Protein kinase C phosphorylates caldesmon77 and vimentin and enhances albumin permeability across cultured bovine pulmonary artery endothelial cell monolayers.

Cytoskeletal protein (CSP) interactions are critical to the contractile response in muscle and non-muscle cells. Current concepts suggest that activation of the contractile apparatus occurs through selective phosphorylation by specific cellular kinase systems. Because the Ca(2+)-phospholipid-dependent protein kinase C (PKC) is involved in the regulation of a number of key endothelial cell responses, the hypothesis that PKC modulates endothelial cell contraction and monolayer permeability was tested. Phorbol myristate acetate (PMA), a direct PKC activator, and alpha-thrombin, a receptor-mediated agonist known to increase endothelial cell permeability, both induced rapid, dose-dependent activation and translocation of PKC in bovine pulmonary artery endothelial cells (BPAEC), as assessed by gamma-[32P]ATP phosphorylation of H1 histone in cellular fractions. This activation was temporally associated with evidence of agonist-mediated endothelial cell contraction as demonstrated by characteristic changes in cellular morphology. Agonist-induced activation of the contractile apparatus was associated with increases in BPAEC monolayer permeability to albumin (approximately 200% increase with 10(-6) MPMA, approximately 400% increase with 10(-8) M alpha-thrombin). To more closely examine the role of PKC in activation of the contractile apparatus, PKC-mediated phosphorylation of two specific CSPs, the actin- and calmodulin-binding protein, caldesmon77, and the intermediate filament protein, vimentin, was assessed. In vitro phosphorylation of both caldesmon and vimentin was demonstrated by addition of exogenous, purified BPAEC PKC to unstimulated BPAEC homogenates, to purified bovine platelet caldesmon77, or to purified smooth muscle caldesmon150. Caldesmon77 and vimentin phosphorylation were observed in intact [32P]-labeled BPAEC monolayers stimulated with either PMA or alpha-thrombin, as detected by immunoprecipitation. In addition, BPAEC pretreatment with the PKC inhibitor, staurosporine, prevented alpha-thrombin- and PMA-induced phosphorylation of both cytoskeletal proteins, attenuated morphologic evidence of contraction, and abolished agonist-induced barrier dysfunction. These results demonstrate that agonist-stimulated PKC activity results in cytoskeletal protein phosphorylation in BPAEC monolayer, an event which occurs in concert with agonist-mediated endothelial cell contraction and resultant barrier dysfunction.     

Phosphorylation and dephosphorylation of human platelet surface proteins by an ecto-protein kinase/phosphatase system.

We have characterized a novel ecto-protein kinase activity and a novel ecto-protein phosphatase activity on the membrane surface of human platelets. Washed intact platelets, when incubated with [gamma-32P]ATP in Tyrode's buffer, showed the phosphorylation of a membrane surface protein migrating with an apparent molecular mass of 42 kDa on 5-15% SDS polyacrylamide gradient gels. The 42 kDa protein could be further resolved on 15% SDS gels into two proteins of 39 kDa and 42 kDa. In this gel system, it was found that the 39 kDa protein became rapidly phosphorylated and dephosphorylated, whereas the 42 kDa protein was phosphorylated and dephosphorylated at a much slower rate. NaF inhibited the dephosphorylation of these proteins indicating the involvement of an ecto-protein phosphatase. The platelet membrane ecto-protein kinase responsible for the phosphorylation of both of these proteins was identified as a serine kinase and showed dependency on divalent cations Mg2+ or Mn2+ ions. Ca2+ ions potentiated the Mg(2+)-dependent ecto-protein kinase activity. The ecto-protein kinase rapidly phosphorylated histone and casein added exogenously to the extracellular medium of intact platelets. Following activation of platelets by alpha-thrombin, the incorporation of [32P]phosphate from exogenously added [gamma-32P]ATP by endogenous protein substrates was reduced by 90%, suggesting a role of the ecto-protein kinase system in the regulation of platelet function. The results presented here demonstrate that both protein kinase and protein phosphatase activities reside on the membrane surface of human platelets. These activities are capable of rapidly phosphorylating and dephosphorylating specific surface platelet membrane proteins which may play important roles in early events of platelet activation and secretion.     

