Tyrosine kinase inhibitor emodin suppresses growth of HER-2/neu-overexpressing breast cancer cells in athymic mice and sensitizes these cells to the inhibitory effect of paclitaxel.
(17/13395)
Overexpression of the HER-2/neu proto-oncogene, which encodes the tyrosine kinase receptor p185neu, has been observed in tumors from breast cancer patients. We demonstrated previously that emodin, a tyrosine kinase inhibitor, suppresses tyrosine kinase activity in HER-2/neu-overexpressing breast cancer cells and preferentially represses transformation phenotypes of these cells in vitro. In the present study, we examined whether emodin can inhibit the growth of HER-2/neu-overexpressing tumors in mice and whether emodin can sensitize these tumors to paclitaxel, a commonly used chemotherapeutic agent for breast cancer patients. We found that emodin significantly inhibited tumor growth and prolonged survival in mice bearing HER-2/neu-overexpressing human breast cancer cells. Furthermore, the combination of emodin and paclitaxel synergistically inhibited the anchorage-dependent and -independent growth of HER-2/neu-overexpressing breast cancer cells in vitro and synergistically inhibited tumor growth and prolonged survival in athymic mice bearing s.c. xenografts of human tumor cells expressing high levels of p185neu. Both immunohistochemical staining and Western blot analysis showed that emodin decreases tyrosine phosphorylation of HER-2/neu in tumor tissue. Taken together, our results suggest that the tyrosine kinase activity of HER-2/neu is required for tumor growth and chemoresistance and that tyrosine kinase inhibitors such as emodin can inhibit the growth of HER-2/neu-overexpressing tumors in mice and also sensitize these tumors to paclitaxel. The results may have important implications in chemotherapy for HER-2/neu-overexpressing breast tumors. (+info)
Tyrosine 319, a newly identified phosphorylation site of ZAP-70, plays a critical role in T cell antigen receptor signaling.
(18/13395)
Following T cell antigen receptor (TCR) engagement, the protein tyrosine kinase (PTK) ZAP-70 is rapidly phosphorylated on several tyrosine residues, presumably by two mechanisms: an autophosphorylation and a trans-phosphorylation by the Src-family PTK Lck. These events have been implicated in both positive and negative regulation of ZAP-70 activity and in coupling this PTK to downstream signaling pathways in T cells. We show here that Tyr315 and Tyr319 in the interdomain B of ZAP-70 are autophosphorylated in vitro and become phosphorylated in vivo upon TCR triggering. Moreover, by mutational analysis, we demonstrate that phosphorylation of Tyr319 is required for the positive regulation of ZAP-70 function. Indeed, overexpression in Jurkat cells and in a murine T cell hybridoma of a ZAP-70 mutant in which Tyr319 was replaced by phenylalanine (ZAP-70-Y319F) dramatically impaired anti-TCR-induced activation of the nuclear factor of activated T cells and interleukin-2 production, respectively. Surprisingly, an analogous mutation of Tyr315 had little or no effect. The inhibitory effect of ZAP-70-Y319F correlated with a substantial loss of its activation-induced tyrosine phosphorylation and up-regulation of catalytic activity, as well as with a decreased in vivo capacity to phosphorylate known ZAP-70 substrates, such as SLP-76 and LAT. Collectively, our data reveal the pivotal role of Tyr319 phosphorylation in the positive regulation of ZAP-70 and in TCR-mediated signaling. (+info)
Vascular endothelial growth factor receptor KDR tyrosine kinase activity is increased by autophosphorylation of two activation loop tyrosine residues.
(19/13395)
Vascular endothelial growth factor is an important physiological regulator of angiogenesis. The function of this endothelial cell selective growth factor is mediated by two homologous tyrosine kinase receptors, fms-like tyrosine kinase 1 (Flt-1) and kinase domain receptor (KDR). Although the functional consequence of vascular endothelial growth factor binding to the Flt-1 receptor is not fully understood, it is well established that mitogenic signaling is mediated by KDR. Upon sequencing several independent cDNA clones spanning the cytoplasmic region of human KDR, we identified and confirmed the identity of a functionally required valine at position 848 in the ATP binding site, rather than the previously reported glutamic acid residue, which corresponds to an inactive tyrosine kinase. The cytoplasmic domain of recombinant native KDR, expressed as a glutathione S-transferase fusion protein, can undergo autophosphorylation in the presence of ATP. In addition, the kinase activity can be substantially increased by autophosphorylation at physiologic ATP concentrations. Mutation analysis indicates that both tyrosine residues 1054 and 1059 are required for activation, which is a consequence of an increased affinity for both ATP and the peptide substrate and has no effect on kcat, the intrinsic catalytic activity of the enzyme. KDR kinase catalyzes phosphotransfer by formation of a ternary complex with ATP and the peptide substrate. We demonstrate that tyrosine kinase antagonists can preferentially inhibit either the unactivated or activated form of the enzyme. (+info)
Similarities and differences in RANTES- and (AOP)-RANTES-triggered signals: implications for chemotaxis.
