Donna Boland

A Panel of CAb Antibodies Recognize Endogenous and Ectopically Expressed ING1 Protein

Nine monoclonal antibodies (MAbs) against human and rodent ING1 protein have been generated using an IL6-secreting mouse myeloma line. These antibodies are all effective in recognizing ING1 protein in ELISAs, Western blot assays, and by indirect immunofluorescence. Combining different CAb monoclonal antibodies in a Western blot assay also allows detection of the very low levels of endogenous ING1 found in fibroblast cells in culture and the identification of at least two isoforms of ING1 in normal human diploid fibroblasts and established brain cancer cell lines.

Binding and structural properties of oxytocin receptors in isolated rat epididymal adipocytes

Oxytocin initiates its insulin-like action in adipocytes through oxytocin-specific receptors. We have studied binding and structural properties of these receptors with the radioligand [3H]oxytocin. Steady-state binding was reached after 45 min, at 21 degrees C, and 10 min at 37 degrees C. Scatchard analyses of equilibrium binding data indicated a single class of oxytocin binding sites at 21 degrees C (KD = 3.3 nM, RT = 6 X 10(4) sites/cell) and 2 binding sites at 37 degrees C (KD = 1.5 nM, RT = 6 X 10(4) sites/cell; and KD = 20 nM, RT = 30 X 10(4) sites/cell). Insulin, insulin-like growth factor I, and epidermal growth factor increased oxytocin binding (approximately 20-40%), whereas adenosine, a regulator of oxytocin action, did not affect oxytocin binding. Binding activity of oxytocin was impaired by pretreatment of the hormone or adipocytes with dithiothreitol. Dithiothreitol treatment of adipocytes preferentially inactivated high-affinity binding sites. N-ethyl maleimide inhibited oxytocin binding in adipocytes more than dithiothreitol. In contrast to the inhibitory effects of dithiothreitol and N-ethyl maleimide, proteases (trypsin, chymotrypsin and papain) were not able to inhibit fat cell binding activity. These results suggested that in isolated adipocytes: there are high-affinity and low-affinity receptors, but the low-affinity receptors are absent at 21 degrees C; the binding of oxytocin can be regulated by insulin, and growth factors; and the oxytocin receptors contain disulfide bridges and free thiols that are essential for the maintenance of oxytocin binding.

The 180000 molecular weight plasma membrane insulin receptor substrate is a protein tyrosine phosphatase and is elevated in diabetic plasma membranes

Wheat germ agglutinin-purified non-diabetic and diabetic human placenta membranes were or were not depleted of EGF receptor with monoclonal anti-EGF receptor antibody B1D8, and subsequently phosphorylated. Phosphorylated insulin receptor beta-subunit was lower and pp180 was higher in diabetic placenta membranes than in non-diabetic membranes. Phosphorylated-beta-subunit was also lower in diabetic (streptozotocin-induced) rat liver whereas the amount of pp180 was dependent on membrane protein concentration. When rat liver tyrosine-phosphorylated proteins were incubated 30 min, 4 degrees C with EDTA-terminated 32P-phosphorylation reaction mixtures of wheat germ agglutinin-purified rat liver proteins, less phosphorylated proteins were immunoprecipitated with antiphosphotyrosine. The decrease in tyrosine-phosphorylated products suggested that pp180 was a protein tyrosine phosphatase. Taken together, the results suggest that diabetic plasma membranes contain more tyrosine phosphatase than non-diabetic membranes.

Domain recognition of the ING1 tumor suppressor by a panel of monoclonal antibodies.

The inhibitor of growth (ING) family of proteins play key roles in cell cycle arrest, apoptosis, cell aging, and the DNA damage response. To date, several domains including the plant homeodomain (PHD), lamin interacting domain (LID), and nuclear localization sequence (NLS) have been identified in the ING family of proteins that contribute to their function. To better understand the functional attributes of the ING proteins, we have developed and further characterized a panel of monoclonal IgGs that we call CAbs 1-9 based on their recognition sites, strength of binding affinity, and their specificity for ING1. All of the nine CAbs recognize the C-terminal half of the p33(ING1b) protein, which is fully conserved among all ING1 isoforms, being encoded by a common exon. Two of the nine CAbs bind a fragment that includes the PHD, which is the most conserved domain among ING family proteins (ING1-5), and one CAb cross-reacts with all ING family proteins that are encoded by different genes. Five of the nine CAbs recognized a fragment of ING1, which includes the NLS. Another two, CAb3 and CAb9, show affinity against an inter-domain sequence between the LID and the NLS. The sequence between the LID and NLS is less conserved among the ING proteins and, as expected, CAbs 3 and 9 were completely specific for ING1. Understanding the domains recognized by the different CAbs should further the functional analysis of the ING proteins that are known to participate in a wide variety of protein complexes, both in the cytoplasm and in the nucleus where they bind epigenetic histone marks via their PHD regions and lamin A via their LID domains.

