Epidermal growth factor stimulates tyrosine phosphorylation of specific proteins in permeabilized human fibroblasts.

Giugni, Terrence D.; James, L; Haigler, Harry T.

doi:10.1016/s0021-9258(18)95705-4

Cited by 74 publications

(11 citation statements)

References 55 publications

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“…We also concentrated our study on endonexin II because it has Ca 2÷ and phospholipid binding properties similar to lipocortin 1, but it is not phosphorylated by the EGF receptor/kinase or other known protein kinases (41,42). We focused our study on normal diploid HFF because they express relatively high levels of both lipocortin 1 and endonexin II, because they have a well-defined growth response to EGF (4), and because lipocortin 1 phosphorylation in intact HFF ceils has been characterized (14). We found that the level of lipocortin 1 expression increased three to fourfold when quiescent HFF cells were stimulated to proliferate.…”

Section: Discussionmentioning

confidence: 99%

“…An aliquot of each sample was removed and precipitated with TCA to determine the amount of [35S]methionine incorporated into total cell protein. Immunoprecipitations were then performed on 50 #g total protein was described (14). The antigen was elnted from the protein A-Sepharose (Sigma Chemical Co.) with Laemmli SDS sample buffer (27) and analyzed by SDS-PAGE.…”

Section: Metabolic Labeling and Lmmunoprecipitationmentioning

confidence: 99%

“…Two of the proteins, annexin I (also called lipocortin 1) and annexin II (also called calpactin 1), are phosphorylated on a conserved tyrosine residue in the amino terminal domain by the EGF receptor/kinase (8,20,33) and by pp60 s~ (13,15), respectively. The phosphorylation of lipocortin 1 by the EGF receptor/kinase exhibits many characteristics expected of a physiological substrate for a protein tyrosine kinase: (a) the EGF receptor/kinase affinity for lipocortin 1 in vitro is very high, apparent Km = 50 nM (20); (b) the phosphorylation occurs in intact cells in a growth and EGF-dependent manner (14,39) and; (c) the stoichiometry of phosphorylation in cultured diploid fibroblasts changes from <1% in quiescent cells to ~25% in EGF-stimulated cells (D. D. Since lipocortin 1 appears to be a physiological substrate for the EGF receptor kinase and thus has been implicated in the regulation of cell growth, we have investigated its expression as a function of growth in human diploid foreskin fibroblasts (HFF).t For comparison, we also measured the expression of annexin V (also called endonexin II), a protein with Ca 2+ and phospholipid binding properties similar to those of lipocortin 1 (11,19,25,26,29,42). Endonexin II does not have a tyrosine residue in its amino terminal domain and is not a protein kinase substrate (41).…”

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Expression of annexins as a function of cellular growth state.

Schlaepfer

Haigler

1990

The Journal of Cell Biology

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160

106

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Abstract. Annexins are a structurally related family of Ca 2+ binding proteins of undetermined biological function. Annexin I (also called lipocortin 1) is a substrate for the EGF-stimulated tyrosine kinase and is postulated to be involved in mitogenic signal transduction. To investigate further the involvement of lipocortin 1 in cell proliferation, we measured lipocortin 1 levels in normal diploid human foreskin fibroblasts (HFF) to determine whether its expression changed as a function of growth status. For comparison, the expression of annexin V (also called endonexin II) was measured in HFF cells. Endonexin II is a protein with similar Ca z+ and phospholipid binding properties as lipocortin 1, but it is not a substrate for tyrosine kinases. Quiescent HFF cell cultures were induced to proliferate by either subculture to lower cell density, EGF stimulation, or serum stimulation. In all three protocols, proliferating HFF cells contained three-to fourfold higher levels of lipocortin 1 and three-to fourfold lower levels of endonexin II than quiescent HFF cells. In contrast, the expression of annexin II (also called calpactin I) and annexin IV (also called endonexin I) remained relatively unchanged in growing and quiescent HFF cells. Lipocortin 1 synthesis rate was eightfold higher and its turnover rate was 1.5-fold slower in proliferating compared to quiescent HFF cells. Endonexin II synthesis rate remained constant but its turnover rate was 2.2-fold faster in proliferating compared to quiescent HFF cells. In a separate set of experiments, annexin expression levels were measured in cultures of rat PC-12 cells, a pheochromocytoma that ceases proliferation and undergoes reversible differentiation into nondividing neuronlike cells in response to nerve growth factor (NGF). After NGF treatment, PC-12 cells expressed fivefold higher levels of endonexin II and 32-fold higher levels of calpactin 1. Lipocortin 1 and endonexin I were not expressed in PC-12 cells. In summary, lipocortin 1 expression exhibited a positive correlation with cell proliferation in HFF cells. The increased expression of endonexin II in quiescent HFF cells and differentiating PC-12 cells implies that this protein may play a more prominent role in nondividing cells.NEXINS are a family of structurally related proteins that bind to certain phospholipids in a Ca2+-dependent manner (for reviews, see references 7 and 12). The phospholipids to which they bind are preferentially located on the cytosolic face of the plasma membrane. Since they were independently discovered by several different laboratories that were interested in different biological problems, these proteins have been given several unrelated, and often overlapping names including lipocortins, calpactins, synexin, chromobindins, endonexins, calelectrins, calcimedins, and placental anticoagulant proteins. The relationships of these proteins are illustrated in Table I of reference 21. Each annexin has an amino terminal domain that has only limited sequence similarity with the others, while all...

