Phorbol esters modulate the Ras exchange factor RasGRP3.

RasGRP represents the prototype of a new class of guanine nucleotide exchange factors that activate small GTPases. The guanyl nucleotide-releasing protein (GRP) family members contain catalytic domains related to CDC25, the Ras exchange factor of Saccharomyces cerevisiae. They also contain a motif resembling a pair of calcium-binding EF-hands and a C1 domain similar to the diacylglycerol interaction domain of protein kinase C. The sequence of KIAA0846, identified in a human brain cDNA library, encodes a member of the GRP family that we refer to as RasGRP3. We show here that RasGRP3 bound phorbol esters with high affinity. This binding depended on anionic phospholipids, which is characteristic of phorbol ester binding to C1 domain proteins. In addition, phorbol esters also caused activation of the RasGRP3 exchange activity in intact cells, as determined by an increase in RasGTP and phosphorylation of the extracellular-regulated kinases. Finally, both phorbol 12-myristate 13-acetate and the diacylglycerol analogue 1,2-dioctanoyl-sn-glycerol induced redistribution of RasGRP3 to the plasma membrane and/or perinuclear area in HEK-293 cells, as demonstrated using a green fluorescent fusion protein. We conclude that RasGRP3 serves as a PKC-independent pathway to link the tumor-promoting phorbol esters with activation of Ras GTPases.

History of tobacco use among Kentucky women diagnosed with invasive cervical cancer: 1997-1998.

An association between cigarette smoking and cervical cancer has been demonstrated by numerous epidemiologic studies. However, just because there is an association does not mean that there is an etiologic connection between tobacco use and cervical cancer, although some studies do indicate such a relationship. There are numerous potential explanations, including smoking as a behavior being associated with other behaviors that place women at increased risk of HPV infection. In a state like Kentucky, where the prevalence of smoking is so very high, one would expect that the disease burden from cervical cancer would also be high. A review of available data on invasive cervical cancer cases from the Kentucky Cancer Registry shows that this is indeed the case, with the incidence rate of invasive cervical cancer in Kentucky being as much as 40% higher than the SEER rate during the 1991-1998 time period. An analysis of available tobacco use history data from the KCR on women diagnosed with invasive cervical cancer during 1997-1998 shows that 61% of them indicated a history of tobacco use.

RasGRP links T-cell receptor signaling to Ras.

Stimulation of the T-cell receptor (TCR) alters a number of intracellular signaling pathways including one that involves protein tyrosine kinases, phospholipase C-gamma1 (PLC-gamma1), diacylglycerol (DAG), and calcium messengers. By a divergent pathway, TCR-stimulated protein tyrosine kinase activity is thought to result independently in recruitment of the Ras activator Sos to the plasma membrane, leading to Ras activation. Here we show that RasGRP, a Ras activator that contains calcium-binding EF hands and a DAG-binding domain, is expressed in T cells. A PLC-gamma1 inhibitor diminished activation of Ras following TCR stimulation. Membranes from TCR-stimulated Jurkat T cells exhibited increased RasGRP and increased Ras-guanyl nucleotide association activity that was inhibited by antibodies directed against RasGRP. Overexpression of RasGRP in T cells enhanced TCR-Ras-Erk signaling and augmented interleukin-2 secretion in response to calcium ionophore plus DAG analogues phorbol ester myristate or bryostatin-1. Thus, RasGRP links TCR and PLC-gamma1 to Ras-Erk signaling, a pathway amenable to pharmacologic manipulation.

RasGRP is essential for mouse thymocyte differentiation and TCR signaling.

The Ras signaling pathway plays a critical role in thymopoiesis and T cell activation, but the mechanism of Ras regulation is controversial. At least one mode of Ras regulation in T cells involves the messenger diacylglycerol (DAG). RasGRP, a Ras activator with a DAG-binding C1 domain, is expressed in T cells and thymocytes. Here we show that thymi of RasGRP-null mutant mice have approximately normal numbers of immature thymocytes but a marked deficiency of mature, single-positive (CD4+CD8- and CD4-CD8+) thymocytes. In Ras signaling and proliferation assays, mutant thymocytes showed a complete lack of response to DAG analogs or T cell receptor (TCR) stimulation by antibodies. Thus, TCR and DAG are linked through RasGRP to Ras signaling.

