Leukocyte Tyrosine Kinase Research Articles

Anaplastic Lymphoma Kinase Is Activated through the Pleiotrophin/Receptor Protein Tyrosine Phosphatase (RPTP)β/ζ Signaling Pathway: A Unique Mechanism of Activation of Receptor Protein Tyrosine Kinases.

Anaplastic Lymphoma Kinase (ALK) is a receptor-type transmembrane tyrosine kinase (RTK) of the insulin receptor superfamily that structurally is most closely related to leukocyte tyrosine kinase. It was first discovered as a chimeric protein (NPM-ALK) of nucleophosmin and the C-terminal (kinase) domain of ALK in anaplastic large cell lymphomas (ALCL). NPM-ALK is constitutively active and generates the oncogenic signals that are the pathogenic mechanisms of these highly malignant cancers. The full-length ALK also is believed to have an important role in the pathogenesis of other human malignancies, since its expression is found in rhabdomyosarcomas, neuroblastomas, neuroectodermal tumors, glioblastomas, breast carcinomas, and melanomas. Recently it was proposed that pleiotrophin (PTN the protein, Ptn the gene) is the ligand that stimulates ALK to transduce signals to activate downstream targets. However, this proposal contrasted with earlier studies that demonstrated Receptor Protein Tyrosine Phosphatase (RPTP)β/ζ is the functional receptor for PTN. PTN was shown to inactivate RPTPβ/ζ and thereby permit the activity of different tyrosine kinases to increase tyrosine phosphorylation of the substrates of RPTPβ/ζ at the sites that are dephosphorylated by RPTPβ/ζ in cells not stimulated by PTN. Subsequent studies identified β-catenin, β-adducin, Fyn, GIT1/Cat-1, P190RhoGAP, and histone deacetylase 2 (HDAC-2) as downstream targets of the PTN/RPTPβ/ζ signaling pathway and demonstrated that their levels of tyrosine phosphorylation increase in PTN-stimulated cells. This diversity of PTN-regulated targets is one basis for the pleiotrophic activities of PTN. We now demonstrate that tyrosine phosphorylation of ALK is increased in PTN-stimulated cells through the PTN/RPTPβ/ζ signaling pathway. It is furthermore shown that ALK is activated in PTN-stimulated cells when it is expressed in cells without its extracellular domain, that β-catenin is a substrate of ALK, that the tyrosine phosphorylation site in β-catenin phosphorylated by ALK is the same site dephosphorylated by RPTPβ/ζ, and that PTN-stimulated tyrosine phosphorylation of β-catenin requires expression of ALK. The data suggest a unique mechanism to activate ALK; the data support a mechanism in which β-catenin is phosphorylated in tyrosine through the coordinated inactivation of RPTPβ/ζ, the activation of the tyrosine kinase activity of ALK, and the phosphorylation of β-catenin by ALK at the same site regulated by RPTPβ/ζ in PTN-stimulated cells. Since PTN often is inappropriately expressed in the same malignancies that express ALK, the data suggest a mechanism through which ALK signaling may contribute to those malignancies that express full length ALK through the activity of PTN to signal constitutively the same pathways as NPM-ALK in ALCL.

Activation and Inhibition of Anaplastic Lymphoma Kinase Receptor Tyrosine Kinase by Monoclonal Antibodies and Absence of Agonist Activity of Pleiotrophin

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase that is transiently expressed in specific regions of the central and peripheral nervous systems, suggesting a role in its normal development and function. The nature of the cognate ligands of ALK in vertebrate is still a matter of debate. We produced a panel of monoclonal antibodies (mAbs) directed against the extracellular domain of the human receptor. Two major species of ALK (220 and 140 kDa) were identified in transfected cells, and the use of our mAbs established that the 140-kDa species results from a cleavage of the 220-kDa form. Two mAbs, in the nm range, induced the differentiation of PC12 cells transiently transfected with ALK. In human embryonic kidney 293 cells stably expressing ALK, these two mAbs strongly activated the receptor and subsequently the mitogen-activated protein kinase pathway. We further showed for the first time that activation of ALK also resulted in a specific activation of STAT3. In contrast, other mAbs presented the characteristics of blocking antibodies. Finally, in these cell systems, a mitogenic form of pleiotrophin, a proposed ligand of ALK, failed to activate this receptor. Thus, in the absence of clearly established ligand(s) in vertebrates, the availability of mAbs allowing the activation or the inhibition of the receptor will be essential for a better understanding of the biological roles of ALK.

