Biomedical subjects

M F Verderame

Publications and source records attributed to M F Verderame.

27 records · Page 2Linked to original sources

PubMed1995

cDNA cloning and characterization of a Cek7 receptor protein-tyrosine kinase ligand that is identical to the ligand (ELF-1) for the Mek-4 and Sek receptor protein-tyrosine kinases.

We have isolated a murine cDNA encoding a ligand for the Cek7 receptor protein-tyrosine kinase (RPTK), a member of the Eph/Eck RPTK subfamily. Sequence analysis predicts an open reading frame of 209 amino acids with a predicted molecular mass of 24 kDa. The Cek7 ligand shows a 48% sequence identity at the protein level to B61, a ligand for the related Eck RPTK, 30% to the Cek5 ligand, 59% to the recently cloned Ehk1-L, and identity to ELF-1, a recently described ligand for the Mek4 and Sek RPTKs. The expressed Cek7 ligand is functionally active as it induces autophosphorylation of the Cek7 RPTK.

Amino Acid Sequence↗

PubMed1995

Transformation and pp60v-src autophosphorylation correlate with SHC-GRB2 complex formation in rat and chicken cells expressing host-range and kinase-active, transformation-defective alleles of v-src.

The biochemical properties of several pp60v-src substrates believed to participate in src-mediated transformation were examined in cells expressing a kinase-active, transformation-defective v-src allele (v-src-F172 delta/Y416F) and its parental allele, v-src-F172 delta, a host-range--dependent allele that transforms chicken cells to a fusiform morphology, but does not transform rat cells. Because pp60v-src-F172 delta is dependent on autophosphorylation for transforming ability, these alleles provide a unique opportunity to examine the role of pp60v-src autophosphorylation in regulating substrate interactions. Increased pp125FAK tyrosine phosphorylation and high levels of pp60v-src-associated phosphotidylinositol-3' kinase activity were detected specifically in chicken cells exhibiting round, refractile transformation but not in cells transformed to a fusiform morphology. Increased pp125FAK kinase activity, but not increased pp125FAK tyrosine-phosphorylation correlated with pp60v-src autophosphorylation and increased anchorage-independent growth. Thus, pp125FAK and PI3'K may participate in morphological transformation by v-src. Furthermore, association of phosphorylated SHC with the adapter GRB2 correlated with increased anchorage-independent growth (and autophosphorylation) in both rat and chicken cells independent of the morphological phenotype induced. Therefore, host-range dependence for transformation may be regulated through association of SHC with GRB2, thus implicating SHC as a crucial substrate for src-dependent transformation.

Adaptor Proteins, Signal Transducing↗

PubMed1994

Identification of a complete Cek7 receptor protein tyrosine kinase coding sequence and cDNAs of alternatively spliced transcripts.

Receptor protein tyrosine kinases (RPTK) are critical components of signal transduction pathways in multicellular organisms. Identification of new RPTK constitutes an initial step in understanding the variety of signalling pathways in which these proteins participate. In this study, a cDNA containing a complete coding sequence for Cek7 (chicken RPTK) has been cloned from a chicken embryo expression library using anti-phosphotyrosine antibodies (Ab). Cek7 is a member of the EPH (human RPTK) subfamily of RPTK; this subfamily is characterized by extracellular domains containing an immunoglobulin-like motif, a Cys-rich region and two fibronectin type-III repeats. Analysis of additional cDNAs revealed that two positions of alternative splicing in primary transcripts may produce several isoforms of this RPTK; cDNAs corresponding to three isoforms of this receptor are reported. These isoforms are predicted to have altered extracellular ligand-binding domains and/or altered cytoplasmic juxtamembrane regions. The nucleotide sequence of cek7 cDNAs identified in this study diverges at the 3' end from the sequence found in a recently described partial cek7 cDNA [Sajjadi and Pasquale, Oncogene 8 (1993) 1807-1813]. Therefore, a third position of alternative splicing may produce Cek7 RPTK with divergent C-terminal tails. RNA blot analysis revealed expression of this receptor at highest levels in the central nervous system and eyes of 10-day-old chicken embryos.

