Cloning of PI3 kinase-associated p85 utilizing a novel method for expression/cloning of target proteins for receptor tyrosine kinases.

A novel method has been developed to allow cloning of protein targets for receptors with tyrosine kinase activity. By utilizing the carboxy-terminal tail of EGF receptor (EGFR) as a probe to screen lambda gt11 expression libraries, several EGFR-binding proteins have been cloned; two have been analyzed and contain unique SH2 and SH3 domains. One gene (GRB-1) has been fully sequenced, is expressed in various tissues and cell lines, and has a molecular mass of 85 kd. Interestingly, GRB-1 encodes the human counterpart of the PI3 kinase-associated protein p85. Advantages of this technique include the ease of cloning tyrosine kinase receptor targets present at low levels and the ability to identify proteins that are related in their capacity to bind activated receptors but contain no significant DNA sequence homology. This method, termed CORT (for cloning of receptor targets), offers a general approach for the identification and cloning of various receptor targets.     

Disposition of fucose in brain

Abstract— Labelled fucose administered to rats in vivo was rapidly incorporated into brain glycoproteins, but not into any other brain constituents, including glycolipids and acid mucopolysaccharides. Maximum incorporation of tritium-labelled fucose into brain glyco-proteins occurred 3–6 h after intraperitoneal injection in young or adult rats, and the half-time for the turnover of glycoprotein-fucose in young rats was approximately 2 weeks. Within 3 h after the administration of either [1-³H]fucose or fucose generally labelled with tritium, 75 per cent of the total acid-soluble radioactivity in plasma and brain was found to be volatile, and by 24 h after injection more than 90 per cent of the acid-soluble radioactivity was volatile. The tritium in labelled fiicose appears to undergo arapid exchange reaction with hydrogen atoms in body water, although the tritium in fucose glycosidically- linked to glycoproteins is biologically stable. The rapid disappearance of labelled free fucose from the plasma and tissues of the rat precludes the possibility of any significant degree of reutilization of labelled precursor, and provides support for other data indicating that the turnover of fucose in brain glycoproteins is relatively slow in comparison to that of hexosamine and sialic acid. Activities of α-L-fucosidase in rat brain, with pH optima at 40 and 6.0, had essentially the same K_m (4 × 10^?4 M and 3.2 × 10^?4 M, respectively) with p-nitrophenyl-α-L-fucopyranoside as substrate. Activities of both were competitively inhibited by L-fucose. However, the K_t measured at pH 4 (1.9 × 10^?2) was almost ten times greater than that measured at pH 6 (1.5 × 10^?4).     

一种新的肝细胞生成素（HPO）转录本及其生物学活性

利用 5′RACE技术从人胎肝组织中分离一种新形式的肝细胞生成素 (HPO 2 0 5 )cDNA ,其编码蛋白质氨基酸序列的N端较已报道的人肝细胞生成素HPO(hepatopoietin)多 80个氨基酸 ,推测其蛋白质分子量为 2 3kD。RT PCR检测HPOmRNA在多种肝癌细胞中表达 ,Western印迹可检测到 2 3kDHPO 2 0 5表达 ,表明此种形式HPO在自然状态下存在。将构建的HPO 2 0 5真核表达载体转染入COS 7细胞 ,其表达蛋白质能够刺激HepG2肝癌细胞DNA合成 ;将HPO 2 0 5、HPO和荷空表达载体分别转染入低水平表达HPO的Bel 740 2肝癌细胞株 ,发现HPO 2 0 5比HPO具有较强的激活MAPK磷酸化的活性。细胞周期分析稳定转染HPO 2 0 5 ,HPO细胞的增殖周期也支持这一结论。这些结果表明HPO 2 0 5具有刺激肝源性细胞增殖的活性 ,并提示HPO 2 0 5可能较HPO有更强的生物学活性     

Interferon and Interferon Inducers in the Treatment of Malignancies

The mechanism of the antitumor action of polyinosinic-polycytidylic acid is probably multifaceted. The compound induces the synthesis of interferon, and interferon probably is active against some tumors. Poly I:poly C alters protein and RNA synthesis in tissue culture. It specifically inhibits such macromolecule synthesis in tumors in vivo, while having less inhibitory action on synthesis in normal organs, or it may actually enhance. Finally, poly I:poly C strongly enhances graft vs. host rejection mechanisms, which may play a role in the rejection of some tumors.     

