Epidermal growth factor stimulated phosphorylation of a 120-kilodalton endogenous substrate protein in rat hepatocytes

Okamoto, Motozumi; Karasik, Avraham; Mf, White; Kahn, C. Ronald

doi:10.1021/bi00492a023

Cited by 7 publications

(2 citation statements)

References 43 publications

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“…An EGF-dependent enhancement of tyrosine phosphorylation of the EGFR and an unidentified 120-kDa protein have been detected in rat hepatocyte cell suspensions by Okamoto et al (18). Their figure 1 indicated that under some extraction conditions an EGF-dependent 55-kDa phosphoprotein could be detected but it was not commented upon.…”

Section: Resultsmentioning

confidence: 98%

Epidermal growth factor stimulates tyrosine phosphorylation in the neonatal mouse: association of a M(r) 55,000 substrate with the receptor.

Donaldson

Cohen

1992

Proc. Natl. Acad. Sci. U.S.A.

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Administration of epidermal growth factor (EGF) to neonatal mice resulted in rapid tyrosine phosphorylation of a number of specific substrates in liver, kidney, lung, bladder, skin, and brain as detected by Western blot analysis of tissue homogenates with anti-phosphotyrosine antibodies. In the liver, three prominent EGF-dependent substrates of Mr 170,000, 120,000, and 55,000 were detected. A number of less prominent EGF-dependent substrates also were noted. Maximal tyrosine phosphorylation of ppl7O, pp120, and pp55 occurred within 5 min of subcutaneous injection and the levels of these phosphoproteins remained elevated for at least 45 min.Direct hepatic injection of EGF resulted in the tyrosine phosphorylation of these substrates within 60 sec of treatment. Tyrosine-phosphorylated ppl70 was identified as the EGF receptor (EGFR). The tyrosine-phosphorylated pp55 substrate appeared to be associated with EGFR; both ppS5 and EGFR were adsorbed to EGF-Affi-Gel, wheat germ lectin-Sepharose, and anti-EGFR antibodies bound to protein A-Sepharose. ppS5 was not immunoreactive with anti-EGFR antiserum by Western blot analysis, indicating that it was not a fragment of the receptor. These results were confirmed by repeating the liver experiments using 32P-labeled neonatal mice. Increased amounts of 32P-labeled ppl70 and pp55 were detected in anti-EGFR immunoprecipitates from liver extracts of EGFtreated animals as compared with controls. Phospho amino acid analysis of the 32P-labeled phosphoproteins revealed that EGF stimulated both serine and tyrosine phosphorylation in pp55 as well as in EGFR. The neonatal mouse may be a useful model for the study ofsignal transduction mediated by a variety of growth factors.The administration of epidermal growth factor (EGF) to newborn or adult animals evokes a wide variety of morphological and physiological responses. These include such diverse effects as increased cell proliferation in skin and other organs as well as inhibition ofgastric acid secretion (reviewed in refs. 1-3).At the cellular level, the receptor for EGF (EGFR) is a 170-kDa transmembrane glycoprotein with intrinsic proteintyrosine kinase activity. The binding of EGF to its receptor results in the activation of its tyrosine kinase activity and the phosphorylation of a number of intracellular substrates as well as the receptor itself (reviewed in refs. 4-6).Many studies have addressed the question of the nature and function of protein substrates that are phosphorylated on tyrosine in the presence of an activating ligand. Among the substrates that have been implicated are phospholipase Ca1, GTPase-activating protein (GAP), protein-serine kinases, phosphatidylinositol 3-kinase, structural proteins such as vinculin and talin, and proteins of unknown physiological function such as lipocortin (reviewed in refs. 7-9). Almost all of these studies have employed a variety of cell culture systems to detect these putative signal-transducing molecules.We have begun studies to examine both the normal physiological role of EGF ...

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Section: Resultsmentioning

confidence: 98%

Epidermal growth factor stimulates tyrosine phosphorylation in the neonatal mouse: association of a M(r) 55,000 substrate with the receptor.

