Showing papers by "Kari Alitalo published in 1994"

Repression of cyclin D1: a novel function of MYC.

Abstract: Constitutive expression of human MYC represses mRNA levels of cyclin D1 in proliferating BALB/c-3T3 fibroblasts. We expressed a series of mutant alleles of MYC and found that downregulation of cyclin D1 is distinct from previously described properties of MYC. In particular, we found that association with Max is not required for repression of cyclin D1 by MYC in vivo. Conversely, the integrity of a small amino-terminal region (amino acids 92 to 106) of MYC is critical for repression of cyclin D1 but dispensable for transformation of established RAT1A cells. Runoff transcription assays showed that repression occurs at the level of transcription initiation. We cloned the promoter of the gene for human cyclin D1 and found that it lacks a canonical TATA element. Transcription starts at an initiator element similar to that of the adenovirus major late promoter; this element can be directly bound by USF in vitro. Expression of MYC represses the cyclin D1 promoter via core promoter elements and antagonizes USF-mediated transactivation. Taken together, our data define a new pathway for gene regulation by MYC and show that the cyclin D1 gene is a target gene for repression by MYC.

Signalling properties of FLT4, a proteolytically processed receptor tyrosine kinase related to two VEGF receptors.

Abstract: The FLT4, FLT1 and KDR/FLK1 genes encode structurally similar endothelial cell receptor tyrosine kinases. Recently it has been shown that the FLT1 and KDR/FLK-1 proteins function as high-affinity receptors for vascular endothelial growth factor (VEGF). Here we show that FLT4 does not act as a receptor for VEGF, as VEGF did not show specific binding to the FLT4 tyrosine kinase or induce its autophosphorylation. Also, FLT4 did not interact with KDR in response to VEGF. However, when fused with the ligand binding domain of the colony stimulating factor-1 receptor (CSF-1R), the FLT4 tyrosine kinase was specifically activated by CSF-1. The activated FLT4 tyrosine kinase domain was found to interact with the Src homology 2 domains of the SHC and GRB2 adaptor proteins in vitro and with SHC in cells. CSF-1 stimulation of the CSF-1R/FLT4 receptor chimera induced thymidine incorporation in serum-starved NIH3T3 fibroblasts, but not in porcine aortic or murine lung capillary endothelial cells, although tyrosyl phosphorylation of the receptor and SHC occurred in these cells as well. These results suggest that the endothelial cell FLT4 receptor tyrosine kinase transmits signals for an as yet unidentified growth factor.

BMX, a novel nonreceptor tyrosine kinase gene of the BTK/ITK/TEC/TXK family located in chromosome Xp22.2.

Abstract: The Bmx sequence was identified and cloned during our search for novel tyrosine kinase genes expressed in human bone marrow cells. Bmx cDNA comprises a long open reading frame of 675 amino acids, containing one SH3, one SH2 and one tyrosine kinase domain, which are about 70% identical with Btk, Itk and Tec and somewhat less with Txk tyrosine kinase sequences. The amino terminal sequences of these four tyrosine kinases are about 40% identical and each contains a so-called pleckstrin homology domain. The 2.7 kb Bmx mRNA was expressed in endothelial cells and several human tissues by Northern blotting and an 80 kD Bmx polypeptide was detected in human endothelial cells. Immunoprecipitates of COS cells transfected with a Bmx expression vector and NIH3T3 cells expressing a Bmx retrovirus contained a tyrosyl phosphorylated Bmx polypeptide of similar molecular weight. The BMX gene was located in chromosomal band Xp22.2 between the DXS197 and DXS207 loci. Interestingly, chromosome X also contains the closest relative of BMX, the BTK gene, implicated in X-linked agammaglobulinemia. The BMX gene thus encodes a novel nonreceptor tyrosine kinase, which may play a role in the growth and differentiation of hematopoietic cells.

The mouse tie receptor tyrosine kinase gene: expression during embryonic angiogenesis.

