Bovine fetuin is an inhibitor of insulin receptor tyrosine kinase

Fetuin has been identified earlier as the bovine homolog of the human plasma protein, alpha2-Heremans Schmid glycoprotein (alpha2-HSG). Although bovine fetuin shares over 70% amino acid sequence similarity with alpha2-HSG and rat fetuin, no common function(s) have been identified. We report that immunoaffinity purified bovine fetuin acts as an inhibitor of insulin receptor tyrosine kinase activity (IR-TKA) with half-maximal inhibition at 1.5 microM. In vitro, bovine fetuin (1.5 microM) blocked insulin-induced autophosphorylation of the human IR completely and the half-maximal inhibitory effect was observed at 0.5 microM. Incubation of HIRcB cells (rat1 fibroblasts transfected with wild-type human insulin receptor cDNA) with bovine fetuin (1.5 microM) inhibited insulin-induced tyrosine phosphorylation of the IR beta-subunit by 40%. In addition, bovine fetuin (2 microM) completely blocked insulin-stimulated DNA synthesis in H-35 rat hepatoma cells. Our results, together with earlier reports on rat fetuin and human alpha2-HSG, indicate a common IR-TK inhibitory function for fetuin homologs.     

ICAAR, a novel member of a new family of transmembrane, tyrosine phosphatase-like proteins

We have isolated a cDNA from human foetal brain cDNA library which encodes a putative transmembrane protein bearing an intracellular protein tyrosine phosphatase (PTPase) like domain. The PTPase like domain contains an alanine to aspartate amino acid change relative to other PTPases in the catalytic core domain. This amino acid change is found in only three other known proteins, islet cell autoantigens; human, murine and rat IA-2, murine IA-2b and its rat orthologue phogrin, which have a similar overall structure to ICAAR, and the recently identified X-linked myotubular myopathy (MTM1) gene. ICAAR, IA-2 and IA-2b clearly represent a new family of PTP-like proteins for which catalytic activity has yet to be demonstrated. An abundant ICAAR mRNA is detectable in the brain and pancreas but not in the other normal human tissues surveyed. We have localised ICAAR to human chromosome 7q36.     

p125Fak focal adhesion kinase is a substrate for the insulin and insulin-like growth factor-I tyrosine kinase receptors

The focal adhesion kinase p125(Fak) is a widely expressed cytosolic tyrosine kinase, which is involved in integrin signaling and in signal transduction of a number of growth factors. In contrast to tyrosine kinase receptors such as the platelet-derived growth factor and the hepatocyte growth factor receptors, which induce p125(Fak) phosphorylation, insulin has been shown to promote its dephosphorylation. In this study, we compared p125(Fak) phosphorylation in insulin-stimulated cells maintained in suspension or in an adhesion state. We found that, in nonattached cells, insulin promotes p125(Fak) phosphorylation, whereas dephosphorylation occurred in attached cells. This was observed in Rat-1 fibroblasts overexpressing the insulin receptor, as well as in Hep G2 hepatocytes and in 3T3-L1 adipocytes expressing more natural levels of insulin receptors. Insulin-induced p125(Fak) phosphorylation correlated with an increase in paxillin phosphorylation, indicating that p125(Fak) kinase activity may be stimulated by insulin. Mixing of purified insulin or insulin-like growth factor-I (IGF-I) receptors with p125(Fak) resulted in an increase in p125(Fak) phosphorylation. Using a kinase-deficient p125(Fak) mutant, we found that this protein is a direct substrate of the insulin and IGF-I receptor tyrosine kinases. This view is supported by two additional findings. (i) A peptide corresponding to p125(Fak) sequence comprising amino acids 568-582, which contains tyrosines 576 and 577 of the kinase domain regulatory loop, is phosphorylated by the insulin receptor; and (ii) p125(Fak) phosphorylation by the insulin receptor is prevented by addition of this peptide. Finally, we observed that p125(Fak) phosphorylation by the receptor results in its activation. Our results show that the nature of the cross-talk between the insulin/IGF-I receptors and p125(Fak) is dependent on the cell architecture, and hence the interaction of the insulin/IGF-I signaling system with the integrin system will vary accordingly.     