Modulation of proliferation in epidermal keratinocyte cultures by lowered oxygen tension

The modulation of proliferation and differentiation in primary epidermal keratinocyte cultures by lowered gas phase oxygen tensions was studied. Neonatal mouse epidermal keratinocyte cultures were grown in an Heraeus type B 5060 EK/O2 incubator in oxygen tensions between 5% and 15% (within the physiologic range); the oxygen tension of ambient air being 21%. Cell morphology was studied using histochemical stains and electron microscopy. Differentiation was assessed using autoradiography of SDS PAGE gels of six serially extracted cell protein fractions with [3H]leucine as a marker. Autoradiographs using [14C]glucosamine and 32Pi as markers were also assessed as a measure of other cell functions. Proliferation was studied using autoradiography of [3H]thymidine ([3H]TdR) pulse-labeled cultures and [3H]TdR incorporation into isolated DNA fractions. The results of these studies showed that lowering the oxygen tension in the gas phase reversibly inhibited cell proliferation. There was a direct arithmetic relationship between the proliferative rate of the cultures and the oxygen tension. No change in differentiation as defined by [3H]leucine indexing of protein synthesis was seen. Other markers of cell function, such as [14C]glucosamine glycosylation and [32P] phosphorylation of proteins were also unchanged. These results suggest that oxygen tension regulates only proliferation in epidermal keratinocytes. This epidermal response is well adapted to its role in the healing wound, and is an example of a tissue-specific modification of a regulatory function.     

Global quantitative phosphoprotein analysis using Multiplexed Proteomics technology

Systematic parallel analysis of the phosphorylation status of networks of interacting proteins involved in the regulatory circuitry of cells and tissues is certain to drive research in the post-genomics era for many years to come. Reversible protein phosphorylation plays a critical regulatory role in a multitude of cellular processes, including alterations in signal transduction pathways related to oncogene and tumor suppressor gene products in cancer. While fluorescence detection methods are likely to offer the best solution to global protein quantitation in proteomics, to date, there has been no satisfactory method for the specific and reversible fluorescent detection of gel-separated phosphoproteins from complex samples. The newly developed Pro-Q Diamond phosphoprotein dye technology is suitable for the fluorescent detection of phosphoserine-, phosphothreonine-, and phosphotyrosine-containing proteins directly in sodium dodecyl sulfate (SDS)-polyacrylamide gels and two-dimensional (2-D) gels. Additionally, the technology is appropriate for the determination of protein kinase and phosphatase substrate preference. Other macromolecules, such as DNA, RNA, and sulfated glycans, fail to be detected with Pro-Q Diamond dye. The staining procedure is rapid, simple to perform, readily reversible and fully compatible with modern microchemical analysis procedures, such as matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. Pro-Q Diamond dye technology can detect as little as 1-2 ng of beta-casein, a pentaphosphorylated protein, and 8 ng of pepsin, a monophosphorylated protein. Fluorescence signal intensity correlates with the number of phosphorylated residues on the protein. Through combination of Pro-Q Diamond phosphoprotein stain with SYPRO(R) Ruby protein gel stain, Multiplexed Proteomics technology permits quantitative, dichromatic fluorescence detection of proteins in 2-D gels. This evolving discovery platform allows the parallel determination of protein expression level changes and altered post-translational modification patterns within a single 2-D gel experiment. The linear responses of the fluorescence dyes utilized, allow rigorous quantitation of changes over an unprecedented 500-1000-fold concentration range.     

A Method for the Electrophoretic Analysis of Proteins and RNAs of Single Cells*

SYNOPSIS A method is described for the electrophoretic analysis of proteins or RNAs from individual amebae. The method is based on fluorographic autoradiography of semi-micro polyacrylamide gels in which [³⁵S]methionine or [³H]uridine materials from single cells have been subjected to electrophoresis. The method is more sensitive and provides better resolution than previous methods for single cells. It is suitable, also, for quantitation of the separated components.     