Abstract: Angiogenesis, the process by which new blood vessels are formed, is essential in reproduction, development, wound repair and oncogenesis. Endothelial receptor tyrosine kinases are likely to play key roles in the intercellular signalling controlling angiogenesis. We have here analysed the expression of the endothelial receptor tyrosine kinase tie during the earliest stages of vascular development. The mouse tie cDNA was isolated and sequenced and the exon structure of the coding region was determined. The part of the tie gene encoding the extracellular domain was found to be organized in exons specifying the characteristic domains of the Tie polypeptide. Early postimplantation mouse tissues were analysed for tie expression by in situ hybridization. No tie mRNA was detected in 7.5 day mouse embryos. In 8.5 day embryos, tie expression was observed in differentiating angioblasts of the head mesenchyme, in the splanchnopleure and dorsal aorta as well as in migrating endothelial cells of the developing heart. A weak tie signal was also obtained from angioblasts in the blood islands of the yolk sac. Furthermore, tie mRNA was prominent in the endocardium of the embryo and in the endothelial cells forming the lung vasculature. Expression of tie persisted in the lung capillaries of adult mice, but was decreased in the endocardium. These results suggest that the tie receptor tyrosine kinase is involved in angiogenesis and/or maintenance of endothelial cell functions.

Enhanced Expression of the Tie Receptor Tyrosine Kinase Messenger RNA in the Vascular Endothelium of Metastatic Melanomas

Abstract: Angiogenesis of human melanomas has been the focus of intense interest since it was shown that the spread and prognosis of primary tumors is correlated with their vascularization (N Weidner, J P Semple, W R Welch, and J Folkman, N Engl J Med, 324: 1–8, 1991) Basic fibroblast growth factor (bFGF) and its high-affinity receptor FGFR-1 have been implicated in melanoma growth and angiogenesis (R Halaban, Y Funasaka, J Lee, J Rubin, D Ron, and D Birnbaum, Fibroblast Growth Factors in Normal and Malignant Melanocytes, pp 232–243 New York: The New York Academy of Sciences, 1991) We have studied the expression of the Tie endothelial cell receptor tyrosine kinase mRNA in skin and primary cutaneous melanomas as well as in their skin and brain metastases by in situ hybridization The Tie probe hybridized very weakly with the vascular endothelium of capillaries of normal skin, while it was detected in larger arteries and veins as well as in capillaries around sweat glands However, capillaries and medium-sized vessels within cutaneous and brain metastases of melanoma were strongly positive for Tie mRNA In contrast, endothelial cells contained very little or no FGFR-1 transcripts, whereas abundant FGFR-1 mRNA was present in melanoma tumor cells and in fibrovascular stroma In agreement with these findings, a Tie-specific amplified cDNA band was obtained by reverse transcription-polymerase chain reaction from melanoma metastases but not from normal skin These results suggest a role for the Tie receptor in the angiogenesis associated with melanoma metastases

c-Myc induces cellular susceptibility to the cytotoxic action of TNF-alpha.

Abstract: Tumor necrosis factor-alpha (TNF) is a multifunctional cytokine which is cytotoxic for some tumor cells and transformed cells. The molecular mechanisms which render transformed and tumor cells sensitive to the cytotoxic action of TNF are unclear. We show here that an increased expression of the c-Myc oncoprotein strongly increases cellular sensitivity to TNF cytotoxicity. In Rat1A fibroblasts, which are resistant to TNF, the addition of TNF with a concomitant activation of a hormone-inducible c-Myc-estrogen receptor chimera (MycER) resulted in apoptotic cell death. Similarly, c-Myc overexpression enhanced the sensitivity of NIH3T3 fibroblasts to TNF-induced death. The c-Myc and TNF-induced apoptosis was inhibited by ectopic expression of the Bcl2 oncoprotein and by the free oxygen radical scavenging enzyme Mn superoxide dismutase. Furthermore, in highly TNF-sensitive fibrosarcoma cells, antisense c-myc oligodeoxynucleotides caused a specific inhibition of TNF cytotoxicity. Our results suggest that the deregulation of c-Myc, which is common in human tumors and tumor cell lines is one reason why these cells are TNF sensitive.

Tyrosine phosphorylation of CD45 phosphotyrosine phosphatase by p50csk kinase creates a binding site for p56lck tyrosine kinase and activates the phosphatase.