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Section: Discussionmentioning

confidence: 99%

Section: Metabolic Labeling and Lmmunoprecipitationmentioning

confidence: 99%

mentioning

confidence: 99%

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Expression of annexins as a function of cellular growth state.

Schlaepfer

Haigler

1990

The Journal of Cell Biology

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160

106

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“…Immunoprecipitation was performed with MAbs that were negative by the Western blot procedure. The procedure of Giugmi et al (6) was adapted for our immunoprecipitation experiments.…”

Section: Methodsmentioning

confidence: 99%

Protective role of magnesium in the neutralization by antibodies of Chlamydia trachomatis infectivity

et al. 1988

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Neutralization of the infectivity of Chlamydia trachomatis was assessed by using polyclonal antisera and monoclonal antibodies (MAbs). Polyclonal antisera and a species-reactive MAb as well as a subspecies-specific MAb, both of which were directed toward the major outer membrane protein of C. trachomatis, reduced the number of chlamydial inclusion-forming units in an in vitro assay. Neutralization was dependent on the presence of complement. The species-specific MAb reacted with all 15 serovars by a microimmunofluorescence assay and a dot blot enzyme-linked immunosorbent assay with heat-treated elementary bodies. On the other hand, this same MAb reacted with all serovars, except those in the C complex, by the dot blot enzyme-linked immunosorbent assay with viable organisms and neutralized in vitro all 10 serovars tested, except those in the C complex. When neutralization assays were performed in a solution containing Mg2+, neutralization by both polyclonal antisera and MAbs was significantly reduced. A dose response to Mg2+ supplied as MgSO4 revealed that all concentrations tested from 50 to 800 microM had some effect. Concentrations of greater than or equal to 400 microM MgSO4 completely abolished neutralization at the lowest dilution of polyclonal antisera and species-reactive MAb tested. Although Mg2+ also blocked the neutralization effect of the subspecies-specific MAb, this neutralization was not as complete as that observed with the species-reactive MAb. Addition of Mg2+ to the assay over the initial 45 min of incubation of C. trachomatis with MAb and complement showed that the organisms could be rescued to some extent over the first 30 min of incubation, after which time neutralization of infectivity could not be reversed. C. trachomatis treated with Mg2+, the species-reactive MAb, and complement were lethal to mice in an in vivo toxicity and infectivity assay, whereas mice injected with organisms incubated with the same MAb and complement without Mg2+ survived.

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“…The identification of cellular substrates for these kinases is an active area of current research. Recently, lipocortin (Wallner et al, 1986) has been identified as a high-affinity substrate for the EGFstimulated protein-tyrosine kinase (De et al, 1986;Haigler et al, 1987;Giugni et al, 1985;Sawyer & Cohen, 1985) and for PKC (Summers & Creutz, 1985;Michener et al, 1986; Khanna et al, 1986) both in vivo and in vitro.…”

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In vitro protein kinase C phosphorylation sites of placental lipocortin

Schlaepfer¹,

Haigler²

1988

Biochemistry

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Human placental lipocortin is a high-affinity substrate for rat brain protein kinase C in vitro with phosphorylation occurring on serine and threonine residues in a ratio of approximately 2 to 1. Comparison of the ability of various N-terminal-truncated derivatives of lipocortin to serve as phosphorylation substrates, and direct analysis of the N-terminal peptides cleaved from 32P-labeled lipocortin, indicated that threonine-24, serine-27, and serine-28 were the phosphorylation sites. The possibility is discussed that a lysine residue near the carboxy side of the phosphorylation site was involved in lipocortin interaction with the catalytic site of protein kinase C.

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Epidermal growth factor stimulates tyrosine phosphorylation of specific proteins in permeabilized human fibroblasts.

Cited by 74 publications

References 55 publications

Expression of annexins as a function of cellular growth state.

Expression of annexins as a function of cellular growth state.

Protective role of magnesium in the neutralization by antibodies of Chlamydia trachomatis infectivity

In vitro protein kinase C phosphorylation sites of placental lipocortin

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