A C. elegans Ror receptor tyrosine kinase regulates cell motility and asymmetric cell division.

Ror kinases are a family of orphan receptors with tyrosine kinase activity that are related to muscle specific kinase (MuSK), a receptor tyrosine kinase that assembles acetylcholine receptors at the neuromuscular junction. Although the functions of Ror kinases are unknown, similarities between Ror and MuSK kinases have led to speculation that Ror kinases regulate synaptic development. Here we show that the Caenorhabditis elegans gene cam-1 encodes a member of the Ror kinase family that guides migrating cells and orients the polarity of asymmetric cell divisions and axon outgrowth. We find that tyrosine kinase activity is required for some of the functions of CAM-1, but not for its role in cell migration. CAM-1 is expressed in cells that require its function, and acts cell autonomously in migrating neurons. Overexpression and loss of cam-1 function result in reciprocal cell-migration phenotypes, indicating that levels of CAM-1 influence the final positions of migrating cells. Our results raise the possibility that Ror kinases regulate cell motility and asymmetric cell division in organisms as diverse as nematodes and mammals.     

A functional RNAi screen for regulators of receptor tyrosine kinase and ERK signalling

Receptor tyrosine kinase (RTK) signalling through extracellular-signal-regulated kinases (ERKs) has pivotal roles during metazoan development, underlying processes as diverse as fate determination, differentiation, proliferation, survival, migration and growth. Abnormal RTK/ERK signalling has been extensively documented to contribute to developmental disorders and disease, most notably in oncogenic transformation by mutant RTKs or downstream pathway components such as Ras and Raf. Although the core RTK/ERK signalling cassette has been characterized by decades of research using mammalian cell culture and forward genetic screens in model organisms, signal propagation through this pathway is probably regulated by a larger network of moderate, context-specific proteins. The genes encoding these proteins may not have been discovered through traditional screens owing, in particular, to the requirement for visible phenotypes. To obtain a global view of RTK/ERK signalling, we performed an unbiased, RNA interference (RNAi), genome-wide, high-throughput screen in Drosophila cells using a novel, quantitative, cellular assay monitoring ERK activation. Here we show that ERK pathway output integrates a wide array of conserved cellular processes. Further analysis of selected components-in multiple cell types with different RTK ligands and oncogenic stimuli-validates and classifies 331 pathway regulators. The relevance of these genes is highlighted by our isolation of a Ste20-like kinase and a PPM-family phosphatase that seem to regulate RTK/ERK signalling in vivo and in mammalian cells. Novel regulators that modulate specific pathway outputs may be selective targets for drug discovery.     

Epidermal growth factor receptor occupancy inhibits vaccinia virus infection

Vaccinia virus encodes VGF, an early protein of relative molecular mass 19,000 (19K) which, from amino-acid residues 45 to 85, is homologous in 19 residues to epidermal growth factor (EGF), and transforming growth factor-alpha (TGF-alpha). The conserved sequence includes a region of high homology (6 out of 10 amino acids) from residues 71 to 80, corresponding to the third disulphide loop of both EGF and TGF-alpha. This region has recently been shown to contain a binding region of TGF-alpha for the EGF receptor, and this raises the question of whether vaccinia virus utilizes the EGF receptor in order to bind to and infect cells. We now show that occupancy of the EGF receptor inhibits vaccinia virus infection. Inhibition is observed in a dose-dependent fashion by pre-treatment with either EGF or synthetic decapeptide antagonists of EGF's mitogenic activity which correspond to the sequence of the third disulphide loop of VGF or TGF-alpha. The relative ability of the peptides to inhibit vaccinia virus infection parallels their binding affinity to the EGF receptor.     

The Drosophila gene torso encodes a putative receptor tyrosine kinase

The maternal gene torso, required for determination of anterior and posterior terminal structures in the Drosophila embryo, was cloned using P-element tagging. Genetic evidence suggests that the action of the gene product is spatially restricted to the terminal regions; the torso messenger RNA, however, is evenly distributed. Structural similarities of the predicted torso protein with growth-factor receptor tyrosine kinases suggest that the spatial restriction of torso activity results from a localized activation of the torso protein at the anterior and posterior egg pole.     