Multiple GPI-anchored receptors control GDNF-dependent and independent activation of the c-Ret receptor tyrosine kinase

The application of a newly developed thermal desorption method for the analysis of workplace air to the analysis of polar compounds is reported. The method was validated for both pumped and diffusive sampling of test gases containing polar volatile organic compounds (esters, alcohols, ketones or aldehydes) on adsorption tubes and subsequent analysis of these tubes. Carbosieve SIII, Carboxen 569, Carbopack B and Tenax TA were used as solid adsorbents. Analysis was performed by thermal desorption of the analytes from the adsorbent tubes followed by gas chromatography-flame ionisation detection (GC-FID). It could be demonstrated that thermal desorption-GC-FID is feasible also for the analysis of polar compounds and that problems arising from the high concentration levels of some analytes in workplace air could be solved.     

PTP-NP, a new member of the receptor protein tyrosine phosphatase family, implicated in development of nervous system and pancreatic endocrine cells

The regulation of protein tyrosine phosphorylation is an important mechanism for developmental control. We describe here a new member of the protein tyrosine phosphatase (PTP) family, called PTP-NP (for neural and pancreatic). The cDNA sequence indicates a receptor-type transmembrane molecule. At early organogenesis, in situ hybridization with a probe for the PTP-NP extracellular region detects expression confined to the region of the developing pancreas, an organ of medical importance, but poorly understood with regard to molecular mechanisms of developmental control. This localized expression appears early, even before morphological differentiation of the pancreas, and is found in presumptive precursors of the endocrine cells by the earliest times that they can be distinguished. In neural development, an alternate RNA with a different or missing extracellular region is expressed transiently at early stages of neurogenesis and the full-length PTP-NP RNA appears later. To search for a ligand of PTP-NP, a fusion protein probe was made with the extracellular domain fused to an alkaline phosphatase tag. This probe bound strongly to pancreatic islets, providing evidence for a ligand-receptor interaction that could be involved in endocrine cell regulation. The results show PTP-NP is an especially early marker for pancreatic development and suggest it may be a receptor that could control the development of pancreatic endocrine cells.     

Interaction of a receptor tyrosine kinase, EGF-R, with caveolins. Caveolin binding negatively regulates tyrosine and serine/threonine kinase activities

Caveolin, a 21-24-kDa integral membrane protein, is a principal component of caveolae membranes. We and others have suggested that caveolin functions as a scaffolding protein to organize and concentrate certain caveolin-interacting signaling molecules within caveolae membranes. In this regard, it has been shown that a 20-amino acid membrane-proximal region of the cytosolic NH2-terminal domain of caveolin is sufficient to mediate the interaction of caveolin with signaling proteins, namely G-proteins, Src-like kinases, eNOS, and H-Ras. This caveolin-derived protein domain has been termed the caveolin-scaffolding domain. Binding of the caveolin-scaffolding domain functionally suppresses the activity of G-protein alpha subunits, eNOS, and Src-like kinases, suggesting that caveolin binding may also play a negative regulatory role in signal transduction. Here, we report the direct interaction of caveolin with a growth factor receptor, EGF-R, a known caveolae-associated receptor tyrosine kinase. Two consensus caveolin binding motifs have been previously defined using phage display technology. One of these motifs is present within the conserved kinase domains of most known receptor tyrosine kinases (termed region IX). We now show that this caveolin binding motif within the kinase domain of the EGF-R can mediate the interaction of the EGF-R with the scaffolding domains of caveolins 1 and 3 but not with caveolin 2. In addition, the scaffolding domains of caveolins 1 and 3 both functionally inhibit the autophosphorylation of the EGF-R kinase in vitro. Importantly, this caveolin-mediated inhibition of the EGF-R kinase could be prevented by the addition of an EGF-R-derived peptide that (i) contains a well conserved caveolin binding motif and (ii) is located within the kinase domain of the EGF-R and most known receptor tyrosine kinases. Similar results were obtained with protein kinase C, a serine/threonine kinase, suggesting that caveolin may function as a general kinase inhibitor. The implications of our results are discussed within the context of caveolae-mediated signal transduction. In this regard, caveolae-coupled signaling might explain how linear signaling pathways can branch and interconnect extensively, forming a signaling module or network.     

