Isolation and characteristics of HeLa surface proteins

HeLa 71 and 65 cells grown in attached culture possess a coat of extracellular proteins that can be released from the cell by mild EDTA-detachment, with no significant effect on cellular integrity. This suggests that these surface proteins are weakly associated with the cell, possibly through divalent cations. The high affinity of surface proteins for critical divalent cations, shown by their high precipitability by Zn²⁺, Ca²⁺ and Mg²⁺, supports this assumption. Since surface proteins appear to be phosphoproteins, as suggested by significant incorporation of ³²Pi in vitro, it is possible that binding occurs through The amount of surface protein on HeLa 65 cells grown in suspension culture is greatly reduced compared with cells grown in monolayer culture. This may be related to impaired availability of Ca²⁺ in suspension culture medium. In monolayer grown HeLa cells surface proteins are predominantly distributed underneath the cells. The highest amount of these proteins is found on cells prior to growth initiation and steadily decreases as cells approach confluency. As shown by radioactive leucine protein labeling, surface proteins are primarily comprised of proteins synthesized within HeLa cells and released to the outer cell surface. The presence of serum proteins in surface protein matrix is physiologically significant.     

Protein kinase activity and substrates at the surface of intact HeLa cells

Evidence is presented for the location at the surface of HeLa cells of a protein kinase capable of phosphorylating surface as well as extracellular (foreign) proteins. The reaction products have been found to be proteins containing phosphoryl groups as monoesters of seryl and threonyl residues (but not of tyrosine). The enzyme is of the cyclic AMP-independent type, since neither cyclic AMP nor the heat- and acid-stable inhibitor protein (specific for cyclic AMP-dependent protein kinases) influenced its activity. Further, co-substrate ATP could in part be substituted by GTP, and the spectrum of proteins phosphorylated by the ecto-enzyme differed from that phosphorylated by cyclic AMP-dependent protein kinases. Evidence for the ecto-enzymic nature of this protein kinase includes (a) utilization of co-substrate and location of products at the surface of cells carefully controlled as being in an intact state and (b) phosphorylation of exogenous protein (phosvitin; specific serum proteins) by intact cells. Conclusive proof was gained by qualitative and quantitative comparative studies of phosphorylation in cultures with varying degrees of damaged cells either as a whole or after separation into groups of intact and damaged cells by electronic cell sorting. The results of experiments with cell sonicates excluded the possibility that either enzyme or substrates released from damaged cells were simply adsorbing to the cell surface.     

Analysis of the Arabidopsis cell suspension phosphoproteome in response to short-term low temperature and abscisic acid treatment

To shed light on the early protein phosphorylation events involved in plant cell signaling in response to environmental stresses, we studied changes in the phosphorylation status of the Arabidopsis cell suspension proteome after short-term low temperature and abscisic acid (ABA) treatment. We used radioactive pulse-labeling of Arabidopsis cell suspension cultures and two-dimensional (2-D) gel electrophoresis to identify proteins that are differentially phosphorylated in response to these treatments. Changes in the phosphorylation levels of several proteins were detected in response to short-term (5 min or less) cold (4°C) and chilling (12°C) stress and ABA treatment, and we observed that some of these changes were common between these treatments. In addition, we used Pro-Q Diamond phosphoprotein gel stain to study the steady-state protein phosphorylation status under the same treatments. We demonstrated that Pro-Q Diamond effectively stained phosphorylated proteins, however, the overall Pro-Q Diamond 2-D gel staining pattern of proteins extracted from low-temperature and ABA-treated cells was not consistent with the gel patterns obtained by in vivo radioactive labeling of phosphoproteins. These in vivo pulsed-labeling experiments demonstrate that the Arabidopsis phosphoproteome is dynamic in response to short-term low temperature and ABA treatment, and thus represents a strategy for the identification of signaling proteins that could be utilized in the production of chilling or freeze tolerant crop varieties.     