Donaldson

Cohen

1992

Proc. Natl. Acad. Sci. U.S.A.

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“…Analysis of phosphorylated amino-acid residues in polyacrylamide gel fragments as described previously [21]. Gel fragments containing phosphorylated proteins were excised, washed for 12 h at 37°C in 20 % (v/v) methanol, dried at 80°C for 2 h, and digested with 2 ml of 50 mM NH4HC03 containing 100 ,ug of trypsin (pH 8.0).…”

Section: Labelling and Immunoprecipitationsmentioning

confidence: 99%

Specific activity of phosphatidylinositol 3-kinase is increased by insulin stimulation

Okamoto

Hayashi

Kono

et al. 1993

Biochemical Journal

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We investigated whether phosphatidylinositol 3-kinase (PI3K) is phosphorylated and whether its specific activity is increased by insulin stimulation in vivo using Fao cells and antibodies raised against the 85 kDa subunit of PI3K, insulin-receptor substrate-1 (IRS-1), and phosphotyrosine (pTyr). PI3K activity was detected in the immunoprecipitate produced with anti-PI3K at a basal state. The activity was increased 2-3-fold by insulin stimulation, although the protein concentration of kinase in the anti-PI3K immunoprecipitates was the same before and after insulin stimulation. Both anti-pTyr and anti-IRS-1 antibodies immunoprecipitated the kinase activity only after insulin stimulation. After the first immunoprecipitation with anti-pTyr, the supernatant was immunoprecipitated once more with anti-PI3K. PI3K activity in the second immunoprecipitate revealed little difference between the basal and insulin-stimulated states, suggesting that most of the insulin-activated portion of PI3K was precipitated by anti-pTyr. Both IRS-1 and the insulin-receptor beta-subunit (95 kDa) were phosphorylated on tyrosine residues by insulin stimulation and immunoprecipitated with anti-pTyr. However, phosphorylation of neither subunit of PI3K (85 kDa or 110 kDa) was detectable in the immunoprecipitate produced with anti-pTyr. The 185 kDa pTyr-containing protein was immunoprecipitated with anti-PI3K after insulin stimulation, although there was little phosphorylation of the 85 kDa protein. pTyr in the 110 kDa protein immunoprecipitated with anti-PI3K was below detectable levels. These results indicate that the specific activity of PI3K is increased by insulin stimulation without detectable tyrosine phosphorylation of PI3K itself in Fao cells. The majority of the insulin-activated portion of PI3K is associated with pTyr-containing proteins including IRS-1, which suggests that this is important for activation of PI3K by insulin.

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Phosphosite-Specific Antibodies: A Brief Update on Generation and Applications

Brumbaugh¹,

Liao²,

Houchins³

et al. 2017

Methods in Molecular Biology

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Phosphate addition is a posttranslational modification of proteins, and this modification can affect the activity and other properties of intracellular proteins. Different animal species can be used to generate phosphosite-specific antibodies as either polyclonals or monoclonals, and each approach offers its own benefits and disadvantages. The validation of phosphosite-specific antibodies requires multiple techniques and tactics to demonstrate their specificity. These antibodies can be used in arrays, flow cytometry, and imaging platforms. The specificity of phosphosite-specific antibodies is vital for their use in proteomics and profiling of disease.

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Epidermal growth factor stimulated phosphorylation of a 120-kilodalton endogenous substrate protein in rat hepatocytes

Cited by 7 publications

References 43 publications

Epidermal growth factor stimulates tyrosine phosphorylation in the neonatal mouse: association of a M(r) 55,000 substrate with the receptor.

Epidermal growth factor stimulates tyrosine phosphorylation in the neonatal mouse: association of a M(r) 55,000 substrate with the receptor.

Specific activity of phosphatidylinositol 3-kinase is increased by insulin stimulation

Phosphosite-Specific Antibodies: A Brief Update on Generation and Applications

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