Non-radioisotopic method for thein vitro measurement of EGF receptor tyrosine kinase

A non-radioisotopic method was developed for the assay of epidermal growth factor receptor (EGFR). A peptide with twenty amino acid residues around Tyr 1173, the major phosphorylation site of EGFR, was cloned as a GST fusion protein and used as substrate. Anti-phosphoty-rosine monoclonal antibody PY99 was used for the determination of the extent of phosphorylation. Both the specificity and the sensitivity were substantially higher than that of the existing method. Km value of the fusion protein is much lower (10 μmol/L) than that of the synthetic peptide (110 μmol/L). The method can be applied to the measurement of the tyrosine kinase activity of c-erb B2 (Neu/HER2).     

A chimaeric receptor allows insulin to stimulate tyrosine kinase activity of epidermal growth factor receptor

The cell surface receptors for insulin and epidermal growth factor (EGF) appear to share a common evolutionary origin, as suggested by structural similarity of cysteine-rich regions in their extracellular domains and a highly conserved tyrosine-specific protein kinase domain. Only minor similarity is found outside this catalytic domain, as expected for receptors that have different ligand specificities and generate different biological signals. The EGF receptor is a single polypeptide chain but the insulin receptor consists of distinct alpha and beta subunits that function as an alpha 2 beta 2 heterotetrameric receptor complex. Provoked by this major structural difference in two receptors that carry out parallel functions, we have designed a chimaeric receptor molecule comprising the extracellular portion of the insulin receptor joined to the transmembrane and intracellular domains of the EGF receptor to investigate whether one ligand will activate the tyrosine kinase domain of the receptor for the other ligand. We show here that the EGF receptor kinase domain of the chimaeric protein, expressed transiently in simian cells, is activated by insulin binding. This strongly suggests that insulin and EGF receptors employ closely related or identical mechanisms for signal transduction across the plasma membrane.     

Rab5 is a signalling GTPase involved in actin remodelling by receptor tyrosine kinases

Rab5 is a small GTPase involved in the control of intracellular trafficking, both at the level of receptor endocytosis and endosomal dynamics. The finding that Rab5 can be activated by receptor tyrosine kinases (RTK) raised the question of whether it also participates in effector pathways emanating from these receptors. Here we show that Rab5 is indispensable for a form of RTK-induced actin remodelling, called circular ruffling. Three independent signals, originating from Rab5, phosphatidylinositol-3-OH kinase and Rac, respectively, are simultaneously required for the induction of circular ruffles. Rab5 signals to the actin cytoskeleton through RN-tre, a previously identified Rab5-specific GTPase-activating protein (GAP). Here we demonstrate that RN-tre has the dual function of Rab5-GAP and Rab5 effector. We also show that RN-tre is critical for macropinocytosis, a process previously connected to the formation of circular ruffles. Finally, RN-tre interacts with both F-actin and actinin-4, an F-actin bundling protein. We propose that RN-tre establishes a three-pronged connection with Rab5, F-actin and actinin-4. This may aid crosslinking of actin fibres into actin networks at the plasma membrane. Thus, we have shown that Rab5 is a signalling GTPase and have elucidated the major molecular elements of its downstream pathway.     

Human insulin receptor and its relationship to the tyrosine kinase family of oncogenes

We have deduced the entire 1,370-amino-acid sequence of the human insulin receptor precursor from a single complementary DNA clone. The precursor starts with a 27-amino-acid signal sequence, followed by the receptor alpha-subunit, a precursor processing enzyme cleavage site, then the beta-subunit containing a single 23-amino-acid transmembrane sequence. There are sequence homologies to human epidermal growth factor receptor and the members of the src family of oncogene products.     

Insulin receptor tyrosine kinase is defective in skeletal muscle of insulin-resistant obese mice

Obese syndromes of genetic origin or experimentally induced are characterized by resistance to insulin both in vivo (association of hyperglycaemia and hyperinsulinaemia) and in vitro. Thus, skeletal muscle of obese mice, which is the most important target organ for the action of insulin, displays a reduced response to insulin. This hormonal resistance cannot be explained by the moderate decrease in the number of insulin receptors found in obese animals. In fact, it is generally believed that a biochemical event occurring very early after binding of insulin to its receptor, which is the first step in insulin action, is defective in obesity. One of the earliest post-binding events so far recognized, and which is thought to have a key role in cellular signalling by the insulin receptor, is the insulin-stimulated phosphorylation of its receptor. In an effort to localize the defect responsible for the insulin resistance in obesity, we have studied the insulin receptor protein kinase activity and we show here that insulin receptors from skeletal muscles of insulin-resistant obese mice have an altered kinase activity for phosphorylation of both the receptor itself and of exogeneous substrates.     