Effect of growth hormone on protein phosphorylation in isolated rat hepatocytes

Hepatocytes from male rats were incubated with [32P]Pi for 40 min at 37 degrees C, thereby equilibrating the cellular ATP pool with 32P. Subsequent exposure to bovine growth hormone for 10 additional min did not change the specific activity of cellular [gamma-32P]ATP. Two-dimensional gel electrophoresis or chromatofocusing followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to fractionate phosphoproteins solubilized from control or hormone-stimulated cells. Stimulation of hepatocytes with 5 nM growth hormone for 10 min at 37 degrees C affected the phosphorylation of a number of proteins including an Mr 46,000 species of pI 4.7 whose phosphorylation was augmented (2.65 +/- 0.50)-fold. A significant fraction of the maximal effect of growth hormone on phosphorylation of the Mr 46,000 species was elicited by 1-5% receptor occupancy. Bovine growth hormone, which binds to somatogenic receptors with great specificity, or recombinant human growth hormone, which is not contaminated with other hormones, affected phosphorylation of hepatic proteins similarly. The Mr 46,000 phosphoprotein was isolated in a fraction enriched in cytosol after centrifugation of cellular homogenates. Phosphorylation of the Mr 46,000 phosphoprotein was also increased (1.75 +/- 0.35)-fold and (2.15 +/- 0.50)-fold by insulin and glucagon, respectively. These observations are consistent with the possibility that selective changes in the phosphorylation state of cellular proteins may mediate growth hormone actions in cells.     

Quantitative phosphoproteomic analysis reveals a role for serine and threonine kinases in the cytoskeletal reorganization in early T cell receptor activation in human primary T cells

Protein phosphorylation-dephosphorylation events play a primary role in regulation of almost all aspects of cell function including signal transduction, cell cycle, or apoptosis. Thus far, T cell phosphoproteomics have focused on analysis of phosphotyrosine residues, and little is known about the role of serine/threonine phosphorylation in early activation of the T cell receptor (TCR). Therefore, we performed a quantitative mass spectrometry-based analysis of the global phosphoproteome of human primary T cells in response to 5 min of TCR activation with anti-CD3 antibody. Combining immunoprecipitation with an antiphosphotyrosine antibody, titanium dioxide phosphopeptide enrichment, isobaric tag for the relative and absolute quantitation methodology, and strong cation exchange separation, we were able to identify 2814 phosphopeptides. These unique sites were employed to investigate the site-specific phosphorylation dynamics. Five hundred and seventeen phosphorylation sites showed TCR-responsive changes. We found that upon 5 min of stimulation of the TCR, specific serine and threonine kinase motifs are overrepresented in the set of responsive phosphorylation sites. These phosphorylation events targeted proteins with many different activities and are present in different subcellular locations. Many of these proteins are involved in intracellular signaling cascades related mainly to cytoskeletal reorganization and regulation of small GTPase-mediated signal transduction, probably involved in the formation of the immune synapse.     

Sequential phosphorylation of chartin microtubule-associated proteins is regulated by the presence of microtubules

Chartins are a unique class of three families of microtubule-associated proteins, each consisting of several isoforms possessing varying degrees of phosphorylation. The most highly phosphorylated chartin isoforms are highly enriched in neuronal cell fractions containing microtubules and there is evidence that their phosphorylation may play a role in promoting neurite outgrowth. The present work describes the relationship between the phosphorylation state of chartins and the presence of intact microtubules in long-term cultures of NGF-treated, neurite-bearing PC12 cells. Cultures were depleted of microtubules by exposure to high concentrations of depolymerizing agents for 2-24 h. Radiolabeling of cellular proteins with [32P]orthophosphate or [35S]methionine revealed that both the ongoing and steady-state phosphorylation of chartins is markedly altered under these conditions. Two-dimensional isoelectric focusing by SDS-PAGE of whole cell extracts demonstrated that the more acidic, highly phosphorylated isoforms are diminished with a concomitant increase in the more basic, less phosphorylated isoforms. These phosphorylation changes were relatively specific for the chartins and were not observed for phosphorylated MAP 1.2, phospho-beta-tubulin, or most other phosphoproteins. Thus, the phosphorylation state of chartins, but not of other phosphoproteins, is regulated by the presence of native microtubules. Despite depolymerization of microtubules, neurites remained extended for at least 24 h. Neurite elongation, however, was arrested. Microtubules, therefore, may be required for extension, but not for short-term maintenance of well-established neurites. Taxol, which promotes tubule assembly and stability, does not, conversely, drive phosphorylation of the chartins. Instead, taxol appeared to decrease the turnover of phosphate in microtubule-associated, acidic chartin isoforms. These data suggest several models as to how chartin phosphorylation is regulated in neurite-bearing cells and indicate that phosphorylation of cytoplasmic and microtubule-associated chartins occurs via different mechanisms.     