CENP-A is essential for cardiac progenitor cell proliferation

Centromere protein A (CENP-A) is a homolog of histone H3 that epigenetically marks the heterochromatin of chromosomes. CENP-A is a critical component of the cell cycle machinery that is necessary for proper assembly of the mitotic spindle. However, the role of CENP-A in the heart and cardiac progenitor cells (CPCs) has not been previously studied. This study shows that CENP-A is expressed in CPCs and declines with age. Silencing CENP-A results in a decreased CPC growth rate, reduced cell number in phase G₂/M of the cell cycle, and increased senescence associated β-galactosidase activity. Lineage commitment is not affected by CENP-A silencing, suggesting that cell cycle arrest induced by loss of CENP-A is a consequence of senescence and not differentiation. CENP-A knockdown does not exacerbate cell death in undifferentiated CPCs, but increases apoptosis upon lineage commitment. Taken together, these results indicate that CPCs maintain relatively high levels of CENP-A early in life, which is necessary for sustaining proliferation, inhibiting senescence, and promoting survival following differentiation of CPCs.     

Changes in the subcellular localization of replication initiation proteins and cell cycle proteins during G1- to S-phase transition in mammalian cells

DNA replication in eukaryotic cells is restricted to the S-phase of the cell cycle. In a cell-free replication model system, using SV40 origin-containing DNA, extracts from G1 cells are inefficient in supporting DNA replication. We have undertaken a detailed analysis of the subcellular localization of replication proteins and cell cycle regulators to determine when these proteins are present in the nucleus and therefore available for DNA replication. Cyclin A and cdk2 have been implicated in regulating DNA replication, and may be responsible for activating components of the DNA replication mitiation complex on entry into S-phase. G1 cell extracts used for in vitro replication contain the replication proteins RPA (the eukaryotic single-stranded DNA binding protein) and DNA polymerase as well as cdk2, but lack cyclin A. On localizing these components in G1 cells we find that both RPA and DNA polymerase are present as nuclear proteins, while cdk2 is primarily cytoplasmic and there is no detectable cyclin A. An apparent change in the distribution of these proteins occurs as the cell enters S-phase. Cyclin A becomes abundant and both cyclin A and cdk2 become localized to the nucleus in S-phase. In contrast, the RPA-34 and RPA-70 subunits of RPA, which are already nuclear, undergo a transition from the uniform nuclear distribution observed during G1, and now display a distinct punctate nuclear pattern. The initiation of DNA replication therefore most likely occurs by modification and activation of these replication initiation proteins rather than by their recruitment to the nuclear compartment.     

Extractability of glycoproteins and mucopolysaccharides of brain

Very little is known about the localization and functions of the glycoproteins and mucopolysaccharides of nervous tissue. There have been two major approaches to the study of these substances in brain. The first involves the isolation of glycopeptides and mucopolysaccharides after digestion of the lipid-free protein residue from whole brain with proteolytic enzymes (Margolis , 1967; Di Benedetta et al., 1969; Margolis and Margolis , 1970; Katzman , 1972). This approach has the advantage that sufficient tissue is used to permit analysis of the structure and metabolism of the carbohydrate components of these macromolecules. However, any differentiation of the various glycoproteins and mucopolysaccharides based on such features as their anatomical location, association with proteins, lipids or other membrane components, and the properties conferred by their non-carbohydrate portion, is unavoidably lost as a consequence of the procedures used for their isolation. On the other hand, several laboratories have attempted to study the glycoproteins and mucopolysaccharides of nervous tissue by treating brain (or subcellular fractions) with various detergents, and then examining the extracts for the pattern of separation obtained by polyacrylamide gel electrophoresis in terms of carbohydrate staining reactions or the incorporation of labelled precursors (Bosmann , Case and Shea , 1970; Duiton and Barondes , 1970; Quarles and Brady , 1971; Waehneldt , Morgan and Gombos , 1971). This approach has the advantages of relatively high sensitivity and the ability to study intact glycoproteins rather than glycopeptides produced by proteolytic enzyme digestion. However, it is presently impossible to identify any of the numerous and often poorly resolved bands thus obtained with glycoproteins or mucopolysaccharides of known structure and chemical composition, or in many cases even to identify the various complex carbohydrates as being glycoproteins, glycolipids or acid mucopolysaccharides. In an attempt to obtain some indication of the degree of anatomical heterogeneity of these compounds in nervous tissue, we have sequentially treated whole rat brain with several solvents to obtain intact glycoproteins and mucopolysaccharides. After removal of lipids and digestion with pronase, the composition of the glycopeptides and mucopolysaccharides has been analyzed.     

