RasGRP4 is a novel Ras activator isolated from acute myeloid leukemia

Although a number of genetic defects are commonly associated with acute myeloid leukemia (AML), a large percentage of AML cases are cytogenetically normal. This suggests a functional screen for transforming genes is required to identify genetic mutations that are missed by cytogenetic analyses. We utilized a retrovirus-based cDNA expression system to identify transforming genes expressed in cytogenetically normal AML patients. We identified a new member of the Ras guanyl nucleotide-releasing protein (RasGRP) family of Ras guanine nucleotide exchange factors, designating it RasGRP4. Subsequently, cDNA sequences encoding rodent and human RasGRP4 proteins were deposited in GenBank. RasGRP4 contains the same protein domain structure as other members of the RasGRP family, including a Ras exchange motif, a CDC25 homology domain, a C1/diacyglycerol-binding domain, and putative calcium-binding EF hands. We show that expression of RasGRP4 induces anchorage-independent growth of Rat1 fibroblasts. RasGRP4 is a Ras-specific activator and, interestingly, is highly expressed in peripheral blood leukocytes and myeloid cell lines. Unlike other RasGRP proteins, RasGRP4 is not expressed in the brain or in lymphoid cells. We demonstrated that 32D myeloid cells expressing RasGRP4 have elevated levels of activated Ras compared with control cells, and phorbol 12-myristate 13-acetate (PMA) treatment greatly enhanced Ras activation. PMA induced membrane localization of RasGRP4 and 32D cells expressing RasGRP4 were capable of cytokine-independent proliferation in the presence of PMA. We conclude that RasGRP4 is a member of the RasGRP family of Ras guanine nucleotide exchange factors that may play a role in myeloid cell signaling growth regulation pathways that are responsive to diacylglycerol levels.     

RasGRP1 represents a novel non-protein kinase C phorbol ester signaling pathway in mouse epidermal keratinocytes

The mouse skin model of carcinogenesis has been instrumental in our appreciation of the multistage nature of carcinogenesis. In this system, tumor promotion is a critical step in the generation of tumors and is usually achieved by treatment with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Although it is generally assumed that protein kinase C (PKC) is the sole receptor for TPA in this system, we sought to evaluate whether non-PKC pathways could also contribute to the effects of phorbol esters in skin. We documented expression of the high affinity non-PKC phorbol ester receptor and Ras activator RasGRP1 in mouse primary keratinocytes. Overexpression of RasGRP1 in keratinocytes increased the level of active GTP-loaded Ras. TPA treatment further elevated this Ras activation in a PKC-independent manner and induced the translocation and down-regulation of RasGRP1. Overexpression of RasGRP1 in keratinocytes also caused apoptosis. Finally, induction of keratinocyte differentiation by elevation of extracellular calcium suppressed expression of endogenous RasGRP1, whereas overexpression of RasGRP1 inhibited expression of the differentiation markers keratins 1 and 10 induced by high calcium in the medium. Taken together, our results demonstrate that RasGRP1 is an additional diacylglycerol/phorbol ester receptor in epidermal keratinocytes and suggest that activation of this novel receptor may contribute to some of the phorbol ester- and Ras-mediated effects in mouse epidermis.     

The human homolog of the mouse brown gene maps to the short arm of chromosome 9 and extends the known region of homology with mouse chromosome 4.

The mouse brown locus encodes a tyrosinase-related protein, TRP-1. The human homolog of TRP-1 was recently cloned from a melanoma cDNA library and sequenced. We have made oligonucleotide primers corresponding to the human TRP1 3' untranslated region and used them to map the human TRP1 gene by species-specific PCR in human/rodent somatic cell hybrids. By this means, the human TRP1 gene has been mapped to the short arm of chromosome 9.     

cDNA Cloning and Chromosomal Mapping of a Novel Human GAP (GAP1M), a GTPase-Activating Protein of Ras

We have previously isolated a novel Ras GTPase-activating protein (Ras GAP), Gap1^m, from rat brain. Gap1^mis considered to be a negative regulator of the Ras signaling pathways, like other Ras GAPs, neurofibromin, which is a gene product of the neurofibromatosis type I gene, and p120GAP. In this study we have isolated a human cDNA of this Gap and mapped the gene. The gene encodes a protein of 853 amino acids that shows 89% sequence identity to rat Gap1^m. The human gene was mapped to chromosome 3 by PCR analysis on a panel of human–mouse hybrid cells. FISH analysis refined the location of the gene further to 3q22–q23.     

Nucleotide sequence of mouse L19 ribosomal protein cDNA isolated in screening with tre oncogene probes.

A cDNA library was prepared from cytoplasmic poly(A)RNA from mouse NIH-3T3 cells carrying a transfected human tre oncogene. Screening with tre gene probes identified a tre cDNA clone 11-4 and a co-purifying weakly hybridizing cDNA clone 11-5. The 11-5-specific RNA was expressed in both nontransfected and tre-transfected NIH-3T3 cells, showing it is of mouse rather than tre gene origin. Its nucleotide sequence was 717 bp long and contained, starting from the first nucleotide, an open reading frame of 588 bp followed by a 3' noncoding region and 26 A residues at the 3' terminus. Comparison with the GenBank data base revealed 93.7% homology with cDNA encoding the rat L19 ribosomal protein. Furthermore, the 196-amino-acid polypeptide deduced from 11-5 was of the same length and contained only one amino acid difference compared with the rat L19 protein. Comparison with the weakly hybridizing tre gene probe showed stretches of homology that were, however, too short to be taken into consideration. We conclude that the 11-5 sequence encodes the mouse L19 ribosomal protein.     

Differential splicing in mouse thymus generates two forms of terminal deoxynucleotidyl transferase.