(20/13395)
Chemokines are a family of proinflammatory cytokines that attract and activate specific types of leukocytes. Chemokines mediate their effects via interaction with seven transmembrane G protein-coupled receptors (GPCR). Using CCR5-transfected HEK-293 cells, we show that both the CCR5 ligand, RANTES, as well as its derivative, aminooxypentane (AOP)- RANTES, trigger immediate responses such as Ca2+ influx, receptor dimerization, tyrosine phosphorylation, and Galphai as well as JAK/STAT association to the receptor. In contrast to RANTES, (AOP)-RANTES is unable to trigger late responses, as measured by the association of focal adhesion kinase (FAK) to the chemokine receptor complex, impaired cell polarization required for migration, or chemotaxis. The results are discussed in the context of the dissociation of the late signals, provoked by the chemokines required for cell migration, from early signals. (+info)
A novel epitope for the specific detection of exogenous prion proteins in transgenic mice and transfected murine cell lines.
(21/13395)
Prion diseases are closely linked to the conversion of host-encoded cellular prion protein (PrPC) into its pathological isoform (PrPSc). PrP conversion experiments in scrapie infected tissue culture cells, transgenic mice, and cell-free systems usually require unique epitopes and corresponding monoclonal antibodies (MAbs) for the immunological discrimination of exogenously introduced and endogenous PrP compounds (e.g., MAb 3F4, which is directed to an epitope on hamster and human but not on murine PrP). In the current work, we characterize a novel MAb designated L42 that reacts to PrP of a variety of species, including cattle, sheep, goat, dog, human, cat, mink, rabbit, and guinea pig, but does not bind to mouse, hamster, and rat PrP. Therefore, MAb L42 may allow future in vitro conversion and transgenic studies on PrPs of the former species. The MAb L42 epitope on PrPC includes a tyrosine residue at position 144, whereas mouse, rat, and hamster PrPs incorporate tryptophane at this site. To verify this observation, we generated PrP expression vectors coding for authentic or mutated murine PrPCs (i.e., codon 144 encoding tyrosine instead of tryptophan). After transfection into neuroblastoma cells, MAb L42 did not react with immunoblotted wild-type murine PrPC, whereas L42 epitope-tagged murine PrPC was strongly recognized. Immunoblot and fluorescence-activated cell sorting data revealed that tagged PrPC was correctly posttranslationally processed and translocated to the cell surface. (+info)
Unexpected crucial role of residue 225 in serine proteases.
(22/13395)
Residue 225 in serine proteases of the chymotrypsin family is Pro or Tyr in more than 95% of nearly 300 available sequences. Proteases with Y225 (like some blood coagulation and complement factors) are almost exclusively found in vertebrates, whereas proteases with P225 (like degradative enzymes) are present from bacteria to human. Saturation mutagenesis of Y225 in thrombin shows that residue 225 affects ligand recognition up to 60,000-fold. With the exception of Tyr and Phe, all residues are associated with comparable or greatly reduced catalytic activity relative to Pro. The crystal structures of three mutants that differ widely in catalytic activity (Y225F, Y225P, and Y225I) show that although residue 225 makes no contact with substrate, it drastically influences the shape of the water channel around the primary specificity site. The activity profiles obtained for thrombin also suggest that the conversion of Pro to Tyr or Phe documented in the vertebrates occurred through Ser and was driven by a significant gain (up to 50-fold) in catalytic activity. In fact, Ser and Phe are documented in 4% of serine proteases, which together with Pro and Tyr account for almost the entire distribution of residues at position 225. The unexpected crucial role of residue 225 in serine proteases explains the evolutionary selection of residues at this position and shows that the structural determinants of protease activity and specificity are more complex than currently believed. These findings have broad implications in the rational design of enzymes with enhanced catalytic properties. (+info)
Structural identification of sulfated tyrosine in human urine.
(23/13395)
A reliable HPLC method was used for the identification of positional isomerism and stereoisomerism of sulfated tyrosine residues in human urine. Upon separation of human urine by ion-pair HPLC on a reverse-phase column, p-tyrosine-O-sulfate (p-TyrS) was identified. Differentiation of the L and D forms was done by using a column with a chiral stationary phase. It was concluded that L-p-tyrosine (L-p-Tyr) which is the predominant tyrosine isomer in the human body, was sulfated and excreted in human urine as a normal constituent. The sulfated forms of D-p-Tyr and m-Tyr could not be detected under these analytical conditions. (+info)
Metabolic stabilization of muscle nicotinic acetylcholine receptor by rapsyn.
(24/13395)
Although the metabolic half-life of muscle endplate acetylcholine receptor (AChR) changes during development and after denervation in the adult, little is known about the molecular mechanisms that influence receptor stability. We have investigated the effect on AChR turnover of its interaction with rapsyn, a 43 kDa peripheral membrane protein that is closely associated with the AChR in muscle cells and is required for its clustering at endplates. Both in transfected COS cells and in cultured myotubes from rapsyn-negative and rapsyn-positive mice, we have found that the presence of rapsyn slows the turnover of AChRs by as much as twofold. The effect was similar for both embryonic (alpha2betadeltagamma) and adult (alpha2betadeltaepsilon) AChRs and for AChRs whose beta subunit lacked a putative tyrosine phosphorylation site. Neither colchicine nor cytochalasin D altered AChR turnover or prevented the rapsyn effect. Mutant rapsyn proteins whose N-terminal myristoylation signal was eliminated, or whose C terminus or zinc-finger domains were deleted, failed to change the rate of receptor turnover. Each of these mutations affects the association of the AChR with rapsyn, suggesting that AChR stability is altered by interaction between the two proteins. Our results suggest that, in addition to its role in AChR clustering, rapsyn also functions to metabolically stabilize the AChR. (+info)