Analyses of cell surface and secreted proteins of primary cultures of mouse extraembryonic membranes

Procedures have been developed for primary culture of 13th day mouse parietal and visceral endoderm, yolk sac mesoderm, and amnion cells. We have analyzed cell surface and secreted proteins of these cultures by labeling the cells with radioactive iodine, glucosamine, or amino acids, and/or by immunofluorescence. Cell surface and secreted proteins of visceral endoderm, yolk sac mesoderm, and amnion cells resemble each other closely, whereas parietal endoderm cells are strikingly different. Unlike the other cell types, parietal endoderm cells synthesize and secrete substantial quantities of a protein tentatively identified as procollagen. These cells also secrete a number of other glycoproteins not observed in the media from the other cultures. It is proposed that the procollagen and one or more of the other unique, secreted glycoproteins are normally constituents of Reichert's membrane. Compared to the other cultures, parietal endoderm cells appear to be deficient in production of LETS protein. However, parietal endoderm—Reichert's membrane complexes analyzed by immunofluorescence directly after dissection from the uterus show an abundant association with LETS protein. It is not clear whether this LETS protein is actually synthesized by the parietal endoderm cells themselves. If so, it is possible that this protein is rapidly degraded after its secretion in parietal endoderm primary cultures. The studies reported here represent a first step in the characterization of cell surface properties of embryonic and extraembryonic cell types. The information already accumulated should be useful in investigations aimed at identification of cells derived from blastocysts and teratocarcinomas in vitro.     

Investigations on myelinogenesisin vitro: I. The occurrence of endogenous protein kinase and its role in the phosphorylation of myelin basic proteins in “Myelin-like membranes” isolated from cerebral cell cultures

The presence of a protein kinase capable of phosphorylating endogenous as well as exogenously added myelin basic proteins has been demonstrated in a myelin-like membrane fraction isolated from reaggregating and surface adhering, primary cultures of cells dissociated from embryonic mouse brain. Only the large and small components of myelin basic proteins were found to be phosphorylated when myelin-like membrane fraction was incubated with [-³²P]ATP. The protein kinase endogenous to the myelin-like membrane fraction was mainly of the cyclic AMP independent type. There was very little cyclic AMP dependent or cyclic GMP dependent protein kinase activities in this myelin-like fraction. Although the myelin basic proteins were the only endogenous proteins phosphorylated, protein kinase of the myelin-like membrane was capable of catalyzing the phosphorylation of exogenous substrates, such as histones.     

A comparison of ribosomal proteins from rabbit reticulocytes phosphorylated in situ and in vitro.

A comparison has been made between the ribosomal proteins phosphorylated in intact cells and proteins isolated from ribosomal subunits after modification in vitro by purified protein kinases and [gamma-32P]ATP. When intact reticulocytes were incubated for 2 h in a nutritional medium containing radioactive inorganic phosphate, one phosphorylated protein was identified as a 40S ribosomal component using two-dimensional polyacrylamide gel electrophoresis followed by electrophoresis in a third step containing sodium dodecyl sulfate. This protein, containing 99% of the total radioactivity associated with ribosomal proteins as observed by two-dimensional electrophoresis, is found in a nonphosphorylated form in addition to several phosphorylated states. These states differ by the number of phosphoryl group attached to the protein. The same 40S protein is modified in vitro by the three cAMP-regulated protein kinases from rabbit reticulocytes. Two additional proteins associated with the 40S subunit are phosphorylated in situ. These proteins migrate as a symmetrical doublet, and contain less than 1% of the radioactive phosphate in the 40S subunit. A number of phosphorylated proteins associated with 60S subunits are observed by disc gel electrophoresis after incubation of whole cells with labeled phosphate. These proteins do not migrate with previously identified ribosomal proteins and are not present in sufficient amounts to be identified as ribosomal structural proteins. Proteins in the large subunit are modified in vitro by cAMP-regulated protein kinases and ATP, and these modified proteins migrate with known ribosomal proteins. However, this phosphorylation has not been shown to occur in intact cells.     

