Biomedical subjects
Publications and source records attributed to D F Stern.
Completing a clinical incident report (CIR) when an unexpected "event" occurs is the first step in identifying practice issues and potential risk management problems. The CIR's subsequent path is explained, including the role that the peer review process plays in identifying substandard care.
The regulation of endothelial nitric oxide synthase (eNOS) by phosphorylation is poorly understood. Here, we demonstrate that eNOS is tyrosine-phosphorylated in bovine aortic endothelial cells (BAEC) using 32P metabolic labeling followed by phosphoamino acid analysis and by phosphotyrosine specific Western blotting. Treatment of BAEC with hydrogen peroxide and the protein tyrosine phosphatase inhibitor, sodium orthovanadate, increases eNOS tyrosine phosphorylation. Utilizing a novel immunoNOS assay, the increase in tyrosine phosphorylation is associated with a 50% decrease in the specific activity of the enzyme. Because eNOS is localized in plasmalemma caveolae, we examined if tyrosine phosphorylated eNOS interacts with caveolin-1, the coat protein of caveolae. Immunoprecipitation of eNOS from bovine lung microvascular endothelial cells resulted in the co-precipitation of caveolin-1. Conversely, immunoprecipitation of caveolin-1 resulted in the co-precipitation of tyrosine-phosphorylated eNOS. Thus, tyrosine phosphorylation is a novel regulatory mechanism for eNOS and caveolin-1 is the first eNOS-associated protein. Collectively, these observations provide a novel regulatory mechanism for eNOS and suggest that tyrosine phosphorylation may influence its activity, subcellular trafficking, and interaction with other caveolin-interacting proteins in caveolae.
The epidermal growth factor (EGF) family hormones amphiregulin (AR), transforming growth factor-alpha (TGF-alpha), and heparin-binding EGF-like growth factor (HB-EGF) are thought to play significant roles in the genesis or progression of a number of human malignancies. However, the ability of these ligands to activate all four erbB family receptors has not been evaluated. Therefore, we have assessed the stimulation of erbB family receptor tyrosine phosphorylation by these hormones in a panel of mouse Ba/F3 cell lines expressing the four erbB family receptors, singly and in pairwise combinations. We also measured the stimulation of interleukin-3-independent survival or proliferation in this panel of Ba/F3 cell lines to compare the patterns of erbB family receptor coupling to physiologic responses induced by these peptides. EGF, TGF-alpha, AR, and HB-EGF all stimulated qualitatively similar patterns of erbB family receptor tyrosine phosphorylation and coupling to physiologic responses. Therefore, EGF, TGF-alpha, AR, and HB-EGF are functionally identical in this model system and behave differently from the EGF family hormones betacellulin and neuregulins.
SPK1/RAD53/MEC2/SAD1 of Saccharomyces cerevisiae encodes an essential protein kinase that is required for activation of replication-sensitive and DNA damage-sensitive checkpoint arrest. We have investigated the regulation of phosphorylation and kinase activity of Spk1p during the cell cycle and by conditions that activate checkpoint pathways. Phosphorylation of Spk1p is induced by treatment of cells with agents that damage DNA or interfere with DNA synthesis. Although only S- and G2-phase cdc mutants arrest with hyperphosphorylated Spk1p, damage-induced phosphorylation of Spk1p can occur in G1 and M as well. Hydroxyurea (HU) induces phosphorylation of kinase-defective forms of Spk1p, demonstrating that this regulated phosphorylation of Spk1p occurs in trans. HU-induced phosphorylation is associated with increased catalytic activity of Spk1p. Furthermore, overexpression of wild-type SPK1, but not checkpoint-defective alleles, delays progression through the G1/S boundary. Damage-dependent phosphorylation of Spk1p requires both MEC1 and MEC3, whereas MEC1 but not MEC3, is required for replication block-induced phosphorylation. These data support the model that Spk1p is an essential intermediate component in a signal transduction pathway coupling damage and checkpoint functions to cell cycle arrest. This regulation is mediated through a protein kinase cascade that potentially includes Mec1p and Tel1p as the upstream kinases.