hSK4, a member of a novel subfamily of calcium-activated potassium channels

The gene for hSK4, a novel human small conductance calcium-activated potassium channel, or SK channel, has been identified and expressed in Chinese hamster ovary cells. In physiological saline hSK4 generates a conductance of approximately 12 pS, a value in close agreement with that of other cloned SK channels. Like other members of this family, the polypeptide encoded by hSK4 contains a previously unnoted leucine zipper-like domain in its C terminus of unknown function. hSK4 appears unique, however, in its very high affinity for Ca2+ (EC50 of 95 nM) and its predominant expression in nonexcitable tissues of adult animals. Together with the relatively low homology of hSK4 to other SK channel polypeptides (approximately 40% identical), these data suggest that hSK4 belongs to a novel subfamily of SK channels.     

Synthesis and biological evaluation of aeroplysinin analogues: a new class of receptor tyrosine kinase inhibitors

Receptor tyrosine kinases (RTKs), such as the epidermal growth factor receptor (EGFR) and the platelet-derived growth factor receptor (PDGFR), are critically involved in the transduction of mitogenic signals across the plasma membrane and therefore in the regulation of cell growth and proliferation. Enhanced RTK activity is associated with proliferative diseases such as cancer, psoriasis and atherosclerosis, while decreased function may be associated for instance with diabetes. EGFR and PDGFR are selectively inhibited by analogues of the marine natural product aeroplysinin. The synthetic inhibitors display IC50 values in the low micromolar range and in contrast to the natural product show pronounced inhibitory activity in cultured cells in vivo. The mechanism of inhibition is likely based on a covalent modification of the target enzymes by reaction of epoxy ketone 8 with various nucleophiles.     

Neurturin responsiveness requires a GPI-linked receptor and the Ret receptor tyrosine kinase

Neurturin (NTN) is a recently identified homologue of glial-cell-line-derived neurotrophic factor (GDNF). Both factors promote the survival of a variety of neurons, and GDNF is required for the development of the enteric nervous system and kidney. GDNF signals through a receptor complex consisting of the receptor tyrosine kinase Ret and a glycosyl-phosphatidylinositol (GPI)-linked receptor termed GDNFR-alpha. Here we report the cloning of a new GPI-linked receptor termed NTNR-alpha that is homologous with GDNFR-alpha and is widely expressed in the nervous system and other tissues. By using microinjection to introduce expression plasmids into neurons, we show that coexpression of NTNR-alpha with Ret confers a survival response to neurturin but not GDNF, and that coexpression of GDNFR-alpha with Ret confers a survival response to GDNF but not neurturin. Our findings indicate that GDNF and neurturin promote neuronal survival by signalling through similar multicomponent receptors that consist of a common receptor tyrosine kinase and a member of a GPI-linked family of receptors that determines ligand specificity.     

The epidermal growth factor receptor tyrosine kinase phosphorylates connexin32

To elucidate the role of excessive nitric oxide (NO) via the inducible nitric oxide synthase (iNOS) in experimental allergic encephalomyelitis (EAE), the effect of a selective iNOS inhibitor, aminoguanidine, was investigated using mice with actively induced EAE. Administration of aminoguanidine by intraperitoneal or intracisternal injection from day 2 to day 12 after immunization produced a significant delay in the onset of EAE. On the other hand, administration of aminoguanidine by intraperitoneal or intracisternal injection for 10 days after the onset of EAE enhanced the clinical severity and mortality rate and hastened the onset of relapse significantly. The histological study at day 11 after the onset revealed that more inflammatory cells were present in the central nervous system of mice treated with aminoguanidine as compared with mice without aminoguanidine treatment. These results suggested that NO via iNOS was a pathogenetic factor in the induction phase of EAE, but had an inhibitory role in the progression phase of EAE. Although the effect of NO synthase inhibitors on EAE has been controversial, the present study suggested that the timing of administration might be an important consideration and might explain the previous contradictory reports.     