ING3 protein expression profiling in normal human tissues suggest its role in cellular growth and self-renewal.

Members of the INhibitor of Growth (ING) family of proteins act as readers of the epigenetic code through specific recognition of the trimethylated form of lysine 4 of histone H3 (H3K4Me3) by their plant homeodomains. The founding member of the family, ING1, was initially identified as a tumor suppressor with altered regulation in a variety of cancer types. While alterations in ING1 and ING4 levels have been reported in a variety of cancer types, little is known regarding ING3 protein levels in normal or transformed cells due to a lack of reliable immunological tools. In this study we present the characterization of a new monoclonal antibody we have developed against ING3 that specifically recognizes human and mouse ING3. The antibody works in western blots, immunofluorescence, immunoprecipitation and immunohistochemistry. Using this antibody we show that ING3 is most highly expressed in small intestine, bone marrow and epidermis, tissues in which cells undergo rapid proliferation and renewal. Consistent with this observation, we show that ING3 is expressed at significantly higher levels in proliferating versus quiescent epithelial cells. These data suggest that ING3 levels may serve as a surrogate for growth rate, and suggest possible roles for ING3 in growth and self renewal and related diseases such as cancer.

In vitro, insulin receptor catalyses phosphorylation of clathrin heavy chain and a plasma membrane 180,000 molecular weight protein

Insulin receptor mutation studies indicate that the receptor tyrosine kinase activity is necessary for receptor endocytosis, and several insulin receptor-containing tissues have a plasma membrane-associated protein (Mr congruent to 180,000, p180) whose tyrosine phosphorylation is receptor catalysed. Since clathrin heavy chain (Mr congruent to 180,000 in dodecyl sulphate gel electrophoresis) is a major component of coated vesicles, the latter functioning in receptor endocytosis, we investigated whether insulin receptors can catalyse clathrin phosphorylation and whether p180 is clathrin. Bovine brain triskelion or coated vesicles and 32P-ATP were added to prephosphorylated insulin receptor preparations (wheat germ agglutinin-purified human placenta membrane proteins). Antiphosphotyrosine immunoprecipitated a phosphorylated 180,000 molecular weight protein. Insulin (10(-7) M) increased the rate of phosphorylation. Monoclonal anti-clathrin antibody immunoprecipitated the phosphorylated 180,000 molecular weight protein, whereas monoclonal anti-insulin receptor antibodies (alpha-IR1, MA10) immunoprecipitated both insulin receptors and the phosphorylated 180,000 molecular weight protein. In the absence of added clathrin, anticlathrin immunoprecipitated no proteins, and alpha-IR1 immunoprecipitated only the insulin receptor. Density gradient (glycerol 7.5-30%, w/v) centrifugation separated human placenta microsomal membrane proteins into endosomal, plasma membrane, cytoplasmic and coated vesicle fractions. Antiphosphotyrosine immunoprecipitated phosphorylated-microsomal proteins that centrifugated into endosomal and plasma membrane fractions. Addition of glycerol gradient fractions to a prephosphorylated insulin receptor preparation, however, gave a tyrosine-phosphorylated 180,000 molecular weight protein when cytoplasmic and coated vesicle fractions were added. Taken together these results suggest: (1) that, in vitro, human placenta insulin receptors can phosphorylate bovine brain and human placenta clathrin heavy chain; (2) that both assembled and unassembled clathrin can be phosphorylated; and (3) that p180, the plasma membrane-associated insulin receptor substrate, is not clathrin heavy chain.

A 180,000 molecular weight glycoprotein substrate of the insulin receptor tyrosine kinase is present in human placenta and in rat liver, muscle, heart and brain plasma membrane preparations

Cell signalling for insulin may include insulin receptor tyrosine kinase catalysing the phosphorylation of one or more cell proteins. Since temporally the insulin receptor will encounter plasma membrane proteins first, we have studied the in vitro phosphorylation of purified plasma membrane preparations. Two proteins were immunoprecipitated with anti-phosphotyrosine antibody from rat liver, muscle, heart and brain membranes and from human placenta membranes: the insulin receptor (detected as a phosphorylated-beta-subunit) and a 180,000 molecular weight protein (pp180). pp180 is a monomeric glycoprotein that in the absence of dithiothreitol migrated in denaturing gels like a 150,000 molecular weight protein. pp180 was a substrate for the insulin receptor: (i) receptor and pp180 phosphorylation followed a similar insulin dose-response, although fold-stimulation of autophosphorylation was greater; and (ii) removal of insulin receptors with monoclonal antibodies prevented subsequent pp180 phosphorylation. Insulin-activated receptors increased the extent, but not the rate, of pp180 phosphorylation; the increased phosphate was incorporated into tyrosine and appeared to do so in three or four of pp180s 12 tryptic phosphopeptides. Some data suggest that pp180 is the same protein in each of the tested tissues. The occurrence of pp180, an insulin receptor substrate, in plasma membranes of several insulin responsive tissues suggests that it has a role in insulin signalling.