Modification of fos proteins: phosphorylation of c-fos, but not v-fos, is stimulated by 12-tetradecanoyl-phorbol-13-acetate and serum.

We have investigated the covalent modification of the proteins encoded by the murine fos proto-oncogene (c-fos) and that of the corresponding gene product of FBJ murine osteosarcoma virus (v-fos). Both proteins are posttranslationally processed in the cell, resulting in forms with lower electrophoretic mobilities than that of the initial translation product on sodium dodecyl sulfate-polyacrylamide gels. Treatment with alkaline phosphatase indicates that most, if not all, of this electrophoretic shift is due to phosphoesterification of both proteins. These phosphoryl groups stoichiometrically modify the v-fos and c-fos proteins on serine residues and turn over rapidly in vivo in the presence of protein kinase inhibitors (half-life, less than 15 min). Direct quantitative comparison of steady-state labeling studies with L-[35S]methionine and [32P]phosphate reveals that the c-fos protein is four- to fivefold more highly phosphorylated than the v-fos protein is. Comparison of tryptic fragments from [32P]phosphate-labeled proteins indicates that although the two proteins have several tryptic phosphopeptides in common, the c-fos protein contains unique major tryptic phosphopeptides that the v-fos protein lacks. These unique sites of c-fos phosphorylation have been tentatively localized to the carboxy-terminal 20 amino acid residues of the protein. Phosphorylation of the c-fos protein, but not the v-fos protein, can be stimulated at least fivefold in vivo by the addition of either 12-tetradecanoyl-phorbol-13-acetate or serum. This increase in the steady-state degree of phosphorylation of c-fos appears to be independent of protein kinase C since phosphorylation is Ca2+ and diacylglycerol independent. The possible role of phosphorylation of these proteins in cellular transformation is discussed.     

Changes in content and cAMP-dependent phosphorylation of specific proteins in granulosa cells of preantral and preovulatory ovarian follicles and in corpora lutea

The responsiveness of granulosa cells to the gonadotropins and cAMP increases as ovarian follicles mature. To determine if this change in response might be related to either the content or cAMP-dependent phosphorylation of specific proteins, we labeled proteins in 30,000 X g supernatant fractions (cytosol) with [gamma-32P] ATP in the presence or absence of cAMP. Using two-dimensional gel electrophoresis, we observed that granulosa cells of preantral follicles exhibited low amounts of cAMP-dependent phosphorylation of two proteins with apparent molecular weights of 54,000-56,000 and 43,000. Using [32P]8-N3cAMP and photoaffinity labeling procedures, the Mr = 54,000-56,000 protein was identified as RII, the regulatory subunit of type II protein kinase. Polychromatic silver staining, as well as the photoaffinity labeling, revealed that RII exists in three forms, each of which was also labeled by [gamma-32P] ATP. Based on the relative isoelectric points and specific silver staining of highly purified actin and phosphorylated actin, the Mr = 43,000 protein has been provisionally identified as actin. Five proteins (Mr = 37,500, 27,500, 22,500, 19,000, and 15,000), in addition to RII and actin, were phosphorylated in cytosol of granulosa cells from preovulatory follicles. By adding increasing concentrations of exogenous catalytic subunit to the cytosols, we demonstrated that the content, as well as the phosphorylation of these proteins, was increased selectively in granulosa cells of antral follicles. By using hypophysectomized rats, we demonstrated that these five proteins are induced by follitropin (FSH). Because they were not present in cytosols of thecal cells or corpora lutea, they appear to be specific markers for granulosa cells. The content and phosphorylation of RII was also dramatically increased in cytosols of granulosa cells from antral follicles, whereas that of actin remained unchanged. These observations indicate that granulosa cell differentiation involves regulation by FSH of specific proteins which are substrates for cAMP-dependent protein kinase. Thus, FSH and cAMP appear to regulate the intracellular content and phosphorylation of a cAMP response system in granulosa cells. The extent to which RII and the five specific phosphoproteins themselves regulate granulosa cell responsiveness remains to be determined.     