Hypothermic stress leads to activation of Ras-Erk signaling.

The small GTPase Ras is converted to the active, GTP-bound state during exposure of vertebrate cells to hypothermic stress. This activation occurs more rapidly than can be accounted for by spontaneous nucleotide exchange. Ras-guanyl nucleotide exchange factors and Ras GTPase-activating proteins have significant activity at 0 degrees C in vitro, leading to the hypothesis that normal Ras regulators influence the relative amounts of Ras-GTP and Ras-GDP at low temperatures in vivo. When hypothermic cells are warmed to 37 degrees C, the Raf-Mek-Erk protein kinase cascade is activated. After prolonged hypothermic stress, followed by warming to physiologic temperature, cultured fibroblasts assume a rounded morphology, detach from the substratum, and die. All of these biologic responses are attenuated by pharmacologic inhibition of Mek. Previously, it had been found that low temperature blocks acute growth factor signaling to Erk. In the present study, we found that this block occurs at the level of Raf activation. Temperature regulation of Ras signaling could help animal cells respond appropriately to hypothermic stress, and Ras-Erk signaling can be manipulated to improve the survival of cells in cold storage.

RasGRP, a Ras activator: mouse and human cDNA sequences and chromosomal positions.

We recently reported the molecular cloning of a novel transforming rat brain cDNA, rbc7, that encodes a Ras activator (Ebinu et al. Science 280, p. 1082, 1998). We proposed that this cDNA is a 5' and 3' truncated version of a larger normal transcript that encodes a predicted 90-kDa protein which we called RasGRP (Ras guanyl nucleotide releasing protein). We have now studied the structure of the mouse and human sequences and confirmed our conclusions about the nature of the 5' truncation. The human gene has been localized to 15q15 by an in situ hybridization technique, while the mouse gene has been positioned on Chr 2 near thrombospondin by linkage analysis. The relatedness of RasGRP to another human sequence and a hypothetical nematode protein are also discussed.

RasGRP, a Ras guanyl nucleotide- releasing protein with calcium- and diacylglycerol-binding motifs.

RasGRP, a guanyl nucleotide-releasing protein for the small guanosine triphosphatase Ras, was characterized. Besides the catalytic domain, RasGRP has an atypical pair of "EF hands" that bind calcium and a diacylglycerol (DAG)-binding domain. RasGRP activated Ras and caused transformation in fibroblasts. A DAG analog caused sustained activation of Ras-Erk signaling and changes in cell morphology. Signaling was associated with partitioning of RasGRP protein into the membrane fraction. Sustained ligand-induced signaling and membrane partitioning were absent when the DAG-binding domain was deleted. RasGRP is expressed in the nervous system, where it may couple changes in DAG and possibly calcium concentrations to Ras activation.

Interaction of activated Ras with Raf-1 alone may be sufficient for transformation of rat2 cells.

v-H-ras effector mutants have been assessed for transforming activity and for the ability of the encoded proteins to interact with Raf-1-, B-Raf-, byr2-, ralGDS-, and CDC25-encoded proteins in the yeast two-hybrid system. Transformation was assessed in rat2 cells as well as in a mutant cell line, rv68BUR, that affords a more sensitive transformation assay. Selected mutant Ras proteins were also examined for their ability to interact with an amino-terminal fragment of Raf-1 in vitro. Finally, possible cooperation between different v-H-ras effector mutants and between effector mutants and overexpressed Raf-1 was assessed. Ras transforming activity was shown to correlate best with the ability of the encoded protein to interact with Raf-1. No evidence for cooperation between v-H-ras effector mutants was found. Signaling through the Raf1-MEK-mitogen-activated protein kinase cascade may be the only effector pathway contributing to RAS transformation in these cells.

ras effector loop mutations that dissociate p120GAP and neurofibromin interactions.