Abstract: Src family protein tyrosine kinases (PTKs) play an essential role in antigen receptor-initiated lymphocyte activation. Their activity is largely regulated by a negative regulatory tyrosine which is a substrate for the activating action of the CD45 phosphotyrosine phosphatase (PTPase) or, conversely, the suppressing action of the cytosolic p50csk PTK. Here we report that CD45 was phosphorylated by p50csk on two tyrosine residues, one of them identified as Tyr-1193. This residue was not phosphorylated by T-cell PTKs p56lck and p59fyn. Tyr-1193 was phosphorylated in intact T cells, and phosphorylation increased upon treatment with PTPase inhibitors, indicating that this tyrosine is a target for a constitutively active PTK. Cotransfection of CD45 and csk into COS-1 cells caused tyrosine phosphorylation of CD45 in the intact cells. Tyrosine-phosphorylated CD45 bound p56lck through the SH2 domain of the kinase. Finally, p50csk-mediated phosphorylation of CD45 caused a severalfold increase in its PTPase activity. Our results show that direct tyrosine phosphorylation of CD45 can affect its activity and association with Src family PTKs and that this phosphorylation could be mediated by p50csk. If this is also true in the intact cells, it adds a new dimension to the physiological function of p50csk in T lymphocytes.

Rak, a novel nuclear tyrosine kinase expressed in epithelial cells

Abstract: We have identified a new tyrosine kinase from human breast cancer cells called Rak (a Russian word for cancer) that shares 5i % identity with c-Src. Sequencing of the full-length complementary DNA revealed that Rak is a tyrosine kinase with a molecular weight of 54,000 that contains 5H2 and SH3 domains, as well as tyrosine residues analogous to the autophosphorylation and regulatory tyrosines of the Src family. Biochemical and site-directed mutagenesis analyses revealed that a carboxy-terminal peptide of p54�’ was phosphorylated by a cytoplasmic tyrosine kinase (CSK) and that, as in the Src family, it is the COOH-terminal tyrosine that is phosphorylated by CSK. However, there were some properties of Rak that are distinct from Src-Iike kinases: (a) expression of Rak was predominantly in epithelialderived cell lines and tissues, especially normal liver and kidney, and cell lines of breast and colon origin; (b) Rak does not harbor the NH2-terminal glycine essential for myristylation and membrane localization; and (c) Rak possessesa putative bipartite nuclear localization signal in the SH2 domain, and subcelluIar fractionation studies revealed that p54rak resides predominantly in the

FLT4, a receptor tyrosine kinase

Abstract: FLT4 gene, expression systems and proteins are provided for use in diagnosis and treatment of conditions related to the tyrosine kinase receptor encoded by the FLT4 gene.

Rapid and efficient purification of Src homology 2 domain-containing proteins: Fyn, Csk and phosphatidylinositol 3-kinase p85.

Abstract: To analyse the regulation of Src family tyrosine kinases in vitro, we have purified Fyn and Csk, a kinase capable of regulating Fyn activity by phosphorylation, from baculovirus-infected insect cells. The proteins were purified by affinity purification over a phosphotyrosine column. Highly purified proteins were eluted from the resin by a salt gradient and further purified by ion-exchange chromatography. This purification scheme was successfully applied to a third, unrelated protein that also contains the Src homology 2 (SH2) domain, namely the 85 kDa subunit of phosphatidylinositol 3-kinase, indicating that this method is versatile and should prove applicable to any protein with an accessible SH2 domain. The binding of Csk to different phosphopeptides was tested, and specificity for the autophosphorylation site of Fyn was demonstrated. Pure Csk was used to phosphorylate Fyn and down-regulate its kinase activity, and the kinetic parameters of both the active and the repressed forms of Fyn were determined. Repression of Fyn activity by Csk reduced binding of Fyn to phosphopeptides to undetectable levels, supporting the model that predicts an intramolecular interaction of the Fyn SH2 domain with a C-terminal phosphotyrosine residue.

TCR/CD3-triggering causes increased activity of the p50csk tyrosine kinase and engagement of its SH2 domain.

Abstract: The majority of known protein tyrosine kinases are integral membrane proteins or bound to cellular membranes via a covalently attached myristic acid. An exception is the newly described p50csk tyrosine kinase, which is believed to control the activity of the src-family of protein tyrosine kinases. This small kinase does not contain a myristylation signal or other known membrane attachment motifs, and indeed appears to be cytosolic. Recently, we found that p50csk specifically phosphorylates the negative regulatory Tyr-505 of the T cell-specific src-family kinase p56lck, and thereby suppresses its catalytic activity. Here we show that p50csk is activated in Jurkat T cells within one minute after stimulation of the cells with anti-CD3 MAbs. In parallel with this activation, p50csk formed a stable complex with one major 72 kDa tyrosine phosphorylated protein and minor polypeptides at 90 and 110 kDa. The isolated SH2 domain of p50csk specifically bound the 72 kDa protein in lysates from activated, but not resting, T or B cells. By several criteria, the 72 kDa protein was not a tyrosine kinase itself and it did not react with antibodies to a panel of known proteins of this molecular size. Our results suggest that p50csk is rapidly engaged via its SH2 domain and participates in the earliest events of T cell activation.

