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.

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 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.

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).

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.