Influence of concanavalin A on protein synthesis and protein release in BHK 21 cells

Addition of concanavalin A to baby-hamster kidney cells (strain BHK 21/C13) grown in Eagle's minimal essential medium devoid of serum but supplemented with insulin as growth-promoting factor, caused a marked reduction of the total protein content of the cells: as early as 1 h after treatment, the amount of protein decreased to about 60–70% of the values found in untreated cultures.Pulse-labelling experiments performed with [³H]leucine demonstrated that the uptake and incorporation of the labelled amino acid was not affected by the lectin up to 6 h after treatment. Pulse-chase experiments gave no evidence for an enhanced degradation of proteins.Examination of the supernatants of concanavalin A-treated cultures as well as their controls, pre-labelled with [³H]fucose and [¹⁴C]leucine revealed that the amount of membrane-derived glycoproteins which were shed into the culture medium was considerably higher in concanavalin A-treated cultures.However, the bulk of protein which accounts for the difference between lectin-treated and untreated cultures consists of intracellular material which was released during the cell harvest procedure. The loss of protein was prevented by α-methyl-d-mannoside (10^?2 M).Scanning electron microscopy of concanavalin A-treated cells showed a change from the smooth surface of the fibroblastic cells to a retracted one as early as 30 min after addition of the lectin. The surface of the altered cells was characterized by the presence of numerous bleb-like protuberances.The viability of the cells was not affected by concanavalin A-treatment during the course of the experiments.Experiments performed with variable concentrations of insulin excluded the possibility that the observed effects might be due to a competition between the lectin and the hormone.     

Variations in some molecular events during the early phases of the Reuber H 35 cell cycle. II.-Chromatin protein phosphorylation and protein kinases

Reuber H 35 hepatoma cells were synchronized by transfer in a serum free medium. Growth was re-initiated by addition of serum. Under these conditions DNA synthesis exhibited a maximum after 24 hours. Chromatin non-histone proteins prepared from cells at various phases of the cell cycle were incubated with [gamma-32P] ATP and the radioactive pattern of protein bound 32P was analysed by electrophoresis on polyacrylamide gels. No radioactive peak was observed in G0. Several peaks appeared 3 hours after the addition of serum. The radioactivity progressively increased until the cells reached the S phase. When most of the cells were in the S phase the radioactivity strongly decreased. Chromatin protein kinase activities were found to increase in late G1 and continued to increase in the S phase. The increase was 65% when phosvitin was the substrate, 100% with casein and histone H1. It is suggested that chromatin phosphorylated proteins could be involved in the mechanism which initiates DNA synthesis in G1 phase cells.     

The membrane (M1) protein of influenza virus occurs in two forms and is a phosphoprotein.

The membrane (M1) protein of influenza virus was found to be heterogenous and to occur in two forms in the virus particle. The two forms of M1 were found in virus which was produced both early and late after infection and in infected cells. The two forms could be separated on polyacrylamide gels under specific conditions. The two components of M1 contained similar tryptic peptides. However, a small proteolytic difference between the two proteins could not be ruled out. Both M1 proteins were present in phosphorylated form in the virus particle. The phosphorylated M1 components were not readily distinguished from phosphorylated nonstructural protein (NS1) when cytoplasm of infected cells was analyzed on polyacrylamide gels. The two phosphorylated M1 components could, however, be detected in infected cells by immunoprecipitation. One M1 component contained only phosphoserine whereas the second contained phosphoserine and a small amount of phosphothreonine as well. In addition to the phosphorylated nucleoprotein and M1, a third phosphorylated protein was routinely detected in virus particles. It was a surface component of the virus, since it could be removed from whole virus with chymotrypsin and contained phosphate at serine residues. The identity of this component was not known.     