Max activity is affected by phosphorylation at two NH2-terminal sites.

Abstract: Max is a nuclear phosphoprotein that has a dose-dependent role in regulation of Myc function. The DNA-binding activity of Max homodimers, but not of Myc/Max heterodimers, has been reported to be inhibited by NH2-terminal phosphorylation. (S. J. Berberich and M. D. Cole, Genes & Dev., 6: 166-176, 1992). Here, we have mapped the NH2-terminal in vivo phosphorylation sites of Max to Ser2 and Ser11 and show that the NH2 termini of the two major alternatively spliced forms of Max (p21max and p22max) are equally phosphorylated despite differences in their amino acid sequences following Ser11. A Max mutant deficient in the NH2-terminal phosphorylation was found to inhibit both basal and Myc-induced transcription of a reporter gene more efficiently than the wild-type protein. Similarly, the ability of Myc and Ras to induce transformation was more severely impaired by the mutant. These results indicate that the NH2-terminal phosphorylation diminishes the ability of Max to negatively interfere with Myc function. However, we found no evidence that Max phosphorylation would be regulated during cell growth or differentiation. Similarly, we observed no major cell cycle-dependent changes in the extent of phosphorylation between cell populations fractionated by centrifugal elutriation or by cell cycle inhibitors.

Myc protein: partners and antagonists

Abstract: One of the first oncogenes identified from human tumors was c-myc, which is frequently activated in Burkitt's lymphomas due to chromosomal translocations. Subsequently, members of the myc oncogene family were found to be amplified in neuroblastoma and small-cell lung cancer. In normal cells, Myc activity has been shown to be both necessary and sufficient for resting cells to enter the cell cycle. Interestingly, it appears that Myc not only drives the cell cycle, but also induces cell death by apoptosis in certain situations. Myc contains a transcriptional activation domain and a basic helix-loop-helix-leucine zipper DNA-binding and dimerization domain. As a heterodimer with a structurally related protein, Max, Myc can bind DNA in a sequence-specific manner. These results suggest that the Myc/Max heterodimer functions as a transcriptional activator of genes that are critical for the regulation of cell growth.

Antibodies recognizing tie receptor tyrosine kinase and uses thereof

Abstract: The present invention relates to an anti-Tie monoclonal antibody, 3C4C7G6, which is useful as a diagnostic tool for detecting angiogenesis-associated with neoplasia, wound healing, and a variety of other angiogenesis associated diseases and for radiological imaging of blood vessels. In addition, the disclosed antibody is useful as a therapeutic agent.

Expression of a rlf/L-myc minigene inhibits differentiation of embryonic stem cells and embroid body formation.

Abstract: Rearrangements of the L-myc proto-oncogene with the cellular gene rlf occur in a subset of human small cell lung carcinoma (SCLC) resulting in the expression of a fusion protein. To investigate whether expression of such a rlf/L-myc fusion protein could contribute to the development of SCLC we constructed a chimeric minigene where the rlf first exon and the L-myc second and third exon are under the control of the rlf promoter thereby recapitulating the events of the rearrangement. Attempts to generate transgenic mice with this minigene showed that mouse embryos containing high copy numbers of the rlf/L-myc minigene fail to develop, suggesting that the expression of a rlf/L-myc fusion protein interferes with early differentiation processes. To investigate the nature of this potential embryonic lethality further, we transfected the rlf/L-myc construct stably into embryonic stem (ES) cells. Transfected ES lines that express the rlf/L-myc construct do not show a higher proliferation rate than the parental ES line but fail to properly develop embroid bodies. In addition, outgrowth and differentiation of cells from embroid bodies was severely impaired in ES cells expressing the rlf/L-myc construct when compared to normal ES cells, again suggesting an interference of rlf/L-myc expression with proper differentiation. Expression of a rlf/L-myc fusion may therefore be of critical importance in tumorigenesis by blocking differentiation and thereby allowing continued proliferation of cells and the acquisition of further mutations leading to a fully malignant tumor.