Effects of phosphorylation on the self-assembly of native full-length porcine amelogenin and its regulation of calcium phosphate formation in vitro

The self-assembly of the predominant extracellular enamel matrix protein amelogenin plays an essential role in regulating the growth and organization of enamel mineral during early stages of dental enamel formation. The present study describes the effect of the phosphorylation of a single site on the full-length native porcine amelogenin P173 on self-assembly and on the regulation of spontaneous calcium phosphate formation in vitro. Studies were also conducted using recombinant non-phosphorylated (rP172) porcine amelogenin, along with the most abundant amelogenin cleavage product (P148) and its recombinant form (rP147). Amelogenin self-assembly was assessed using dynamic light scattering (DLS) and transmission electron microscopy (TEM). Using these approaches, we have shown that self-assembly of each amelogenin is very sensitive to pH and appears to be affected by both hydrophilic and hydrophobic interactions. Furthermore, our results suggest that the phosphorylation of the full-length porcine amelogenin P173 has a small but potentially important effect on its higher-order self-assembly into chain-like structures under physiological conditions of pH, temperature, and ionic strength. Although phosphorylation has a subtle effect on the higher-order assembly of full-length amelogenin, native phosphorylated P173 was found to stabilize amorphous calcium phosphate for extended periods of time, in sharp contrast to previous findings using non-phosphorylated rP172. The biological relevance of these findings is discussed.     

Interaction of phosphatidylinositol 3-kinase-associated p85 with epidermal growth factor and platelet-derived growth factor receptors.

One of the immediate cellular responses to stimulation by various growth factors is the activation of a phosphatidylinositol (PI) 3-kinase. We recently cloned the 85-kDa subunit of PI 3-kinase (p85) from a lambda gt11 expression library, using the tyrosine-phosphorylated carboxy terminus of the epidermal growth factor (EGF) receptor as a probe (E. Y. Skolnik, B. Margolis, M. Mohammadi, E. Lowenstein, R. Fischer, A. Drepps, A. Ullrich, and J. Schlessinger, Cell 65:83-90, 1991). In this study, we have examined the association of p85 with EGF and platelet-derived growth factor (PDGF) receptors and the tyrosine phosphorylation of p85 in 3T3 (HER14) cells in response to EGF and PDGF treatment. Treatment of cells with EGF or PDGF markedly increased the amount of p85 associated with EGF and PDGF receptors. Binding assays with glutathione S-transferase (GST) fusion proteins demonstrated that either Src homology region 2 (SH2) domain of p85 is sufficient for binding to EGF and PDGF receptors and that receptor tyrosine autophosphorylation is required for binding. Binding of a GST fusion protein expressing the N-terminal SH2 domain of p85 (GST-N-SH2) to EGF and PDGF receptors was half-maximally inhibited by 2 and 24 mM phosphotyrosine (P-Tyr), respectively, suggesting that the N-SH2 domain interacts more stably with PDGF receptors than with EGF receptors. The amount of receptor-p85 complex detected in HER14 cells treated with EGF or PDGF. Growth factor treatment also increased the amount of p85 found in anti-PDGF-treated HER14 cells, suggesting that the vast majority of p85 in the anti-P-Tyr fraction is receptor associated but not phosphorylated on tyrosine residues. Only upon transient overexpression of p85 and PDGF receptor did p85 become tyrosine phosphorylated. These are consistent with the hypothesis that p85 functions as an adaptor molecule that targets PI 3-kinase to activated growth factor receptors.