Betacellulin is a member of the epidermal growth factor (EGF) family. These soluble proteins are ligands for one or more of the four receptor tyrosine kinases encoded by the erbB gene family (erbB-1/epidermal growth factor receptor (EGFR), neu/erbB-2/HER2, erbB-3/HER3 and erbB-4/HER4). While evidence suggests that betacellulin is a ligand for the EGFR, the ability of betacellulin to regulate other erbB family receptors has not been analysed. Previously we engineered derivatives of the mouse Ba/F3 hematopoietic cell line to ectopically express erbB family receptors, singly and in pairwise combinations. We have stimulated this panel of cell lines with betacellulin and two other EGF family members, EGF itself and neuregulin-beta (NRG-beta). In the cell lines expressing a single erbB family receptor, betacellulin not only stimulated EGFR tyrosine phosphorylation, but it activated erbB-4 as well. Furthermore, in the double recombinant Ba/F3 derivatives, betacellulin stimulated a complex pattern of receptor phosphorylation distinct from the patterns activated by NRG-beta and EGF. Moreover, betacellulin stimulated a complex pattern of interleukin-3 independence in the Ba/F3 derivatives distinct from those activated by NRG-beta and EGF. These data identify a novel receptor for betacellulin and establish that different EGF family ligands activate distinct patterns of receptor phosphorylation and coupling to cellular signaling pathways.
Overexpression of HER-2/neu in human breast carcinomas correlates with poor prognosis, although its strength as a prognostic indicator varies widely in different reports. Variability may be due to active signalling by HER-2/neu in a subset of the tumours in which it is overexpressed. To study this hypothesis, we have developed an activation state-specific anti-HER-2/neu monoclonal antibody. In this report, we use this antibody to analyse the signalling status of HER-2/neu in a large series of invasive breast carcinomas. Overexpression of HER-2/neu was detected in 9% of 223 cases. Of the cases demonstrating overexpression, active signalling by HER-2/neu was detected in only 35%. The clinicopathological characteristics of these cases are described. This functional assay is predicted to improve the utility of HER-2/ neu as a prognostic indicator.
The neu/erbB-2/HER-2 proto-oncogene is amplified and/or overexpressed in up to 30% of mammary carcinomas and has been variably correlated with poor prognosis. The signaling activity of the encoded receptor tyrosine kinase is regulated by interactions with other type 1 receptors and their ligands. We have used a novel approach, phosphorylation-sensitive anti-Neu antibodies, to quantify signaling by Neu and epidermal growth factor receptor in a panel of frozen sections of mammary carcinoma specimens. We also determined the relationship of Neu, phosphorylated Neu (and epidermal growth factor receptor), and phosphotyrosine to the expression of Neu-related receptors (epidermal growth factor receptor, HER-3, and HER-4) and to prognostic factors (estrogen and progesterone receptor). We found that tyrosine phosphorylation of Neu (and hence signaling activity) is highly variable among mammary carcinomas. Neu and HER-4 were associated with divergent correlates, suggesting that they have profoundly different biological activities. These results have implications for etiology of mammary carcinoma for clinical evaluation of mammary carcinoma patients, and for development of Neu-targeted therapeutic strategies.
Mammary gland development and differentiation is mediated through the combined activities of systemic hormones and locally synthesized growth factors. To determine the in vivo response of mammary epithelium to heregulin (HRG), we implanted Elvax pellets containing HRG alpha or HRG beta within the mammary glands of prepubescent female mice in the presence or absence of exogenous estradiol and progesterone (E/ P). Mice treated in the same way with transforming growth factor alpha (TGF-alpha) were included as a positive control. Each growth factor treatment induced epithelial ductal branching in the presence or absence of E/P. In the absence of E/P, HRG beta did not effect terminal end bud formation, mammary epitheilum branching, or ductal migration. In contrast, TGF-alpha and HRG alpha induced ductal branching and HRG alpha induced ductal migration in the absence of E/P. The overall mammary response to growth factors was potentiated by the concomitant presence of E/P. In every case, the in vivo mammary epithelial responses to HRG alpha were more robust than TGF-alpha. Limited lobuloalveolar development was also observed in growth factor-treated mammary glands when E/P was present. Histological examination of growth factor-induced lobuloalveoli revealed secretory products within the lumen of HRG alpha and HRG beta lobuloalveoli. TGF-alpha-induced lobuloalveoli lacked similar secretory products.