Bovine skeletal growth factor stimulates protein phosphorylation of chicken bone cells in vitro

1. Bovine skeletal growth factor (SGF), a potent bone cell mitogen, stimulated protein phosphorylation in cultured chicken calvarial cells. 2. SDS-PAGE followed by autoradiographic analysis of the cellular proteins indicated that [32P] incorporation was enhanced in several proteins in response to 10 ng/ml of SGF (the maximum stimulatory mitogenic dose for these cells). 3. Under conditions favoring tyrosine kinases, SGF stimulated phosphorylation of at least 6 proteins in crude calvarial cell membrane fraction, and caused a time-dependent stimulation of phosphorylation of angiotensin II by crude calvarial cell membrane fractions. 4. Thus, our data demonstrate that SGF stimulates protein phosphorylation in bone cells, and suggest that at least some of the protein phosphorylation involves tyrosine residues.     

腺苷对大鼠心肌钙蛋白Ⅰ及磷脂酰基醇磷酸化的抑制作用

 用差速离心及等密度梯度离心法从大白鼠心肌细胞分离收缩蛋白质及质膜,分别与[γ-~(32)P]ATP保温以观察细胞成分的磷酸化,以及腺苷和腺苷类似物对磷酸化的影响。结果表明,在收缩蛋白质组分,~(32)P主要参入肌钙蛋白I(Troponin I,29000Da);在质膜组分,~(32)P主要参入磷脂酰肌醇-4-一磷酸(PtdIns4P),亦即ATP使磷脂酰肌醇(Ptd Ins)磷酸化。腺苷对此两种磷酸化都有抑制作用,尤以对PtdIns磷酸化的抑制最强烈。cAMP对肌钙蛋白Ⅰ的磷酸化有刺激作用,这与文献报道相符。作者认为,腺苷和cAMP对肌钙蛋白Ⅰ磷酸化的拮抗作用与腺苷和肾上腺素对心肌调节的拮抗作用有明显的相关性。鉴于近年发现,肌醇磷脂转换在调节细胞活动中起重要作用,腺苷对磷脂酰肌醇磷酸化的抑制作用可能有重要的生物学意义。     

Cellular localization of steroid hormone-regulated proteins during sexual development in Achlya

In the fungus Achlya ambisexualis sexual development in the male strain E87 is controlled by the steroid hormone antheridiol. To investigate the effects of antheridiol on the synthesis and/or accumulation of specific cellular proteins we have analysed [35S]methionine-labeled proteins from control and hormone-treated cells using both one-dimensional (1D) and two-dimensional (2D) PAGE. Since in a total cell extract, hormone-induced changes in specific proteins might not be apparent against a background of more abundant proteins, cells were fractionated prior to protein isolation. It was also necessary to establish a concentration of hormone carrier, in this case methanol, which by itself did not alter the pattern of protein synthesis. Using these approaches the addition of the hormone antheridiol to vegetatively growing cells of Achlya E87 was found to result in changes in the synthesis and/or accumulation of at least 16 specific proteins, which could be localized to the cytoplasmic, nuclear or cell wall/cell membrane fractions. The most prominent changes observed in the hormone-treated cells included the appearance in the cytoplasmic fraction of labeled proteins at 28.4 and 24.3 kD which were not detectable in control cells, and a significant enrichment in the labeling of a 24.3 kD protein in the cell wall/cell membrane fraction. A marked increase in the labeling of 85, 63 and 47 kD proteins in the nuclear fraction from hormone-treated cells was also noted. The molecular weight (MW) and the behavior on 2D gels of the 85 kD hormone-induced protein appeared very similar to that of the 85 kD heat-shock protein reported in Achlya. Quantitive changes in the [35S]methionine labeling of several other proteins were noted in all three cell fractions.     