ras proteins are positively regulated by nucleotide exchange factors and negatively regulated by GTPase-activating proteins (GAPs). Two GAPs have been found in mammalian cells, p120GAP and neurofibromin, the product of the type 1 neurofibromatosis (NF1) gene. A library of substitutions in the effector loop region of ras in an Escherichia coli plasmid expression system was screened for c-Ha-ras species with altered GAP interactions. Several substitutions preferentially disrupted the interaction of ras with p120GAP as compared with the interaction with the recombinant GAP-related domain of neurofibromin (NF1-GRD). The most extreme example, Tyr32His, encoded a ras species that was unaffected by p120GAP but was stimulated normally by NF1-GRD. Tyr32His was weakly transforming in Rat2 cells. Tyr32His ras was primarily GDP-bound in quiescent Rat2 cells, although it rapidly associated with GTP after treatment of cells with epidermal growth factor. These results show that the NF1 product has less stringent requirements than p120GAP for ras effector domain structure and that negative regulation of ras can be achieved in rat fibroblasts by the product of NF1.

RAS signalling is abnormal in a c-raf1 MEK1 double mutant.

A mutant rat cell clone that suppresses the transformation defects of RAS effector loop substitutions is heterozygous for mutations in c-raf1 and MEK1. The mutant cells can be transformed by many otherwise defective RAS effector mutants, including RAS genes with the effector regions of distantly related GTPases, even though the encoded RAS proteins do not interact with either the mutant or wild-type RAF in Saccharomyces cerevisiae. While the significance of the c-raf1 mutation is unclear, the MEK1 mutation increases MEK1 activity and leads to activation of mitogen-activated protein kinase. The mutant MEK1 is coupled to the epidermal growth factor pathway but exhibits decreased physical interaction with RAF. When overexpressed, the MEK1 mutation is transforming and causes hyperphosphorylation of RAF. Signalling from RAS to MEK1 may be mediated by something other than RAF alone, but signalling through MEK1 is probably sufficient for RAS transformation.

Mutations in the structural gene for cytochrome c result in deficiency of both cytochromes aa3 and c in Neurospora crassa.

The cyt-12-12 mutant of Neurospora crassa is characterized by slow growth and a deficiency of spectrophotometrically-detectable cytochromes aa3 and c. Using a sib-selection procedure we have isolated the cyt-12+ allele from a cosmid library of N. crassa genomic DNA. Characterization of the cyt-12+ allele reveals that it encodes the structural gene for cytochrome c. DNA sequence analysis of the cyt-12-12 allele revealed a mutation in the cytochrome c coding sequence that results in replacement of a glycine residue, which is invariant in the cytochrome c of other species, with an aspartic acid. Genetic analysis confirms that cyt-12-12 is allelic with the previously-characterized cyc-1-1 mutant, which was also shown to affect the single locus encoding cytochrome c in N. crassa. We suggest that the amount of functional cytochrome c present in mitochondria influences the level of cytochrome aa3.

Isolation and characterization of a cDNA clone coding for the calcium-binding subunit of calcineurin from bovine brain: an identical amino acid sequence to the human protein.

A cDNA clone encoding the calcium-binding subunit of calcineurin, calcineurin B, was isolated from a bovine brain library by immunoscreening. The 841 bp cDNA has a 56 bp 5'-noncoding region, an open reading frame of 510 bp, and a 275 bp 3'-noncoding sequence. The deduced amino acid sequence of bovine calcineurin B differs from the previously reported protein sequence (Aitken et al., 1984) by three residues. The sequence contained additional valine at the carboxyl terminus and substitutions of Met-11 and Ser-153 (the positions according to Aitken et al., 1984) by cysteine. The amino acid sequence of bovine calcineurin B was found to be identical to that of human calcineurin B sequence (Guerini et al., 1989). In fact, 97.1% homology was observed between the coding regions of human and bovine calcineurin B. In addition, a very high homology of 95.2% was observed for the 3'-noncoding region while the 5'-noncoding region showed 58.9% homology. The beta-galactosidase fusion protein, having the apparent molecular weight of 29 kDa, was detected on Western blots by subunit B-specific monoclonal antibody (Matsui et al., 1985). Northern analysis revealed that there is a single calcineurin B transcript in bovine brain which is 2.3 kb in length. This is in agreement with the observation of only one immunologically detectable subunit B protein in bovine brain (Matsui et al., 1985).