Amplification of the neu/erbB-2/HER-2 gene and/or overexpression of its receptor tyrosine kinase product, p185neu/erbB-2 (p185), occurs in 15-40% of primary human breast tumors and is variably correlated with poor patient prognosis. Variability in predictive accuracy likely results from activation of p185 by agonist(s) in only a subset of tumors in which it is overexpressed, which may greatly affect outcomes. As a first step toward evaluating this hypothesis, we previously produced a polyclonal antibody that specifically recognizes the activated, tyrosine-phosphorylated forms of p185 and the closely related epidermal growth factor receptor (L. Bangalore et al., Proc. Natl. Acad. Sci. USA, 89: 11637-11641, 1992). We now describe the production of a mAb, PN2A, that specifically recognizes tyrosine-phosphorylated p185 and bears no cross-reactivity with closely related receptors. Furthermore, we demonstrate its reactivity in immunohistochemical staining of paraffin-embedded, formalin-fixed tumor samples. In a series of five p185-overexpressing human tumors examined thus far, PN2A reactivity was detected in two, indicating that p185 is phosphorylated and hence actively signaling in a subset. This reagent will facilitate both clinical and research analyses of p185 activity. Furthermore, this work serves as a prototype for similar analyses of other tyrosine phosphoproteins.
Deregulated signaling by the four members of the epidermal growth factor receptor tyrosine kinase family (erbB family) is implicated in the genesis or progression of human cancers. However, efforts to analyze signaling by these receptors have been hampered by the diversity of ligands and extensive interreceptor cross talk. We have expressed the four human erbB family receptors, singly and in pairwise combinations, in a pro-B-lymphocyte cell line (Ba/F3) and investigated the range of interactions activated by the epidermal growth factor homology domain of the agonist neuregulin beta. The results provide the first comprehensive analysis of the response of this receptor family to a single peptide agonist. This peptide induced complex patterns of receptor tyrosine phosphorylation and regulation of Ba/F3 cell survival and proliferation. These data demonstrate the existence of several previously undocumented receptor interactions driven by neuregulin.
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SPK1 was originally discovered in an immunoscreen for tyrosine-protein kinases in Saccharomyces cerevisiae. We have used biochemical and genetic techniques to investigate the function of this gene and its encoded protein. Hybridization of an SPK1 probe to an ordered genomic library showed that SPK1 is adjacent to PEP4 (chromosome XVI L). Sporulation of spk1/+ heterozygotes gave rise to spk1 spores that grew into microcolonies but could not be further propagated. These colonies were greatly enriched for budded cells, especially those with large buds. Similarly, eviction of CEN plasmids bearing SPK1 from cells with a chromosomal SPK1 disruption yielded viable cells with only low frequency. Spk1 protein was identified by immunoprecipitation and immunoblotting. It was associated with protein-Ser, Thr, and Tyr kinase activity in immune complex kinase assays. Spk1 was localized to the nucleus by immunofluorescence. The nucleotide sequence of the SPK1 5' noncoding region revealed that SPK1 contains two MluI cell cycle box elements. These elements confer S-phase-specific transcription to many genes involved in DNA synthesis. Northern (RNA) blotting of synchronized cells verified that the SPK1 transcript is coregulated with other MluI box-regulated genes. The SPK1 upstream region also includes a domain highly homologous to sequences involved in induction of RAD2 and other excision repair genes by agents that induce DNA damage. spk1 strains were hypersensitive to UV irradiation. Taken together, these findings indicate that SPK1 is a dual-specificity (Ser/Thr and Tyr) protein kinase that is essential for viability. The cell cycle-dependent transcription, presence of DNA damage-related sequences, requirement for UV resistance, and nuclear localization of Spk1 all link this gene to a crucial S-phase-specific role, probably as a positive regulator of DNA synthesis.