Identification of Metastasis-Associated Receptor Tyrosine Kinases in Non–Small Cell Lung Cancer

Development of distant metastasis after tumor resection is the leading cause of death in early-stage non-small cell lung cancer (NSCLC). Receptor tyrosine kinases (RTK) are involved in tumorigenesis but only few RTKs have been systematically studied in NSCLC. Here, we provide quantitative real-time reverse transcription-PCR expression data of all RTKs (n=56) in primary tumors of 70 patients with early-stage (I-IIIA) NSCLC. Overall, 33 RTKs were expressed in at least 25% of the patients. Several RTKs were significantly expressed higher in tumors that ultimately metastasized. The hazard risk for metastasis development in stage I/II disease was increased at least 3-fold for tumors with high expression levels of insulin receptor, neurotrophic tyrosine receptor kinase 1, epidermal growth factor receptor, ERBB2, ERBB3, platelet-derived growth factor receptor beta, fibroblast growth factor receptor 1, or leukocyte tyrosine kinase. Relative risks were reduced 3-fold by expression of EPHB6 or DKFZ1. Three members of the epidermal growth factor receptor family were associated with a high risk of metastasis, emphasizing the validity of our data. High ERBB3 expression was significantly associated with decreased survival. Taken together, our genome-wide RTK expression map uncovered the previously unknown value of several RTKs as potential markers for prognosis and metastasis prediction in early-stage NSCLC. The identified RTKs represent promising novel candidates for further functional analyses.

High-throughput analysis of genome-wide receptor tyrosine kinase expression in human cancers identifies potential novel drug targets.

Novel high-throughput analyses in molecular biology allow sensitive and rapid identification of disease-related genes and drug targets. We have used quantitative real-time reverse transcription-PCR reactions (n = 23000) to analyze expression of all human receptor tyrosine kinases (n = 56) in malignant tumors (n = 313) of different origins and normal control samples (n = 58). The different tumor types expressed very different numbers of receptor tyrosine kinases: whereas brain tumors and testicular cancer expressed 50 receptor tyrosine kinases, acute myeloid leukemia (AML) samples expressed only 20 different ones. Specimens of similar tumor origin exhibited characteristic receptor tyrosine kinase expression patterns and were grouped together in hierarchical cluster analyses. When we focused on specific tumor entities, receptor tyrosine kinases were identified that were disease and/or stage specific. Leukemic blasts from AML bone marrow samples differed significantly in receptor tyrosine kinase expression compared with normal bone marrow and purified CD34+ cells. Among the differentially expressed receptor tyrosine kinases, we found FLT3, c-kit, CSF1 receptor, EPHB6, leukocyte tyrosine kinase, and ptk7 to be highly overexpressed in AML samples. Whereas expression changes of some of these were associated with altered differentiation patterns (e.g., CSF1 receptor), others, such as FLT3, were genuinely overexpressed in leukemic blasts. These data and the associated database (http://medweb.uni-muenster.de/institute/meda/research/) provide a comprehensive view of receptor tyrosine kinase expression in human cancer. This information can assist in the definition of novel drug targets.

Gain-of-function polymorphism in mouse and human Ltk: implications for the pathogenesis of systemic lupus erythematosus.

Systemic lupus erythematosus (SLE), a complex multigenic disease, is a typical antibody-mediated autoimmune disease characterized by production of autoantibodies against a variety of autoantigens and immune complex-type tissue inflammation, most prominently in the kidney. Evidence suggests that genetic factors predisposing to aberrant proliferation/maturation of self-reactive B cells initiate and propagate the disease. In SLE-prone New Zealand Black (NZB) mice and their F1 cross with New Zealand White (NZW) mice, B cell abnormalities can be ascribed mainly to self-reactive CD5+ B1 cells. Our genome-wide scans to search for susceptibility genes for aberrant activation of B1 cells in these mice showed evidence that the gene, Ltk, encoding leukocyte tyrosine kinase (LTK), is a possible candidate. LTK is a receptor-type protein tyrosine kinase, belonging to the insulin receptor superfamily, and is mainly expressed in B lymphocyte precursors and neuronal tissues. Sequence and functional analyses of the gene revealed that NZB has a gain-of-function polymorphism in the LTK kinase domain near YXXM, a binding motif of the p85 subunit of phosphatidylinositol 3-kinase (PI3K). SLE patients also had this type of Ltk polymorphism with a significantly higher frequency compared with the healthy controls. Our findings suggest that these polymorphic LTKs cause up-regulation of the PI3K pathway and possibly form one genetic component of susceptibility to abnormal proliferation of self-reactive B cells in SLE.