Histidine phosphorylation of annexin I in airway epithelia

Although [Cl(-)](i) regulates many cellular functions including cell secretion, the mechanisms governing these actions are not known. We have previously shown that the apical membrane of airway epithelium contains a 37-kDa phosphoprotein (p37) whose phosphorylation is regulated by chloride concentration. Using metal affinity (chelating Fe(3+)-Sepharose) and anion exchange (POROS HQ 20) chromatography, we have purified p37 from ovine tracheal epithelia to electrophoretic homogeneity. Sequence analysis and immunoprecipitation using monoclonal and specific polyclonal antibodies identified p37 as annexin I, a member of a family of Ca(2+)-dependent phospholipid-binding proteins. Phosphate on [(32)P]annexin I, phosphorylated using both [gamma-(32)P]ATP and [gamma-(32)P]GTP, was labile under acidic but not alkaline conditions. Phosphoamino acid analysis showed the presence of phosphohistidine. The site of phosphorylation was localized to a carboxyl-terminal fragment of annexin I. Our data suggest that cAMP and AMP (but not cGMP) may regulate annexin I histidine phosphorylation. We propose a role for annexin I in an intracellular signaling system involving histidine phosphorylation.     

Thiophosphorylation and phosphorylation of saponin-permeabilized cultured chromaffin cells

There is increasing evidence that phosphorylation of cellular proteins plays a role in the control of events surrounding secretion in neurons and chromaffin cells. In previous studies, we have used thiophosphorylation of cell proteins as a means of fixing cellular phosphorylation reactions in the phosphorylated state. Thiophosphorylation of permeabilized chromaffin cells with adenosine-5′-O-(3-thiotriphosphate) results in irreversible inhibition of secretion. Thiophosphate is incorporated primarily by two cellular proteins of 58 and 47 kDa. Calcium enhanced thiophosphorylation of the 47 kDa protein but not the 54 kDa protein. This pattern of thiophosphorylation differed markedly from that for phosphorylation under similar treatment conditions. The phosphoprotein composition of the cells depended upon the medium calcium and ATP concentration. In the absence of exogenous ATP, fewer phosphoproteins were seen in calcium stimulated cells than in unstimulated cells. Proteins labelled with ³²P or ³⁵S migrated to the same position on polyacrylamide gels containing sodium dodecyl sulfate. In the presence of exogenous ATP, ³²P incorporation was similar for both control and calcium-stimulated cells and was found primarily in a 64 kDa protein. Incorporation of [³²P]phosphate by calcium-stimulated cells was reduced to the same extent by pretreatment of the cells with either adenosine-5′-O-(3-thiotriphosphate) or ATP.The different electrophoretic banding patterns for thiophosphorylation and phosphorylation are likely due to the irreversibility of the thiophosphorylation reaction and reversibility of the phosphorylation reaction. The inability to turn over thiophosphate groups, in association with changes in secretion, may permit identification of those phosphoproteins that are putatively involved in secretion.     