Specific, irreversible inactivation of the epidermal growth factor receptor and erbB2, by a new class of tyrosine kinase inhibitor

A class of high-affinity inhibitors is disclosed that selectively target and irreversibly inactivate the epidermal growth factor receptor tyrosine kinase through specific, covalent modification of a cysteine residue present in the ATP binding pocket. A series of experiments employing MS, molecular modeling, site-directed mutagenesis, and 14C-labeling studies in viable cells unequivocally demonstrate that these compounds selectively bind to the catalytic domain of the epidermal growth factor receptor with a 1:1 stoichiometry and alkylate Cys-773. While the compounds are essentially nonreactive in solution, they are subject to rapid nucleophilic attack by this particular amino acid when bound in the ATP pocket. The molecular orientation and positioning of the acrylamide group in these inhibitors in relation to Cys-773 entirely support these results as determined from docking experiments in a homology-built molecular model of the ATP site. Evidence is also presented to indicate that the compounds interact in an analogous fashion with erbB2 but have no activity against the other receptor tyrosine kinases or intracellular tyrosine kinases that were tested in this study. Finally, a direct comparison between 6-acrylamido-4-anilinoquinazoline and an equally potent but reversible analog shows that the irreversible inhibitor has far superior in vivo antitumor activity in a human epidermoid carcinoma xenograft model with no overt toxicity at therapeutically active doses. The activity profile for this compound is prototypical of a generation of tyrosine kinase inhibitors with great promise for therapeutic significance in the treatment of proliferative disease.     

EBI1/CCR7 is a new member of dendritic cell chemokine receptor that is up-regulated upon maturation

Dendritic cells (DC) that are stimulated with inflammatory mediators can maturate and migrate from nonlymphoid tissues to lymphoid organs to initiate T cell-mediated immune responses. This migratory step is closely related to the maturation of the DC. In an attempt to identify chemokine receptors that might influence migration and are selectively expressed in mature DC, we have discovered that the chemokine receptor, EBI1/CCR7, is strikingly up-regulated upon maturation in three distinct culture systems: 1) mouse bone marrow-derived DC, 2) mouse epidermal Langerhans cells, and 3) human monocyte-derived DC. The EBI1/CCR7 expressed in mature DC is functional because ELC/MIP-3beta, recently identified as a ligand of EBI1/CCR7, induces a rise in intracellular free calcium concentrations and directional migration of human monocyte-derived mature DC (HLA-DRhigh, CD1a(low), CD14-, CD25+, CD83+, and CD86high) in a dose-dependent manner, but not of immature DC (HLA-DRlow, CD1a(high), CD14-, CD25-, CD83-, and CD86-). In contrast, macrophage inflammatory protein-1alpha (MIP-1alpha), monocyte chemotactic protein-3 (MCP-3), and RANTES are active on immature DC but not on mature DC. Thus, it seems likely that MIP-1alpha, MCP-3, and RANTES can mediate the migration of immature DC located in peripheral sites, whereas ELC/MIP-3beta can direct the migration of Ag-carrying DC from peripheral inflammatory sites, where DC are stimulated to up-regulate the expression of EBI1/CCR7, to lymphoid organs. It is postulated that different chemokines and chemokine receptors are involved in DC migration in vivo, depending on the maturation state of DC.     

Mechanism of down-regulation of c-kit receptor. Roles of receptor tyrosine kinase, phosphatidylinositol 3'-kinase, and protein kinase C

The receptor tyrosine kinase Kit and Kit ligand (KL), encoded at the murine white spotting (W) and steel (Sl) loci, respectively, function in hematopoiesis, melanogenesis, and gametogenesis. To understand the mechanism of turnover of Kit in mast cells, mutant receptors generated in vitro were heterologously expressed in Wsb/Wsh mast cells lacking endogenous c-kit expression, and the effects of mutations on KL-induced internalization and ubiquitination/degradation of Kit were studied. Upon binding of KL, KL.Kit receptor complexes were rapidly internalized, and the turnover was accelerated by ubiquitin-mediated degradation. Inactivation of the Kit kinase resulted in a reduced rate of internalization of KL.Kit complexes, degradation of kinase-inactive receptor complexes was relatively slow, and receptor ubiquitination was absent. But abolishment of KL-induced receptor association and activation of phosphatidylinositol 3'-kinase and of tyrosine 821 autophosphorylation did not affect KL-induced internalization and ubiquitination/degradation of Kit. Furthermore, Kit receptors can be down-regulated by proteolytic cleavage induced by either activation of protein kinase C or by isopropyl alcohol. In summary, KL-induced internalization of KL.Kit complexes and ubiquitination/degradation require an active kinase. By contrast, proteolytic cleavage of Kit mediated by protein kinase C activation is independent of kinase activity.     

