomal phosphoprotein of 30,000 molecular weight was rapidly dephosphorylated in cultured Drosophila melanogaster cells heat shocked at 37 degrees C. The protein was associated with the 40S ribosomal subunit and had an electrophoretic mobility similar to that of purified rat liver protein S6 on basic two-dimensional polyacrylamide gels as well as a similar partial proteolysis peptide map. In logarithmically growing cultures, this D. melanogaster S6 protein appeared to have a single phosphorylated species consisting of 30 to 40% of the total cellular S6. Thus, the nearly complete dephosphorylation of this protein observed in heat shock involves a large fraction of the cellular S6. The significance of this dephosphorylation in the expression of the heat shock response was investigated by examining the phosphorylation status of S6 in recovery from heat shock and in response to chemical inducers of the heat shock response. During recovery from a 30-min heat shock, the recovery of normal protein synthesis was almost complete in 2 to 4 hr, whereas there was no significant rephosphorylation of S6 for 8 h. Two chemical inducers of the heat shock response, canavanine and sodium arsenite, induced the synthesis of heat shock proteins in D. melanogaster cells. Sodium arsenite also caused an inhibition of normal protein synthesis similar to that observed in heat shock. Neither agent, however, caused significant dephosphorylation of S6. These results suggest that the dephosphorylation of S6, although invariably observed in heat-shocked cells, may in some cases be dissociated from both the induction of heat shock protein synthesis and the turnoff of normal protein synthesis which occur in a heat shock response.     

Purification of phosphoproteins by immobilized metal affinity chromatography and its application to phosphoproteome analysis

Prefractionation procedures facilitate the identification of lower-abundance proteins in proteome analysis. Here we have optimized the conditions for immobilized metal affinity chromatography (IMAC) to enrich for phosphoproteins. The metal ions, Ga(III), Fe(III), Zn(II), and Al(III), were compared for their abilities to trap phosphoproteins; Ga(III) was the best. Detailed analyses of the pH and ionic strength for IMAC enabled us to determine the optimal conditions (pH 5.5 and 0.5 m NaCl). When whole cell lysates were fractionated in this way, about one-tenth of the total protein was recovered in the eluate, and the recovery of phosphorylated extracellular signal-regulated kinase (ERK) was more than 90%. Phosphorylated forms of ribosomal S6 kinase (RSK) and Akt were also enriched efficiently under the same conditions. Our Ga(III) IMAC and a commercially available purification kit for phosphoproteins performed similarly, with a slight difference in the spectrum of phosphoproteins. When phosphoproteins enriched from NIH3T3 cells in which ERK was either activated or suppressed were analyzed by two-dimensional fluorescence difference gel electrophoresis, phosphorylated ERK was detected as discrete spots unique to ERK-activated cells, which overlapped with surrounding spots in the absence of prefractionation. We applied the same technique to search for Akt substrates and identified Abelson interactor 1 as a novel potential target. These results demonstrate the efficacy of phosphoprotein enrichment by IMAC and suggest that this procedure will be of general use in phosphoproteome research.     

Electrophoretic Separation of Different Phophosproteins Associated with Ca-ATPase and Na,K-ATPase in Human Red Cell Ghosts

Ca has been found to increase the quantity of ³²P incorporated into red cell ghosts from [γ-³²P]ATP over the levels obtained by incubation with Mg alone or with Mg + Na, in correlation with the effect of Ca on the associated ATPase activities. When the ³²P-labeled ghosts were solubilized in sodium dodecyl sulfate (SDS) and electrophoresed on acrylamide gels only two bands could be detected either by autoradiography or by counting the sliced gels. The faster moving band (P-2) had the same mobility and the same molecular weight (103,000) as the phosphoprotein found either with Mg alone or with Mg + Na. The slower moving band (P-1) was not found in extensively washed ghosts labeled in the absence of Ca. The molecular weight of P-1 is approximately 150,000. P-1 like P-2 was not affected by pretreatment of intact cells with Pronase before labeling indicating that neither the phosphorylating mechanism nor the phosphoprotein are accessible to externally applied Pronase. The demonstration that a Ca-phosphoprotein is separable from the Na-stimulated phosphoprotein suggests that the Ca-ATPase is distinct from and independent of the Na,K-ATPase. The fact that Ca blocks the dephosphorylation by K of the Na-phosphoprotein indicates that caution is required in interpreting results when the activities of the different phosphoproteins have not been separately determined.     

Identification by RNA-protein cross-linking of ribosomal proteins located at the interface between the small and the large subunits of mammalian ribosomes

Protein constituents at the subunit interface of rat liver ribosomes were analysed by cross-linking with the bifunctional reagent, diepoxybutane (distance between reactive groups 4 A). Isolated 40S and 60S subunits were labelled with 125I and recombined with unlabelled complementary subunits. The two kinds of selectively labelled 80S ribosomes were treated with diepoxybutane at low concentration. Radioactive ribosomal proteins covalently attached to the rRNA of the unlabelled complementary subparticles were isolated by repeated gradient centrifugation. The RNA-bound, labelled proteins were identified by two-dimensional gel electrophoresis. The experiments showed that proteins S2, S3, S4, S6, S7, S13, and S14 in the small subunit of rat liver ribosomes are located at the ribosomal interface in close proximity to 28S rRNA. Similarly, proteins L3, L6, L7, and L8 were found at the the interface of the large ribosomal subunit in the close vicinity of 18S rRNA.     

Interactions between cyclic AMP- and phorbol ester-dependent phosphorylation systems in S49 mouse lymphoma cells

High-resolution two-dimensional gel electrophoresis of proteins labeled with either 32Pi or [35S]methionine was used to study interactions between cyclic AMP and tetradecanoyl phorbol acetate (TPA) at the level of intracellular protein phosphorylation. Cultured S49 mouse lymphoma cells were used as a model system, and mutant sublines lacking either the catalytic subunit of cyclic AMP-dependent protein kinase or the guanyl nucleotide-binding "Ns" factor of adenylate cyclase provided tools to probe mechanisms underlying the interactions observed. Three sets of phosphoproteins responded differently to TPA treatment of wild-type and mutant cells: Phosphorylations shown previously to be responsive to activation of intracellular cyclic AMP-dependent protein kinase were stimulated by TPA in wild-type cells but not in mutant cells, a subset of phosphorylations stimulated strongly by TPA in mutant cells was inhibited in wild-type cells, and two novel phosphoprotein species appeared in response to TPA only in wild-type cells. The latter two classes of TPA-mediated responses specific to wild-type cells could be evoked in adenylate cyclase-deficient cells by treating concomitantly with TPA and either forskolin or an analog of cyclic AMP. Three conclusions are drawn from our results: 1) TPA stimulates adenylate cyclase in wild-type cells causing increased phosphorylation of endogenous substrates by cyclic AMP-dependent protein kinase, 2) activated cyclic AMP-dependent protein kinase inhibits phosphorylation (or enhances dephosphorylation) of a specific subset of TPA-dependent phosphoproteins, and 3) cyclic AMP-dependent events facilitate TPA-dependent phosphorylation of some substrate proteins.