Quantification of gel-separated proteins and their phosphorylation sites by LC-MS using unlabeled internal standards: analysis of phosphoprotein dynamics in a B cell lymphoma cell line

Protein phosphorylation plays a critical role in normal cellular function and is often subverted in disease. Although major advances have recently been made in identification and quantitation of protein phosphorylation sites by MS, current methodological limitations still preclude routine, easily usable, and comprehensive quantitative analysis of protein phosphorylation. Here we report a simple LC-MS method to quantify gel-separated proteins and their sites of phosphorylation; in this approach, integrated chromatographic peak areas of peptide analytes from proteins under study are normalized to those of a non-isotopically labeled internal standard protein spiked into the excised gel samples just prior to in-gel digestion. The internal standard intensities correct for differences in enzymatic activities and sample losses that may occur during the processes of in-gel digestion and peptide extraction from the gel pieces. We used this method of peak area measurement with an internal standard to investigate the effects of pervanadate on protein phosphorylation in the WEHI-231 B cell lymphoma cell line and to assess the role of phosphoinositide 3-kinase (PI3K) in these phosphorylation events. Phosphoproteins, isolated from total cell lysates using IMAC or by immunoprecipitation using Tyr(P) antibodies, were analyzed using this method, leading to identification of >400 proteins, several of which were found at higher levels in phosphoprotein fractions after pervanadate treatment. Pretreatment of cells with the PI3K inhibitor wortmannin reduced the phosphorylation level of certain proteins (e.g. STAT1 and phospholipase Cgamma2) while increasing the phosphorylation of several others. Peak area measurement with an internal standard was also used to follow the dynamics of PI3K-dependent and -independent changes in the post-translational modification of both known and novel phospholipase Cgamma2 phosphorylation sites. Our results illustrate the capacity of this conceptually simple LC-MS method for quantification of gel-separated proteins and their phosphorylation sites and for quantitative profiling of biological systems.     

Phosphorylation of p90 and p52 in response to phorbol-esters in Swiss/3T3 cells overexpressing protein kinase C-alpha.

Cell lines stably overexpressing protein kinase C (PKC)-alpha were previously described by us. These cell lines were generated by the introduction of the full length cDNA coding for PKC-alpha into Swiss/3T3 cells. Here we show that activation of PKC-alpha by phorbol-esters induced in these cells specific phosphorylation of two cellular proteins p90 and p52. Phosphorylation of p80 (MARCKS protein), previously identified as a substrate for PKC, was also enhanced. Phosphorylated p90 and p52 proteins were associated with particulate membrane-enriched fractions and were extractable with the use of nonionic detergents. Time course analysis of phorbol-ester induced phosphorylation of p90 and p52 revealed maximal stimulation of phosphorylation after 15-30 min. Phosphamino acid analysis showed that phosphorylation of p90 and p52 occurred mainly on serine residues. Phosphorylation of p52 was also on threonine residues. Whereas, phorbol ester activation induced phosphorylation of both p90 and p52, the mitogens platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) enhanced phosphorylation of p90, but not p52. Thus, our studies showed the involvement of PKC-alpha in the regulation of p90 and p52 phosphorylation and provided direct evidence for the role of PKC-alpha in cellular signaling by PDGF and FGF. Moreover, the fact that phosphorylation of p52 was specific to phorbol ester activation may suggest its involvement in tumor promotion. Characterization of p90 and p52 will enable us to reveal the phosphorylation cascade activated downstream to PKC-alpha and to determine their role in mitogenic signaling and tumor promotion.     

Fluorescence lifetime imaging of receptor tyrosine kinase activity in cells.

We report a highly specific fluorescence lifetime imaging microscopy (FLIM) method for monitoring epidermal growth factor receptor (EGFR) phosphorylation in cells based on fluorescence resonance energy transfer (FRET). EGFR phosphorylation was monitored using a green fluorescent protein (GFP)-tagged EGFR and Cy3-conjugated anti-phosphotyrosine antibodies. In this FRET-based imaging method, the information about phosphorylation is contained only in the (donor) GFP fluorescence lifetime and is independent of the antibody-derived (acceptor) fluorescence signal. A pixel-by-pixel reference lifetime of the donor GFP in the absence of FRET was acquired from the same cell after photobleaching of the acceptor. We show that this calibration, by acceptor photobleaching, works for the GFP-Cy3 donor-acceptor pair and allows the full quantitation of FRET efficiencies, and therefore the degree of exposed phosphotyrosines, at each pixel. The hallmark of EGFR stimulation is receptor dimerisation [1] [2] [3] [4] and concomitant activation of its intracellular tyrosine kinase domain [5] [6] [7]. Trans-autophosphorylation of the receptor [8] [9] on specific tyrosine residues couples the activated dimer to the intracellular signal transduction machinery as these phosphorylated residues serve as docking sites for adaptor and effector molecules containing Src homology 2 (SH2; reviewed in [10]) and phosphotyrosine-binding (PTB) [11] domains. The time-course and extent of EGFR phosphorylation are therefore important determinants of the underlying pathway and resulting cellular response. Our results strongly suggest that secondary proteins are recruited by activated receptors in endosomes, indicating that these are active compartments in signal transduction.     