Receptor tyrosine kinase mutations in myeloid neoplasms.

Receptor tyrosine kinase mutations in myeloid neoplasms.

A major breakpoint cluster domain in murine radiation-induced acute myeloid leukemia.

Cytogenetic and molecular studies have provided evidence of the clustering of chromosome 2 deletion breakpoints in radiation-induced murine acute myeloid leukemia (AML). Moreover, clustering occurs in at least two fragile domains rich in telomere-like arrays. Here we describe a physical map of the distal breakpoint cluster and confirm the presence of inverted head-to-head telomeric sequence arrays. These potentially recombinogenic sequences were not, however, the direct focus for post-irradiation chromosome breakage in AML. Instead, the two arrays bordered a 2.5-kb sequence with properties expected of a nuclear matrix attachment region (MAR). The putative MAR co-localized in the fragile domain with genes important to the hemopoietic system (leukocyte tyrosine kinase, zinc finger protein 106, erythrocyte protein band 4.2, and beta(2)-microglobulin (beta2m)); the beta2m subdomain was a particular focus of breakage. On the basis of these and other data, we suggest that AML-associated chromosome 2 fragility in the mouse is a consequence of domain-specific fragility in genomic domains containing numerous genes critical to the hemopoietic system. Recorded with the permission of the controller of Her Majesty's Stationery Office. Published by Wiley-Liss, Inc.

Identification of Anaplastic Lymphoma Kinase as a Receptor for the Growth Factor Pleiotrophin

Pleiotrophin (PTN) is a secreted growth factor that induces neurite outgrowth and is mitogenic for fibroblasts, epithelial, and endothelial cells. During tumor growth PTN can serve as an angiogenic factor and drive tumor invasion and metastasis. To identify a receptor for PTN, we panned a phage display human cDNA library against immobilized PTN protein as a bait. From this we isolated a phage insert that was homologous to an amino acid sequence stretch in the extracellular domain (ECD) of the orphan receptor tyrosine kinase anaplastic lymphoma kinase (ALK). In parallel with PTN, ALK is highly expressed during perinatal development of the nervous system and down-modulated in the adult. Here we show in cell-free assays as well as in radioligand receptor binding studies in intact cells that PTN binds to the ALK ECD with an apparent Kd of 32 +/- 9 pm. This receptor binding is inhibited by an excess of PTN, by the ALK ECD, and by anti-PTN and anti-ECD antibodies. PTN added to ALK-expressing cells induces phosphorylation of both ALK and of the downstream effector molecules IRS-1, Shc, phospholipase C-gamma, and phosphatidylinositol 3-kinase. Furthermore, the growth stimulatory effect of PTN on different cell lines in culture coincides with the endogenous expression of ALK mRNA, and the effect of PTN is enhanced by ALK overexpression. From this we conclude that ALK is a receptor that transduces PTN-mediated signals and propose that the PTN-ALK axis can play a significant role during development and during disease processes.

Heart-specific activation of LTK results in cardiac hypertrophy, cardiomyocyte degeneration and gene reprogramming in transgenic mice.

Leukocyte tyrosine kinase (LTK) is a receptor-type protein tyrosine kinase belonging to the insulin receptor superfamily. To elucidate its biological role, we generated transgenic mice expressing LTK under the control of cytomegarovirus enhancer and beta-actin promoter. The transgenic mice exhibited growth retardation and most of the transgenic mice died within several months after birth. Interestingly, although LTK was expressed in several major organs, the activation (tyrosine-phosphorylation, kinase activity, and multimerization) of LTK was observed selectively in the heart, where LTK was localized on intracellular membrane, presumably on endoplasmic reticulum. Echocardiography showed that the transgenic heart underwent severe concentric hypertrophy, which resulted in reduced cardiac output, low blood pressure, and increased heart rate. Histological examination of the heart exhibited focal degeneration of cardiomyocytes. These histological changes were considered to be due to apoptosis, based on the finding that the sarcolemmas of the degenerative cardiomyocytes were well preserved. In addition, expression of fetal genes, such as atrial natriuretic peptide and skeletal alpha-actin, was markedly induced in the transgenic heart. These results indicate that a certain tissue-specific mechanism of activating LTK exists in the heart and that the activated LTK resulted in cardiac hypertrophy, cardiomyocyte degeneration and gene reprogramming. These findings will provide novel insights into the activating mechanism and biological role of LTK in vivo.