Alternative Splicing↗

PubMed1994

Autophosphorylation is required for high kinase activity and efficient transformation ability of proteins encoded by host range alleles of v-src.

pp60v-src is a nonreceptor protein tyrosine kinase that can transform both chicken and rodent fibroblasts. The src homology 2 (SH2) domain of this protein serves a critical role in the regulation of protein tyrosine kinase activity. The host range proteins pp60v-src-L, which contains a deletion of a highly conserved residue (Phe-172) in the SH2 domain, and pp60v-src-PPP, which contains a change from a Leu to a Phe at amino acid 186 in the SH2 domain, transform chicken but not rat cells and have slightly reduced kinase activity measured in vitro. The data presented here show that these altered proteins require autophosphorylation on Tyr-416 for high kinase activity and transforming ability. In the absence of autophosphorylation, there is a further decrease of at least threefold in in vitro kinase activity relative to the phosphorylated host range parental protein, no morphological transformation, a reduction in anchorage independent growth, and no disruption of the actin cytoskeleton. In addition, these SH2 mutations abolish the ability of the SH2 domain to bind a phosphorylated peptide that corresponds to the autophosphorylation site of pp60src. Thus, like mutant alleles of c-src encoding transformation competent proteins, and unlike v-src, transformation by pp60v-src-F172 delta and pp60v-src-L186F is dependent on phosphorylation of Y-416 for high kinase activity and transformation ability. The dependence of transformation on phosphotyrosine is not a reflection of an intramolecular interaction between the autophosphorylation site and the SH2 domains since purified SH2 domains are incapable of binding phosphorylated autophosphorylation site peptides in vitro.

Alleles↗

PubMed1994

Highly conserved amino acids in the SH2 and catalytic domains of v-src are altered in naturally occurring, transformation-defective alleles.

We have identified 11 novel point mutations that abolish the transforming capacity of the oncogene v-src. These transformation-defective alleles were originally identified in morphologically flat subclones of rat cells transformed by wild type v-src. Nine of the mutations affect amino acid residues that are highly conserved in the catalytic domain of pp60v-src and completely abolish kinase activity. The other 2 mutations alter conserved residues in the SH2 domain (Phe-172 replaced with Val in one case [F172V] and Leu-186 replaced with Phe in the other [L186F]), drastically reducing, but not eliminating, kinase activity. The enzymatic and transforming functions of one of the SH2 mutants, L186F are host dependent; the mutant protein is active in chicken cells, but inactive in rat cells, as previously observed for some other SH2 mutants. These results are interpreted in relation to the recently described three-dimensional structures of SH2 domains and of the catalytic domain of a protein kinase. In addition, they support a role for the SH2 domain in the regulation of kinase activity.

Alleles↗

PubMed1992

Transforming growth factor beta 1 partially suppresses the transformed phenotype of ras-transformed hepatocytes.

The effect of transforming growth factor beta type 1 (TGF-beta 1) on DNA synthesis, anchorage-dependent and anchorage-independent proliferation, cytoskeletal organization, and gene expression in ras-transformed simian virus 40 (SV40)-immortalized hepatocyte cell lines was measured. An SV40-immortalized cell line (CWSV1), a control neo-transfected and selected cell line (N1), and neo+ras-transfected and selected cell lines (NR3 and NR4) were used for this study. CWSV1 and N1 cells do not grow in soft agarose and are not tumorigenic. The ras-transformed hepatocytes NR3 and NR4 grow in soft agar and are tumorigenic. TGF-beta 1 treatment did not inhibit DNA synthesis or anchorage-dependent growth in the SV40-immortalized hepatocyte cell line CWSV1 or in the ras-transformed hepatocytes. TGF-beta 1 treatment inhibited anchorage-independent growth, increased actin cytoskeleton organization, and altered the morphology of ras-transformed hepatocytes; that is, with regard to all three of these properties, TGF-beta 1-treated ras-transformed hepatocytes more closely resembled the immortalized parent cell line. c-Ha-ras and c-myc RNA levels were not altered in TGF-beta 1-treated NR4 cells. TGF-beta 1 treatment did alter expression of some genes in NR4 cells. The level of expression of alpha 1 integrin RNA was higher in CWSV1 cells than in NR4 cells and increased in NR4 cells when they were treated with TGF-beta 1. Similarly, the levels and profiles of integrins on the cell surface of CWSV1 cells compared to NR4 cells, as determined by cell surface protein iodination, differed and in TGF-beta 1-treated NR4 cells more closely resembled the surface integrin profile for CWSV1 cells.