A new form of TdT mRNA has been identified by screening a mouse thymus cDNA library. It contains an open reading frame of 1527 base pairs corresponding to a protein containing 509 aminoacids, whereas the previously identified mouse TdT mRNA is composed of 1587 base pairs and encodes a protein of 529 aminoacids. Analysis of a mouse genomic clone containing the 3' portion of the TdT gene shows that these twenty additional aminoacids are encoded by an additional exon located between exons X and XI. Both forms of TdT mRNA are present in the thymus and could be generated by alternative splicing. The cDNA reported here corresponds to the major form of TdT mRNA in Balb/c mice and closely resembles human and bovine TdT cDNA. Expression of this cDNA in mammalian cells shows that it encodes a functional protein capable of catalysing N region insertions at the recombination junction of an episomic recombination substrate.     

Distribution of ras guanyl releasing protein (RasGRP) mRNA in the adult rat central nervous system

In the nervous system, Ras signal transduction pathways are involved in cellular differentiation, neuronal survival and synaptic plasticity. These pathways can be modulated by Ras guanyl nucleotide exchange factors (Ras GEFs), which activate Ras protein by catalyzing the exchange of GDP for GTP. RasGRP, a recently discovered Ras GEF is expressed in brain as well as in T cells. In addition to the catalytic domain which catalyzes dissociation of Ras-GDP, RasGRP has a pair of calcium-binding EF hands and a diacylglycerol binding domain. The structure of RasGRP suggests that it serves to link calcium and lipid messengers to Ras signaling pathways. We have used an RNase protection assay to detect RasGRP mRNA in various regions of the rat brain and we have determined the cellular distribution of RasGRP mRNA by in situ hybridization. RasGRP mRNA is widely distributed and is present in both interneurons and projection neurons but not confined to any neuronal type or neurotransmitter phenotype. The presence of RasGRP mRNA in archicortical neurons suggests that this pathway may be important in phylogenetically older regions of the CNS. The restriction of RasGRP mRNA to subsets of neurons suggests that activation of Ras by RasGRP has a specific function in certain neuronal types. We did not detect RasGRP in glial cells.     

Isolation and genomic analysis of the human surf-6 gene: a member of the Surfeit locus

The human Surfeit locus contains at least six tightly clustered genes (Surf-1 to Surf-6) of which five (Surf-1 to Surf-5) have been characterised and found not to share any sequence homology. The organisation and juxtaposition of the Surfeit genes are conserved between human and mouse. The Surf-6 gene that encodes a novel nucleolar-matrix protein with nucleic-acid binding properties has been characterised in mouse. In this work, we have isolated and analysed the human Surf-6 homologue and determined its genomic organisation in the Surfeit locus. The human Surf-6 gene has five exons spread over a distance of 4.3kb and has features of a housekeeping gene being ubiquitously expressed, having its 5' end located within a CpG rich island and lacking a canonical TATA box. The intragenic region between the 3' end of the Surf-5 gene and the 5' end of the Surf-6 gene is 3.2kb and contains a pseudogene of the ribosomal protein gene rpL21. The putative human Surf-6 protein is 361 amino acids long and includes motifs found in both the mouse and fish Surf-6 homologues, which may underlie the functions of Surf-6. Three amino acid polymorphisms have been detected at codons 163, 175 and 311 by SSCP analysis.     

Cloning and expression of feline interleukin 15

A cDNA encoding feline interleukin 15 (IL15) was cloned from the lymph node of a cat infected with feline infectious peritonitis virus. The cDNA is 486 bp in length and encodes a protein of 162 amino acids. Recombinant protein was readily expressed as a GST fusion in Escherichia coli and purified by glutathione affinity chromatography. Expression of recombinant protein in mammalian cells was only accomplished by eliminating the 5' and 3' UTR, replacing the IL15 signal peptide with the tissue plasminogen activator signal peptide, and adding 3' sequence to disrupt presumptive secondary structure of the mRNA. Biologically active feline IL15 was expressed in HEK293T cells and was shown to sustain primary feline lymphocytes, a feline T cell line, and mouse CTLL-2 cells. Proliferation of CTLL-2 cells was induced by the recombinant protein in a dose-dependent manner. Monoclonal and polyclonal antibodies against human IL15 recognized feline IL15 in immunofluorescence and Western blot assays. Additionally, feline IL15 was detectable using a commercially available human IL15 ELISA kit.     

Cloning, expression, and chromosomal localization of the 140-kilodalton subunit of replication factor C from mice and humans.

We have isolated a full-length mouse cDNA encoding a lysine-rich protein of 1,131 amino acids with a calculated molecular mass of 126 kDa. The protein binds in a sequence-unspecific manner to DNA, is localized exclusively in the nucleus, and contains a putative ATP binding site and a stretch of 80 amino acids with homology to the carboxy terminus of prokaryotic DNA ligases. On the basis of the following facts, we conclude that the isolated cDNA encodes the 140-kDa subunit of mouse replication factor C (mRFC140). (i) The sequence around the ATP binding site shows significant homology to three small subunits of human replication factor C. (ii) Polyclonal antibodies raised against the protein encoded by this cDNA cross-react with the 140-kDa subunit of purified human replication factor C (hRFC140) and recognize in mouse cell extracts an authentic protein with an apparent molecular mass of 130 kDa. (iii) Sequence comparison with a human cDNA isolated by using tryptic peptide sequence information from purified hRFC140 revealed 83% identity of the encoded proteins. The mRFC140 gene is ubiquitously expressed, and two mRNAs approximately 5.0 and 4.5 kb long have been detected. The gene was mapped by in situ hybridization to mouse chromosome 5, and its human homolog was mapped to chromosome 4 (p13-p14).