The phosphatidylinositol 3' kinase pathway is required for the survival signal of leukocyte tyrosine kinase.

Leukocyte tyrosine kinase (LTK) is a receptor tyrosine kinase which belongs to the insulin receptor superfamily and is mainly expressed in pre-B lymphocytes and neuronal tissues. Recently, we demonstrated that LTK utilizes Shc and IRS-1 as two major substrates and while both equally activate the Ras pathway, only IRS-1 suppresses apoptosis of hematopoietic cells, suggesting the existence of another unidentified signaling pathway downstream of IRS-1, which is relevant to the anti-apoptotic activity. In the present study, we found that wortmannin, a specific inhibitor of phosphatidylinositol 3' (PI3)-kinase, abolished the survival effects of LTK. Although c-Cbl is found to be phosphorylated by LTK and therefore is a second candidate linking LTK with the PI3-kinase pathway along with IRS-1, we found that the p85 subunit of PI3 kinase directly binds to tyrosine 753 of LTK, which is located within a YXXM motif, a consensus binding amino acid sequence for the SH2 domain of p85, but fails to bind to IRS-1 or c-Cbl. Ba/F3 cells which stably express the EGF receptor-LTK chimeric receptor carrying a mutation at tyrosine 753 fell into apoptotic death even in the presence of EGF, indicating that the PI3 kinase pathway is required for the survival effects of LTK.

ALK, the chromosome 2 gene locus altered by the t(2;5) in non-Hodgkin's lymphoma, encodes a novel neural receptor tyrosine kinase that is highly related to leukocyte tyrosine kinase (LTK)

Anaplastic Lymphoma Kinase (ALK) was originally identified as a member of the insulin receptor subfamily of receptor tyrosine kinases that acquires transforming capability when truncated and fused to nucleophosmin (NPM) in the t(2;5) chromosomal rearrangement associated with non-Hodgkin's lymphoma, but further insights into its normal structure and function are lacking. Here, we characterize a full-length normal human ALK cDNA and its product, and determine the pattern of expression of its murine homologue in embryonic and adult tissues as a first step toward the functional assessment of the receptor. Analysis of the 6226 bp ALK cDNA identified an open reading frame encoding a 1620-amino acid (aa) protein of predicted mass approximately 177 kDa that is most closely related to leukocyte tyrosine kinase (LTK), the two exhibiting 57% aa identity and 71% similarity over their region of overlap. Biochemical analysis demonstrated that the approximately 177 kDa ALK polypeptide core undergoes co-translational N-linked glycosylation, emerging in its mature form as a 200 kDa single chain receptor. Surface labeling studies indicated that the 200 kDa glycoprotein is exposed at the cell membrane, consistent with the prediction that ALK serves as the receptor for an unidentified ligand(s). In situ hybridization studies revealed Alk expression beginning on embryonic day 11 and persisting into the neonatal and adult periods of development. Alk transcripts were confined to the nervous system and included several thalamic and hypothalamic nuclei; the trigeminal, facial, and acoustic cranial ganglia; the anterior horns of the spinal cord in the region of the developing motor neurons; the sympathetic chain; and the ganglion cells of the gut. Thus, ALK is a novel orphan receptor tyrosine kinase that appears to play an important role in the normal development and function of the nervous system.

Growth and Survival Signals transmitted via Two Distinct NPXY Motifs within Leukocyte Tyrosine Kinase, an Insulin Receptor-related Tyrosine Kinase

Leukocyte tyrosine kinase (LTK) is a receptor tyrosine kinase, which belongs to the insulin receptor family and is mainly expressed in pre-B cells and brain. In this study, we show that LTK utilizes insulin receptor substrate-1 (IRS-1) and Shc as major two substrates and possesses two NPXY motifs for them separately, tyrosine 485 of one NPXY motif at the juxtamembrane domain for IRS-1 and tyrosine 862 of another NPXY motif at the carboxyl-terminal domain for Shc. By using Ba/F3 cells expressing epidermal growth factor receptor-LTK chimeric receptors containing a mutation at each NPXY site, we showed that while both NPXY motifs equally contribute to activation of the Ras pathway and generation of mitogenic signals, only tyrosine 485 of LTK transmits cell survival signals. These data suggest that IRS-1 possesses anti-apoptotic function at least in LTK signaling. Moreover, our data indicate that the survival signaling pathway of LTK is distinct from the Ras pathway and the p70(S6) kinase pathway. Our results provide a useful insight in understanding the distinctive roles of Shc and IRS-1 in the signal transduction system of the insulin receptor family, and this anti-apoptotic function of IRS-1 may explain the survival effects of insulin, IGF-1, and interleukin 4.