Effector domain mutations dissociate p21ras effector function and GTPase-activating protein interaction.

The GTPase activity of p21ras is stimulated by GTPase-activating proteins (GAPs) such as p120GAP and the product of the neurofibromatosis 1 gene, which may negatively regulate p21 function. GAPs are also proposed effectors of ras. We have sought activating substitutions in c-H-ras in the region encoding the effector domain, on the rationale that such mutations would dissociate effector function from negative regulation by GAP. One such activating mutation, Pro-34-->Arg, encodes protein that is substantially bound to GTP in vivo. In vitro, this protein is not stimulated by GAPs, and its binding to p120GAP is grossly impaired. The results support the idea that the p21 structural requirements for effector function and GAP interaction are quite different and suggest that some molecule(s) other than p120GAP serves as the ras effector. In contrast to the results obtained with p120GAP, the Pro-34-->Arg p21 species is effectively coupled to the raf-1 product, as judged from electrophoretic mobility shifts of the Raf-1 phosphoprotein.

The murine leukemia inhibition factor gene (Lif) is located on proximal chromosome 11, not chromosome 13.

Lif, the murine gene encoding leukemia inhibition factor (LIF), has been previously localized to proximal Chromosome (Chr) 11. Hilda, the murine gene encoding "human interleukin in DA cells" (HILDA) has been localized to Chr 13. Since these two growth factors are identical, the proposal for two different structural loci is intriguing. To address this issue, blot hybridization methods have been used to establish the position of the structural gene sequence unambiguously. DNAs from somatic cell hybrids, recombinant inbred mice, and backcross mice have been probed with a sequence that encodes LIF/HILDA. The results support the assignment of this sequence to proximal Chr 11. These studies also establish a synteny group, including Lif and Tcn-2, the structural gene for transcobalamin 2, that is conserved between man and mouse.

Nucleotide sequence of the Varkud mitochondrial plasmid of Neurospora and synthesis of a hybrid transcript with a 5' leader derived from mitochondrial RNA.

The Mauriceville and Varkud mitochondrial plasmids of Neurospora are closely related, closed circular DNAs (3.6 and 3.7 kb, respectively; 1 kb = 10(3) bases or base-pairs), whose characteristics suggest relationships to mitochondrial DNA introns and retrotransposons. Here, we characterized the structure of the Varkud plasmid, determined its complete nucleotide sequence and mapped its major transcripts. The Mauriceville and Varkud plasmids have more than 97% positional identity. Both plasmids contain a 710 amino acid open reading frame that encodes a reverse transcriptase-like protein. The amino acid sequence of this open reading frame is strongly conserved between the two plasmids (701/710 amino acids) as expected for a functionally important protein. Both plasmids have a 0.4 kb region that contains five PstI palindromes and a direct repeat of approximately 160 base-pairs. Comparison of sequences in this region suggests that the Varkud plasmid has diverged less from a common ancestor than has the Mauriceville plasmid. Two major transcripts of the Varkud plasmid were detected by Northern hybridization experiments: a full-length linear RNA of 3.7 kb and an additional prominent transcript of 4.9 kb, 1.2 kb longer than monomer plasmid. Remarkably, we find that the 4.9 kb transcript is a hybrid RNA consisting of the full-length 3.7 kb Varkud plasmid transcript plus a 5' leader of 1.2 kb that is derived from the 5' end of the mitochondrial small rRNA. This and other findings suggest that the Varkud plasmid, like certain RNA viruses, has a mechanism for joining heterologous RNAs to the 5' end of its major transcript, and that, under some circumstances, nucleotide sequences in mitochondria may be recombined at the RNA level.