The G protein-coupled receptor kinase 2 is a microtubule-associated protein kinase that phosphorylates tubulin

The G protein-coupled receptor kinase 2 (GRK2) is a serine/threonine kinase that phosphorylates and desensitizes agonist-occupied G protein-coupled receptors (GPCRs). Here we demonstrate that GRK2 is a microtubule-associated protein and identify tubulin as a novel GRK2 substrate. GRK2 is associated with microtubules purified from bovine brain, forms a complex with tubulin in cell extracts, and colocalizes with tubulin in living cells. Furthermore, an endogenous tubulin kinase activity that copurifies with microtubules has properties similar to GRK2 and is inhibited by anti-GRK2 monoclonal antibodies. Indeed, GRK2 phosphorylates tubulin in vitro with kinetic parameters very similar to those for phosphorylation of the agonist-occupied beta2-adrenergic receptor, suggesting a functionally relevant role for this phosphorylation event. In a cellular environment, agonist occupancy of GPCRs, which leads to recruitment of GRK2 to the plasma membrane and its subsequent activation, promotes GRK2-tubulin complex formation and tubulin phosphorylation. These findings suggest a novel role for GRK2 as a GPCR signal transducer mediating the effects of GPCR activation on the cytoskeleton.     

The Eek receptor, a member of the Eph family of tyrosine protein kinases, can be activated by three different Eph family ligands

The Eph family of receptors, the largest subgroup within the tyrosine protein kinase receptor family, are comprised of at least thirteen members, many of which are predominantly expressed in the developing and adult nervous system. In this study, we have isolated a full-length cDNA, encoding the mouse homologue of a previous partially characterized Eek protein, a member of Eph receptor tyrosine kinase family. In a comparison of the amino acid sequences of various Eph family members, Eek is most similar to Ehk-3/MDK1, Sek/Cek8, Ehk-2, Hek/Mek4/Cek4, and Bsk/Ehk1/Rek7/Cek7, which are predominantly expressed in the nervous system. Additionally, we have used a low-stringency PCR cloning technique to identify ligands, related to B61, that may interact with Eek. Three different GPI-linked ligands, namely Elf-1/Cek7-L, Ehk1-L/Efl-2/Lerk3 and AL-1/RAGS, were isolated from mouse brain. To study the functional interactions between these ligands and the Eek receptors, we have constructed chimeric ligands consisting of the Fc portion of human IgG fused to their carboxyl-terminus. These chimeric ligands bound to, and activated both the Eek receptors and the Eek-TrkB chimeric receptors expressed in NIH3T3 cells. These findings suggest that Eek receptor can be activated by at least three different GPI-linked ligands.     

Structure and expression of a murine homologue of sky receptor tyrosine kinase gene

We examined effects of adenine nucleotide on ischemic myocardial stunning in dogs. Pentobarbitalanesthetized open-chest dogs were subjected to 20-min ligation of the left anterior descending coronary artery (LAD), followed by reperfusion for 30 min. Either saline, 5 mM 8-bromo-5'-AMP (tributyryl-AMP), or 30 mM N6, 2', 3'-tributyryl-5'-AMP (tributyryl-AMP), 5 mM 5-amino-4-imidazole carboxamide riboside (AICAr) as a positive reference, was infused at 0.1 ml/kg/min in the left femoral vein throughout the experiment. The myocardial contractile function was measured by ultrasonometry. The tissue levels of high-energy phosphates in the reperfused heart were determined. Myocardial contractile function assessed by % segment shortening (%SS) in the saline-infused group decreased during ischemia and returned toward the preischemic level during reperfusion but incompletely. A significant improvement in the %SS during reperfusion was observed in the 8-bromo-AMP- and AICAr-infused groups but not in the tributyryl-AMP-infused group. The magnitude of the protective effect of the drugs on myocardial contractility during reperfusion was 8-bromo-AMP > AICAr > tributyryl-AMP = saline. Only in the 8-bromo-AMP-infused group were the levels of ATP, ADP, and total adenine nucleotides in the reperfused heart significantly higher than those in the saline-infused group. The present result indicates that 8-bromo-AMP improves the ability of the heart to recover from ischemia and reperfusion associated with a significant restoration of ATP.     