An Epidermal Growth Factor Receptor-Leukocyte Tyrosine Kinase Chimeric Receptor Generates Ligand-dependent Growth Signals through the Ras Signaling Pathway

Leukocyte tyrosine kinase (LTK) is a receptor tyrosine kinase that belongs to the insulin receptor family. LTK is mainly expressed in pre B cells and brain. Previously we cloned the full-length cDNA of human LTK, but no ligands have so far been identified, and hence, very little is known about the physiological role of LTK. To analyze the function of the LTK kinase, we constructed chimeric receptors composed of the extracellular domain of epidermal growth factor receptor and the transmembrane and the cytoplasmic domains of LTK and established cell lines that stably express these chimeric molecules. When cultured in medium containing EGF, growth of these cell lines was stimulated, and these fusion proteins became autophosphorylated and associated with Shc in vivo in a ligand-dependent manner. By treatment with EGF, Shc was associated with the Grb2/Ash-Sos complex. Our analyses demonstrate that LTK associates with Grb2/Ash through an internal adaptor, Shc, depending on a ligand stimulation. The LTK binding site for Shc was tyrosine 862 at the carboxyl-terminal domain and to a lesser extent tyrosine 485 at the juxtamembrane domain. Both of them are located in NP/AXY motif which is consistent with binding sites for Shc. These findings demonstrate that LTK can activate the Ras pathway in a ligand-dependent manner and that at least one of the functions of this kinase is involved in the cell growth.

Differently spliced cDNAs of human leukocyte tyrosine kinase receptor tyrosine kinase predict receptor proteins with and without a tyrosine kinase domain and a soluble receptor protein.

Leukocyte tyrosine kinase (LTK) is a tyrosine kinase that has been suggested to be specific for hematopoietic cells and neuronal cells and reported as an unusual membrane protein lacking an extracellular domain. Here we report the cloning of a human LTK cDNA clone containing the complete open reading frame of a putative receptor tyrosine kinase protein. The extracellular domain of the receptor protein is larger than previously predicted. Furthermore, we have cloned a set of cDNAs representing differently spliced human LTK mRNAs. These cDNAs predict a truncated receptor protein lacking the tyrosine kinase domain and a soluble receptor protein that has neither a transmembrane nor a tyrosine kinase domain. Our results suggest that the LTK gene produces not only the putative receptor tyrosine kinase for unknown ligand but also multiple protein products that may have different functions.

Leukocytes express a novel gene encoding a putative transmembrane protein-kinase devoid of an extracellular domain.

Tyrosine-specific phosphorylation of proteins is a key to the control of diverse pathways leading to cell growth and differentiation. The protein-tyrosine kinases described to date are either transmembrane proteins having an extracellular ligand binding domain or cytoplasmic proteins related to the v-src oncogene. Most of these proteins are expressed in a wide variety of cells and tissues; few are tissue-specific. Previous studies have suggested that lymphokines could mediate haematopoietic cell survival through their action on glucose transport, regulated in some cells through the protein-tyrosine kinase activity of the insulin receptor. We have investigated the possibility that insulin receptor-like genes are expressed specifically in haematopoietic cells. Using the insulin receptor-related avian sarcoma oncogene v-ros as a probe, we have isolated and characterized the complementary DNA of a novel gene, ltk (leukocyte tyrosine kinase). The ltk gene is expressed mainly in leukocytes, is related to several tyrosine kinase receptor genes of the insulin receptor family and has unique structural properties: it apparently encodes a transmembrane protein devoid of an extracellular domain. Two candidate ltk proteins have been identified with antibodies in the mouse thymus, and have properties indicating that they are integral membrane proteins. These features suggest that ltk could be a signal transduction subunit for one or several of the haematopoietic receptors.