Novel putative receptor tyrosine kinase encoded by the melanoma-inducing Tu locus in Xiphophorus

Malignant melanoma in Xiphophorus fish hybrids is caused by the activity of a dominant oncogene Tu. By combining genetic and molecular approaches, we have isolated the melanoma oncogene. We show that its level of expression correlates with the degree of malignancy of the tumour. The corresponding proto-oncogene is developmentally regulated. The Tu gene codes for a novel receptor tyrosine kinase which is closely related to the receptor for epidermal growth factor.     

Jeb signals through the Alk receptor tyrosine kinase to drive visceral muscle fusion

The Drosophila melanogaster gene Anaplastic lymphoma kinase (Alk) is homologous to mammalian Alk, a member of the Alk/Ltk family of receptor tyrosine kinases (RTKs). We have previously shown that the Drosophila Alk RTK is crucial for visceral mesoderm development during early embryogenesis. Notably, observed Alk visceral mesoderm defects are highly reminiscent of the phenotype reported for the secreted molecule Jelly belly (Jeb). Here we show that Drosophila Alk is the receptor for Jeb in the developing visceral mesoderm, and that Jeb binding stimulates an Alk-driven, extracellular signal-regulated kinase-mediated signalling pathway, which results in the expression of the downstream gene duf (also known as kirre)--needed for muscle fusion. This new signal transduction pathway drives specification of the muscle founder cells, and the regulation of Duf expression by the Drosophila Alk RTK explains the visceral-mesoderm-specific muscle fusion defects observed in both Alk and jeb mutant animals.     

Diminished in vitro tyrosine kinase activity of the EGF receptor of senescent human fibroblasts

Fibroblastic cultures derived from normal human tissues undergo a finite number of population doublings when serially subcultivated in vitro (see refs 1, 2 for reviews). Epidermal growth factor (EGF) serves as a mitogen for early doubling level cultures of the human fetal lung-derived cell strain, WI-38, under serum-free conditions. The ability of cells from late doubling level cultures to respond mitogenically to EGF is lost, however, despite undiminished binding of EGF throughout the replicative lifespan. The ultimate effects of EGF, that is DNA synthesis and mitosis (see ref. 4 for review), occur after a sequence of events initiated by binding of ligand to specific cellular receptors. The receptor for EGF has been characterized as a 145,000-165,000 (145 K-165 K) molecular weight doublet, and, like the receptors for platelet-derived growth factor and insulin, and the transforming proteins of certain of the RNA tumour viruses, is a tyrosine-specific protein kinase with autophosphorylating activity. Moreover, several of the cellular target molecules of tyrosine phosphorylation have been found to be substrates for two or more of these kinases. The hypothesis that tyrosine phosphorylation underlies a common mechanism of growth control prompted us to ask whether the loss of responsiveness to EGF by late doubling level WI-38 cells is accompanied by altered expression of the EGF receptor, and specifically whether changes occur in the ability of receptors from populations of cells of various in vitro ages to catalyse tyrosine autophosphorylation. We show here that autophosphorylating activity is absent from the EGF receptor of cells which have lost their mitogenic responsiveness to EGF.     

No T-cell tyrosine protein kinase signalling or calcium mobilization after CD4 association with HIV-1 or HIV-1 gp120.

The T lymphocyte surface protein CD4 is an integral membrane glycoprotein noncovalently associated with the tyrosine protein kinase p56lck. In normal T cells, surface association of CD4 molecules with other CD4 molecules or other T-cell surface proteins, such as the T-cell antigen receptor, stimulates the activity of the p56lck tyrosine kinase, resulting in the phosphorylation of various cellular proteins at tyrosine residues. Thus, the signal transduction in T cells generated through the surface engagement of CD4 is similar to that observed for the class of growth factor receptors possessing endogenous tyrosine kinase activity. As CD4 is also the cellular receptor for the human immunodeficiency virus (HIV), binding of the virus or gp120 (the virus surface protein responsible for specific CD4+ T-cell association) could mimic the types of immunological interactions that have previously been found to stimulate p56lck and trigger T-cell activation pathways. We have evaluated this possibility and report here that binding of HIV-1 or the virus glycoprotein gp120 to CD4+ human T cells fails to elicit detectable p56lck-dependent tyrosine kinase activation and signalling, alterations in the composition of cellular phosphotyrosine-containing proteins, or changes in intracellular Ca2+ concentration.     

Phytochrome signalling is mediated through nucleoside diphosphate kinase 2.