Intrinsic protein kinase activity in mitochondrial oxidative phosphorylation complexes

Mitochondrial protein phosphorylation is a well-recognized metabolic control mechanism, with the classical example of pyruvate dehydrogenase (PDH) regulation by specific kinases and phosphatases of bacterial origin. However, despite the growing number of reported mitochondrial phosphoproteins, the identity of the protein kinases mediating these phosphorylation events remains largely unknown. The detection of mitochondrial protein kinases is complicated by the low concentration of kinase relative to that of the target protein, the lack of specific antibodies, and contamination from associated, but nonmatrix, proteins. In this study, we use blue native gel electrophoresis (BN-PAGE) to isolate rat and porcine heart mitochondrial complexes for screening of protein kinase activity. To detect kinase activity, one-dimensional BN-PAGE gels were exposed to [γ-(32)P]ATP and then followed by sodium dodecyl sulfate gel electrophoresis. Dozens of mitochondrial proteins were labeled with (32)P in this setting, including all five complexes of oxidative phosphorylation and several citric acid cycle enzymes. The nearly ubiquitous (32)P protein labeling demonstrates protein kinase activity within each mitochondrial protein complex. The validity of this two-dimensional BN-PAGE method was demonstrated by detecting the known PDH kinases and phosphatases within the PDH complex band using Western blots and mass spectrometry. Surprisingly, these same approaches detected only a few additional conventional protein kinases, suggesting a major role for autophosphorylation in mitochondrial proteins. Studies on purified Complex V and creatine kinase confirmed that these proteins undergo autophosphorylation and, to a lesser degree, tenacious (32)P-metabolite association. In-gel Complex IV activity was shown to be inhibited by ATP, and partially reversed by phosphatase activity, consistent with an inhibitory role for protein phosphorylation in this complex. Collectively, this study proposes that many of the mitochondrial complexes contain an autophosphorylation mechanism, which may play a functional role in the regulation of these multiprotein units.     

Evidence against insulin-stimulated phosphorylation of calmodulin in 3T3-L1 adipocytes

To examine the possibility that insulin might stimulate calmodulin phosphorylation in intact cells, we compared autoradiographs of two-dimensional gels of [35S]methionine- and 32P-labeled proteins from 3T3-L1 adipocytes, before and after immunoprecipitation with anti-calmodulin antiserum. Insulin stimulated the phosphorylation of one or two proteins of approximately 22 kDa and pI 4.6; this increased phosphorylation was accompanied by an apparent shift in the position of the analogous [35S]methionine-labeled proteins towards the anode. In contrast, insulin had no effect on the phosphorylation state of another protein of 18-22 kDa and pI 4.6. This protein was very heavily labeled with [35S]methionine, co-migrated exactly with purified calmodulin, reacted specifically with two anti-calmodulin antibodies by Western blotting, and was specifically immunoprecipitated with the anti-calmodulin antiserum. Similar amounts of [35S]methionine-labeled calmodulin were immunoprecipitated from control and insulin-stimulated cells, arguing against the possibility that insulin-stimulated phosphorylation of calmodulin changed its affinity for the antibody. We conclude that calmodulin is phosphorylated to a negligible extent in serum-deprived 3T3-L1 adipocytes and that insulin does not stimulate its phosphorylation under conditions in which it stimulates the phosphorylation of one or more neighboring proteins.