Cell-cycle parameters of soybean (Glycine max L.) cells growing in suspension culture: Suitability of the system for genetic studies

Summary Suspension cultures of Glycine max (L.) Merr. were grown at 22 and 33°. The doubling times of dividing cells were 35 and 25 h, respectively. G₂ was 6.2 and 6.7 h, and S was 13.8 and 6.5 h. G₁ was calculated as 13 and 10 h, respectively. These values were determined by labeling cells with ³H thymidine and measuring the appearance of radioactive mitotic figures. Treatment with 5-fluorodeoxyuridine (FudR) inhibited DNA synthesis and, as a result, cells accumulated in S. Such cells were viable and, upon removal of the FudR, proceeded synchronously into mitosis. Treatment with 5-bromodeoxyuridine, following FudR synchronization, sensitized the cells to white light. Thus cells capable of synthesizing DNA could be killed. 20–30% of the cells in suspension cultures growing at 22 or 33° were not able to synthesize DNA. Nevertheless, these non-dividing (Q) cells were able to synthesize RNA and protein at a reduced rate. The proteins synthesized appear to be a particular subset of the proteins made by normal cells. The results are analyzed in relation to the use of this suspension cell culture system for isolating conditional lethal mutants of plant cells.     

Evidence for an ectophosvitin kinase activity that phosphorylates a 123 kDa endogenous substrate on a human colonic adenocarcinoma cell (HT 29)

When HT 29 cells grown as a monolayer were incubated in a synthetic medium in presence of 0.1 microM [gamma 32P]-ATP, the radioactivity was incorporated predominantly into three major endogenous polypeptides of 123 kDa, 50 kDa and 46 kDa. The radioactive proteins could be detected as soon as 30 s after the addition of the labelled ATP. When exogenous substrates such as casein or phosvitin were added in the synthetic medium, these proteins became phosphorylated. The phosvitin-kinase activity was released in the culture medium following an incubation of the cells with phosvitin. Depletion of the enzymatic activity from the cell surface as well as competition between phosvitin and endogenous substrates led specifically to the inhibition of the 123 kDa polypeptide phosphorylation. At low density, endogenous phosphorylation increased with the cell number, whereas on the contrary it decreased at high cell density. We concluded that the surface of HT 29 cells expressed several protein kinase activities. We have characterized one of them as an ectophosvitin kinase which phosphorylated specifically a 123 kDa polypeptide and whose expression or accessibility varied according to cell density.     

Association of the epidermal growth factor receptor kinase with the detergent-insoluble cytoskeleton of A431 cells

The epidermal growth factor receptor (EGF-R) on human epidermoid carcinoma cells, A431, was found to be predominantly associated with the detergent-insoluble cytoskeleton, where it retained both a functional ligand-binding domain and an intrinsic tyrosine kinase activity. The EGF-R was constitutively associated with the A431 cytoskeleton; this association was not a consequence of adventitious binding. The EGF-R was associated with cytoskeletal elements both at the cell surface, within intracellular vesicles mediating the internalization of the hormone-receptor complex, and within lysosomes. The EGF-R became more stably associated with cytoskeletal elements after its internalization. The cytoskeletal association of the EGF-R was partially disrupted on suspension of adherent cells, indicating that alteration of cellular morphology influences the structural association of the EGF-R, and that the EGF-R is not intrinsically insoluble. Cytoskeletons prepared from EGF-treated A431 cells, when incubated with gamma-32P-ATP, demonstrated enhanced autophosphorylation of the EGF-R in situ as well as the phosphorylation of several high molecular weight proteins. In this system, phosphorylation occurs between immobilized kinase and substrate. The EGF-R and several high molecular weight cytoskeletal proteins were phosphorylated on tyrosine residues; two of the latter proteins were phosphorylated transiently as a consequence of EGF action, suggesting that EGF caused the active redistribution of the protein substrates relative to protein kinases. The ability of EGF to stimulate protein phosphorylation in situ required treatment of intact cells at physiological temperatures; addition of EGF directly to cytoskeletons had no effect. These data suggest that the structural association of the EGF-R may play a role in cellular processing of the hormone, as well as in regulation of the EGF-R kinase activity and in specifying its cellular substrates.     