Because plants are sessile, they have developed intricate strategies to adapt to changing environmental variables, including light. Their growth and development, from germination to flowering, is critically influenced by light, particularly at red (660 nm) and far-red (730 nm) wavelengths. Higher plants perceive red and far-red light by means of specific light sensors called phytochromes(A-E). However, very little is known about how light signals are transduced to elicit responses in plants. Here we report that nucleoside diphosphate kinase 2 (NDPK2) is an upstream component in the phytochrome signalling pathway in the plant Arabidopsis thaliana. In animal and human cells, NDPK acts as a tumour suppressor. We show that recombinant NDPK2 in Arabidopsis preferentially binds to the red-light-activated form of phytochrome in vitro and that this interaction increases the activity of recombinant NDPK2. Furthermore, a mutant lacking NDPK2 showed a partial defect in responses to both red and farred light, including cotyledon opening and greening. These results indicate that NDPK2 is a positive signalling component of the phytochrome-mediated light-signal-transduction pathway in Arabidopsis.     

A Tyr/Ser protein phosphatase encoded by vaccinia virus.

Protein tyrosine phosphorylation is associated with alterations in receptor activity, cellular proliferation and modulation of the cell cycle. Inappropriate tyrosine phosphorylation can lead to unrestrained cell growth and oncogenesis. Enzymes important in tyrosine dephosphorylation have also been described. Protein tyrosine phosphatases (PTPases) consist of two families. There is a receptor-like family of PTPases with an extracellular domain, transmembrane-spanning region and typically two repeated phosphatase domains. Proteins of the non-receptor-like family have a single catalytic phosphatase domain, show a substrate specificity for Tyr phosphate and will not hydrolyse Ser or Thr phosphate. Here we report that the vaccinia virus genome contains an open reading frame which shares amino-acid sequence identity with the PTPases. The purified protein encoded by the vaccinia virus H1 open reading frame expressed in bacteria hydrolyses substrates containing phosphotyrosine and phosphoserine. Mutagenesis of an essential Cys in the vaccinia phosphatase abolishes catalytic activity directed towards both substrates, suggesting that hydrolysis proceeds by a common mechanism. Understanding the function of the H1-encoded protein will help to define the role of the phosphatase in viral replication and pathogenesis.     

Jelly belly protein activates the receptor tyrosine kinase Alk to specify visceral muscle pioneers

The secreted protein Jelly belly (Jeb) is required for an essential signalling event in Drosophila muscle development. In the absence of functional Jeb, visceral muscle precursors are normally specified but fail to migrate and differentiate. The structure and distribution of Jeb protein implies that Jeb functions as a signal to organize the development of visceral muscles. Here we show that the Jeb receptor is the Drosophila homologue of anaplastic lymphoma kinase (Alk), a receptor tyrosine kinase of the insulin receptor superfamily. Human ALK was originally identified as a proto-oncogene, but its normal function in mammals is not known. In Drosophila, localized Jeb activates Alk and the downstream Ras/mitogen-activated protein kinase cascade to specify a select group of visceral muscle precursors as muscle-patterning pioneers. Jeb/Alk signalling induces the myoblast fusion gene dumbfounded (duf; also known as kirre) as well as org-1, a Drosophila homologue of mammalian TBX1, in these cells.     

MAP2 kinase and 70K S6 kinase lie on distinct signalling pathways.

Activation of protein synthesis is required for quiescent cells to transit the cell cycle, and seems to be mediated in part by phosphorylation of the 40S ribosomal protein, S6. A mitogen-activated S6 kinase of relative molecular mass 70,000 (70K) has been isolated from mouse fibroblasts as well as from avian, rat and rabbit tissues. Comparison of complementary DNA sequences shows that this enzyme is distinct from S6 kinase II (92K) found in Xenopus eggs and fibroblasts. Both kinases are activated by serine/threonine phosphorylation, suggesting that at least one serine/threonine kinase links receptor tyrosine kinases with S6 kinases. A candidate for this link is MAP2 kinase, which is rapidly activated by tyrosine/threonine phosphorylation following mitogenic stimulation. Incubation of MAP2 kinase from insulin-treated 3T3-L1 adipocytes with phosphatase-inactivated S6 kinase II from Xenopus leads to partial reactivation and phosphorylation of the enzyme. These and other findings have led to the suggestion that MAP2 kinase also activates the 70K S6 kinase. Here we refute this idea by showing that the two kinases lie on distinct signalling pathways.