Actins↗

PubMed1989

A mutation in v-src that removes a single conserved residue in the SH-2 domain of pp60v-src restricts transformation in a host-dependent manner.

The v-src oncogene of Rous sarcoma virus (RSV) is able to transform both avian and mammalian cells, but the mutant allele v-src-L displays a host range dependence for transformation, transforming chicken but not rat cells with wild-type efficiency. This host range restriction can be detected by measuring growth in low serum, saturation density, and anchorage independent growth. In addition, rat cells expressing v-src-L do not form tumors in syngeneic rats or nude mice, but RSV carrying the mutant allele causes tumors in chicks, although at a reduced efficiency and with increased latency. To determine the lesion responsible for this phenotype, we sequenced the entire v-src gene from the parental B77 strain of RSV, as well as the mutant allele. v-src-L is missing 3 nucleotides present in the wild-type parent, RSV B31, eliminating Phe-172, an invariant residue in a conserved region of src-related proteins known as SH-2. The kinase activity of pp60v-src-L was indistinguishable from that of the wild type in chicken cells but was significantly reduced in rat cells as assayed by an in vitro immune complex assay; in vivo phosphorylation of one specific substrate, p36 (calpactin I heavy chain); and total phosphotyrosine-containing proteins. In addition, the pattern of phosphotyrosine-containing proteins in rat cells was qualitatively different when cells containing pp60v-src-L were compared with cells with wild-type pp60v-src, even though both pp60v-src proteins were membrane associated. The data are consistent with a role for the SH-2 region in substrate specificity.

Alleles↗

PubMed1986

Expression of 100,000-Mr simian virus 40 (SV40) tumor antigen in mouse fibroblasts transfected with replication-defective SV40 genomes.

Simian virus 40 early region mutants which are partially or completely replication defective were tested for their ability to transform postcrisis mouse fibroblasts. All mutants tested were capable of generating anchorage-independent transformants. We have previously reported the presence of a variant tumor antigen of 100,000 Mr (100K protein) generated upon transformation by wild-type simian virus 40 virions which correlates with anchorage-independent growth (Chen et al., Mol. Cell. Biol. 1:994-1006, 1981). In this study, none of the mutants tested produced the 100K variant protein at early (before the fifth) passage. Long-term passage (greater than 20 weeks) permitted the expression of this 100K variant in half of the transformants. Thus the phenotype of these mutants is different from both wild-type simian virus 40 (frequently production of 100K by the third passage, and always by the tenth passage) and the origin-minus class of mutants (no production of 100K at any passage).

Animals↗

PubMed1983

94,000- and 100,000-molecular-weight simian virus 40 T-antigens are associated with the nuclear matrix in transformed and revertant mouse cells.

A small fraction of the 94,000-molecular-weight multifunctional large T-antigen of simian virus 40 was associated with the nuclear protein matrix derived from simian virus 40-transformed mouse cells. The interaction between this fraction of T-antigen and the matrix was largely or entirely independent of nuclear DNA. Similar amounts of T-antigen were retained by the nuclei of transformed and revertant cell lines. A 100,000-molecular-weight variant of T-antigen, which has been found to correlate specifically with anchorage-independent growth, was present in the nuclear protein matrix of a transformed cell line. A T-antigen-containing revertant selected for the reacquisition of a high serum requirement and an anchorage requirement for growth retained T-antigen in association with its matrix.

Animals↗