Differences in Protein Patterns in Suspension Cultures of Taxus cuspidata Induced by Cerium

The changes in soluble proteins induced by Ce⁴⁺ were analyzed in suspension cultures of Taxus cuspidata using two-dimensional polyacrylamide gel electrophoresis. The ultrastructure of cells obviously changed at day 4 after addition of Ce⁴⁺. Large amount of nuclear DNA fragments of about 200 bp were observed. Thirteen protein spots were different between the cultures grown with and without Ce⁴⁺ at day 4 as well as at day 6 after addition of Ce⁴⁺. This revised version was published online in July 2006 with corrections to the Cover Date.     

Cell density and cell shape-related regulation of vimentin and cytokeratin synthesis : Inhibition of vimentin synthesis and appearance of a new 45 kD cytokeratin in dense epithelial cell cultures

The pattern of the intermediate type filament protein synthesis was examined in cultured bovine mammary gland epithelial (BMGE) cells under conditions of varied cell shape and cell-cell contact. In dense monolayer and suspension cultures BMGE cells expressed a new cytokeratin of 45 kD identified as a member of the acidic subfamily of cytokeratins. This polypeptide has a phosphorylated component and is dissociated from the cytokeratins complex in the presence of 6.5 M urea. The mRNA of the new cytokeratin accumulated in dense cell cultures, as revealed by in vitro translation in a cell-free system. In BMGE-H cells that express also vimentin, the synthesis of vimentin decreased dramatically in dense cell cultures, while the synthesis of the 45 kD cytokeratin was maximal under these conditions. The results suggest that the expression of certain cytokeratins and that of vimentin can be coordinately regulated by factors in the cellular environment that effect cell shape and cell surface contacts.     

Cell adhesion-dependent differences in endogenous protein phosphorylation on the surface of various cell lines

Endogenous phosphorylation of intact cells was studied with four mouse, hamster and human cell lines using [gamma-32P]ATP and [gamma-32P]GTP as exogenous substrates. With all four cell lines distinct differences in the phosphoprotein patterns could be demonstrated for cells grown in suspension culture compared to cells grown in monolayers. Two major, apparently ubiquitous phosphoproteins with molecular weights of 135 000 (128 000 in HeLa cells) and 105 000, representing up to 60% of total phosphorylation, were phosphorylated only in cells grown in suspension. These phosphoproteins and the kinase(s) were located on the surface of the suspension cells. Evidence showed that phosphorylation was apparently not a true endogenous reaction, that rather it occurred by cell-cell collision, showing exponentially increasing 32P incorporation with increasing cell population density. Phosphorylation of pp135 and pp105 was established with ATP as well as with GTP and was not dependent on cyclic nucleotides cyclic AMP, cyclic GMP and cyclic CMP. The substrate-attached cells of all four cell lines have protein kinases on the cell surface. The lack of pp135 and pp105 phosphorylation may be due to the fact that these phosphoproteins are not expressed at all on the surface of substrate-attached cells or that these phosphoproteins are already fully phosphorylated.     

Expression of carbohydrate binding protein 35 in human fibroblasts: variations in the levels of mRNA, protein, and isoelectric species as a function of replicative competence