Rabbits were immunized with a synthetic phosphopeptide corresponding to a major autophosphorylation site of p185neu/erbB2 to determine the feasibility of producing tyrosine-phosphopeptide-specific antibodies. A series of adsorption and affinity chromatography steps were used to select antibodies with the desired reactivity. Immunoblot experiments showed that the resulting serum is highly specific for tyrosine-phosphorylated forms of p185 and the related epidermal growth factor receptor. The serum recognized these two receptors selectively when compared to five other receptor tyrosine kinases and several phosphorylated substrates. The serum is compatible with tissue-based assays since it detected tyrosine phosphorylation of the epidermal growth factor receptor in immunofluorescence experiments on permeabilized cells. The generality of the procedures used means that similar anti-tyrosine phosphopeptide sera can be produced that recognize other tyrosine kinases and substrates. Such sera will have numerous applications in research and clinical settings.
The neu proto-oncogene encodes a receptor tyrosine kinase (p185) that is closely related to the epidermal growth factor receptor. It has been proposed that receptor tyrosine kinases are activated through oligomerization. Because this clustering model predicts that oligomerization of receptors is sufficient to activate them, we determined if p185 can be activated by introducing an extra cysteine proximal to the transmembrane domain. This should induce inter-receptor disulfide bonding and, according to the clustering model, activate the receptor. This amino acid substitution enhanced recovery of both normal and transforming neu proteins as dimers, with normal p185 recovered predominantly as monomers and transforming p185* as dimers. However, the cysteine substitution did not affect the transforming activity of the two proteins.
The neu proto-oncogene encodes a protein highly homologous to the epidermal growth factor receptor. The neu protein (p185) has a molecular weight of 185,000 Daltons and, like the EGF receptor, possesses tyrosine kinase activity. neu is activated in chemically induced rat neuro/glioblastomas by substitution of valine 664 with glutamic acid within the transmembrane domain. The activated neu* protein (p185*) has an elevated tyrosine kinase activity and a higher propensity to dimerize, but the mechanism of this activation is still unknown. We have used site-directed mutagenesis to explore the role of specific amino acids within the transmembrane domain in this activation. We found that the lateral position and rotational orientation of the glutamic acid in the transmembrane domain does not correlate with transformation. However, the primary structure in the vicinity of Glu664 plays a significant role in this activation. Our results suggest that the Glu664 activation involves highly specific interactions in the transmembrane domain of p185.
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A Saccharomyces cerevisiae lambda gt11 library was screened with antiphosphotyrosine antibodies in an attempt to identify a gene encoding a tyrosine kinase. A subclone derived from one positive phage was sequenced and found to contain an 821-amino-acid open reading frame that encodes a protein with homology to protein kinases. We tested the activity of the putative kinase by constructing a vector encoding a glutathione-S-transferase fusion protein containing most of the predicted polypeptide. The fusion protein phosphorylated endogenous substrates and enolase primarily on serine and threonine. The gene was designated SPK1 for serine-protein kinase. Expression of the Spk1 fusion protein in bacteria stimulated serine, threonine, and tyrosine phosphorylation of bacterial proteins. These results, combined with the antiphosphotyrosine immunoreactivity induced by the kinase, indicate that Spk1 is capable of phosphorylating tyrosine as well as phosphorylating serine and threonine. In in vitro assays, the fusion protein kinase phosphorylated the synthetic substrate poly(Glu/Tyr) on tyrosine, but the activity was weak compared with serine and threonine phosphorylation of other substrates. To determine if other serine/threonine kinases would phosphorylate poly(Glu/Tyr), we tested calcium/calmodulin-dependent protein kinase II and the catalytic subunit of cyclic AMP-dependent protein kinase. The two kinases had similar tyrosine-phosphorylating activities. These results establish that the functional difference between serine/threonine- and tyrosine-protein kinases is not absolute and suggest that there may be physiological circumstances in which tyrosine phosphorylation is mediated by serine/threonine kinases.
P185 is a receptor-like protein encoded by the neu/erbB-2 proto-oncogene. A point mutation in the transmembrane domain renders this protein oncogenic. We report here that incubation of cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulates the phosphorylation of the normal neu protein (p185) and the oncogenic neu protein (p185*). The increased phosphorylation occurs mainly on serine and threonine residues. Phosphate labeling experiments showed that TPA causes a reduction of basal phosphotyrosine in p185 but not p185*. Immunoblotting with antiphosphotyrosine antibody yielded similar results. TPA also inhibited tyrosine phosphorylation of p185* in an in vitro immune complex kinase assay. These data suggest that protein kinase C, the receptor for TPA, regulates p185 function through serine or threonine phosphorylation.