Identification of a novel receptor tyrosine kinase expressed in acute myeloid leukemic blasts

Using the polymerase chain reaction with degenerate oligonucleotides derived from conserved motifs within the catalytic kinase domain of protein tyrosine kinases, and RNA extracted from embryonic stem cells, sequences that encode a segment of the kinase domain of several potentially novel receptor tyrosine kinases (RTKs) have been identified. One of these was selected for further study because in Northern analysis it hybridized to RNA from multipotential hematopoietic cell lines, but not from lines representative of lineage-committed cells. A cDNA for this receptor, designated developmental tyrosine kinase (DTK), was isolated and encodes a protein with structural similarities to AXL. Together these receptors form a new class of RTK. DTK is expressed in a number of human leukemic cell lines, and in the blasts of 6 of 11 patients with acute myeloid leukemia (AML) analyzed. The structure of DTK suggests that it may function as a cell adhesion molecule, and mediate cell-to-cell or cell-matrix interactions between hematopoietic cells and their respective microenvironments.     

Cloning and characterization of Krct, a member of a novel subfamily of serine/threonine kinases

Protein kinases frequently play key roles in the normal regulation of growth and development in eukaryotic organisms. As a consequence, aberrant expression or mutations in this family of molecules frequently result in transformation. Previously, we have conducted a screen to identify protein kinases that are expressed in the mouse during mammary gland development and in breast cancer cell lines. We now describe the molecular cloning, characterization and expression of Krct, a novel serine/threonine protein kinase unrelated to previously defined families of protein kinases. At the mRNA level, Krct is widely expressed throughout murine development and in adult tissues. Despite its ubiquitous expression, Krct is expressed preferentially within specific cellular compartments in multiple tissues, in particular within the testis and gastrointestinal tract. At the amino acid level, Krct is most closely related to four previously undescribed kinases in Saccharomyces cerevisiae, Arabidopsis thaliana and Caenorhabditis elegans. Together, these kinases appear to define a novel subfamily of serine/threonine protein kinases. Krct possesses an unusually long 5'-untranslated region containing multiple upstream initiation codons and, in this regard, is similar to many proto-oncogenes that regulate normal growth and differentiation. In addition, Krct is located on mouse chromosome 11 closely linked to the epidermal growth factor receptor and, therefore, is likely to be co-amplified in a variety of human tumors.     

Phosphatidylinositol-3' kinase is not required for mitogenesis or internalization of the Flt3/Flk2 receptor tyrosine kinase

Flt3/Flk2 is a receptor tyrosine kinase that is expressed on early hematopoietic progenitor cells. Flt3/Flk2 belongs to a family of receptors, including Kit and colony-stimulating factor-1R, which support growth and differentiation within the hematopoietic system. The Flt3/Flk2 ligand, in combination with other growth factors, stimulates the proliferation of hematopoietic progenitors of both lymphoid and myeloid lineages in vitro. We report that phosphatidylinositol 3'-kinase (PI3K) binds to a unique site in the carboxy tail of murine Flt3/Flk2. In distinction to Kit and colony-stimulating factor-1R, mutant receptors unable to couple to PI3K and expressed in rodent fibroblasts or in the interleukin 3-dependent cell line Ba/F3 provide a mitogenic signal comparable to wild-type receptors. Flt3/Flk2 receptors that do not bind to PI3K also normally down-regulate, a function ascribed to PI3K in the context of other receptor systems. These data point to the existence of other unidentified pathways that, alone or in combination with PI3K, transduce these cellular responses following the activation of Flt3/Flk2.