We have compared the expression and localization of carbohydrate binding protein 35 (CBP35) in human SL66 fibroblasts of different replicative capacities. When serum-starved, quiescent young (passage 11, corresponding to approximately 18 cumulative population doublings) SL66 cells were treated with serum, there was a marked stimulation in the expression of CBP35. This was revealed both by an increase in the percentage of cells positively stained with anti-CBP35 under immunofluorescence and by an increase in the amount of the protein in immunoblots, as well as by an increase in the level of accumulated mRNA in Northern blots. The rise in the expression of CBP35 in proliferating cells was manifested most clearly in the nuclear fraction, with elevation in the levels of the nonphosphorylated (pI8.7) protein, as well as the phosphorylated (pI8.2) derivative. In contrast, older (passage 27-35, 55-68 cumulative population doublings) cultures of SL66 fibroblasts appear to have lost the normal proliferation-dependent regulation of CBP35 expression. The level of CBP35 was high in quiescent high-passage cells and decreased somewhat after serum stimulation. Furthermore, the unphosphorylated (pI 8.7) form of the lectin could not be detected in either the nucleus or the cytoplasm of high-passage SL66 cells. Finally, the level of the mRNA for CBP35 was high in quiescent cultures of high-passage cells, but undetectable 17 h after serum stimulation. These results establish that the expression of CBP35 becomes altered as human fibroblasts acquire reduced replicative capacity.     

Stimulation of Ca2+ uptake and protein phosphorylation in tumor cells by fibronectin

Suspensions derived from attached HeLa cells transported 45Ca2+ considerably faster than those derived from spinner cultures grown in liquid medium. Incubation of spinner cells with fibronectin or cold-insoluble globulin in the presence of 5% calf serum at 37 degrees C for 1 to 2 h greatly increased the rate of Ca2+ flux into the cells. Suspensions of cells transformed by Rous sarcoma virus transported Ca2+ much more slowly than cell suspensions of the parent strain of normal rat kidney. Incubation of the transformed cells or Ehrlich ascites tumor cells with fibronectin increased the rate of Ca2+ uptake, while no effect was seen on Ca2+ transport by this treatment of normal kidney cells grown in tissue cultures. A 45,500-dalton protein was found to interact firmly with Ca2+ that entered into attached HeLa cells or fibronectin-treated spinner cells. This Ca2+-associated protein was detected by lithium dodecyl sulfate gel electrophoresis at 0 degrees C after 30 s of exposure to radioactive Ca2+. In tumor cells without fibronectin treatment, the radioactive band was not seen under the same conditions, even after 10 min incubation with 45Ca2+. In fibronectin-treated tumor cells, addition of Ca2+ to buffered solutions resulted in increased phosphorylation of a protein in the 45,000-dalton region. The phosphorylated protein band which appears to be associated with the cytoskeleton can be resolved by isoelectric focusing into four polypeptide chains. The relation of these observations to the cascade of protein kinases involved in the phosphorylation of the beta-subunit of the (Na+-K+)-ATPase is discussed.     

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

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

Phosphorylation of Escherichia coli proteins during the SOS response

• 1.1. The phosphorylation of Escherichia coli proteins was analyzed comparatively before and after induction of the SOS response in a temperature-sensitive mutant strain.
• 2.2. The presence of phosphorylated proteins was evidenced by gel electrophoresis and autoradiography after labelling with radioactive orthophosphate in vivo or radioactive adenosine triphosphate in vitro.
• 3.3. Significant changes in the intensity of protein labelling were observed upon induction of the SOS functions: six proteins were found to be more phosphorylated while two others were less phosphorylated. Moreover, five additional proteins appeared to become phosphorylated exclusively during the SOS response. The molecular mass and isoelectric point of these various proteins were determined.
• 4.4. For most proteins, the changes in the pattern of protein phosphorylation were concomitant with variations in the amount of protein synthesized.
• 5.5. The changes in the pattern of phosphoproteins observed during the SOS response were not due to the temperature shift required experimentally for expressing the SOS phenotype.
• 6.6. Phosphorylation was found to be catalyzed by protein kinases that modify amino acid residues at hydroxyl groups in protein substrates.
• 7.7. Both in vivo and in vitro studies brought evidence that neither RecA nor LexA, the two key regulatory proteins of the SOS functions, were capable of undergoing phosphorylation.