In the absence of antibody IgGsorb precipitates human placenta phosphotyrosine-containing proteins

Lectin-purified human placenta plasma membrane proteins were phosphorylated in vitro. Mixing the reaction mixture with IgGsorb and incubation of the resultant pellet with p-nitrophenyl phosphate demonstrated the presence of phosphorylated-insulin receptor beta-subunit and a phosphorylated-180 kDa protein in acrylamide gel electrophoresis. The same two proteins were detected in the electrophoretic analyses of anti-phosphotyrosine immunoprecipitated phosphorylation reaction mixtures. In the absence of antibody, the amount of phosphorprotein in the IgGsorb pellet was dependent on the amount of IgGsorb added. IgGsorb did not precipitate 125I-labeled lectin-purified human placenta protein. Further, 10 mM O-phosphotyrosine completely inhibited the precipitation of phosphorylated human placenta proteins. These data suggest that IgGsorb specifically bound and precipitated phosphotyrosine-containing proteins in soluble human placenta plasma membranes.

REVERSE PHASE CHROMATOGRAPHY OF TRYPSIN DIGESTS OF A PLASMA MEMBRANE AND A CYTOPLASMIC INSULIN RECEPTOR SUBSTRATE

The 180,000 molecular weight protein from [32P]phosphorylated wheat germ agglutinin-purified rat liver plasma membranes was digested with trypsin. NIH 3T3 HIR 3.5 cells were [32P]phosphate-labelled in the presence of 10(-7) M insulin, and the 185,000 molecular weight cytoplasmic protein was digested with trypsin. Digests were applied to a C18-mu Bondapak column, eluted with acetonitrile gradients, and radioactivity in the eluate was monitored. The chromatogram for the cytoplasmic protein was similar but not identical to chromatograms of trypsin digests of insulin receptor substrates from other cultured cells. Thirteen and seven phosphopeptides were obtained from the plasma membrane and cytoplasmic substrate, respectively. One phosphopeptide from the two digests eluted at the same acetonitrile concentration; however, dissimilarity in elution profiles and dissimilarity in relative yields of individual phosphopeptides, suggest that the primary structures of tyrosine phosphorylation sites in the two insulin receptor substrates are different.

Plasma membrane p180, which insulin receptor phosphorylates in vivo, is not a tyrosine kinase

The earliest substrates to the transmembrane insulin receptor tyrosine kinase, that would function in insulin signalling, are likely to be associated with the plasma membrane. Rat liver plasma membrane 180,000 M(r) protein (p180) is a substrate to the insulin receptor in vitro [Goren et al. (1990) Cellular Signalling 2, 537-555]. The question as to whether p180 is a substrate in vivo was addressed. Half ml 0.9% NaCl or 500 micrograms insulin was injected into rat livers. Purified plasma membrane glycoproteins from the livers were assayed for in vitro phosphorylation reaction products and endogenous tyrosine-phosphorylated proteins. Membranes from insulin-injected rat livers contained phosphorylated p180 and phosphorylated insulin receptor beta-subunit, whereas saline-injected rat liver membranes contained neither. These data suggested that p180 is an in vivo substrate to the insulin receptor. In vitro p180 is tyrosine-phosphorylated in the absence of insulin. p180, therefore, may be the epidermal growth factor (EGF) receptor or another tyrosine kinase that could be part of a phosphorylation cascade initiated by insulin. Two different experiments suggested that p180 is not the EGF receptor: (i) two-dimensional gel electrophoresis (first dimension--non-equilibrium pH-gradient gel electrophoresis) indicated that p180 is a more basic glycoprotein than EGF receptor; and (ii) based on reverse-phase high pressure liquid chromatography, the tryptic-phosphopeptides of carboxymethyl-Sepharose-purified phosphorylated-p180 were different from those of A431 cell phosphorylated-EGF receptor. Similarly, two different experiments demonstrated that p180 is not a tyrosine kinase: (i) gel-permeation chromatography separated the insulin receptor from p180 and only insulin receptor was autophosphorylated in vitro; and (ii) membrane proteins not bound to immobilized ATP contained p180. Thus, p180 can associate with the insulin receptor and be phosphorylated in vitro and in vivo; however, p180 does not function in an insulin receptor-mediated phosphorylation cascade.