The ras gene product (p21) is a GTP-binding protein and has been thought to transduce signals regulating proliferation or differentiation of cells. Like other GTPbinding proteins, p2l-GTP is an active conformation, which can transduce the signals downstream, whereas p21GDP is an inactive one. Recently, we have shown that p2FGTP levels increased in cells treated with fetal bovine serum or plateletderived growth factor to initiate DNA synthesis. In this paper, we report that epidermal growth factor can also increase the amounts of p2l-GTP in the cells. Effects of epidermal growth factor and platelet-derived growth factor are not additive. In contrast, mutant [Val'2Jp21, which has transforming activity, responded neither to platelet-derived growth factor nor to epidermal growth factor. We also found that the ratio of p21-GTP to p2l-GDP increased 3-to 4-fold in transformants carrying activated erbB-2/neu or v-src oncogenes. These results strongly suggest an important role of p21 in transduction of signals for both normal proliferation and malignant transformation through growth factor receptors with tyrosine kinase activity or related oncogene products.Mammalian ras genes are thought to be involved in signal transduction pathways of proliferation or differentiation in many types of cells (1). The ras gene product (p21) is a GTP-binding protein, and it has been predicted that the ligand (GDP or GTP)-induced conformation of the protein determines its activity as proposed in previous studies on elongation factor (EF)-Tu (2) and guanine nucleotide-binding proteins (G proteins) (3). This contention has been supported by the following observations: (i) mutated p21 proteins with increased transforming activity preferentially bind GTP rather than GDP both in vitro (1) and in vivo (4-7), and (ii) p21-GTP but not p21-GDP is biologically active when injected into cells (7,8). Recently, we have detected more p21-GTP in vigorously growing cells than in quiescent cells, and in addition, it has been revealed that the amount of p21GTP actually increased when quiescent cells were stimulated to initiate DNA synthesis with fetal bovine serum or plateletderived growth factor (PDGF) (9). To our knowledge, these results are the first evidence for the formation of an active p21-GTP complex in response to the stimulus of growth factors.The receptor for PDGF has intrinsic tyrosine kinase activity, which is essential for mitogenesis (10). However, substrates of the receptor-tyrosine kinase that are important for the mitogenic signaling have not yet been fully clarified. Recently, it has been reported that GTPase-activating protein (GAP) (7,11,12) is a substrate for various tyrosine kinases including the PDGF receptor, the epidermal growth factor (EGF) receptor, and products of oncogenes such as src,fms, fps, and abl (13)(14)(15). This suggests the involvement of GAP in signal transduction pathways from these kinases, although alteration of GAP activity by phosphorylation of tyrosine residues has not been observed. Since GAP may be a ...

The ras gene product (p21) is a GTP-binding protein and is thought to play an important role in signal transduction of growth and differentiation in many types of mammalian cells. The p2FGTP complex is an active conformation, as described previously for polypeptide chain elongation factors (EF-Tu and EF-G) and heterotrimeric GTPbinding proteins (G proteins). In the study reported here, we measured the amounts ofp21-bound guanine nucleotides under various conditions in the G54 cell line, a derivative of Swiss 3T3 cells that overexpresses normal c-Ha-ras. More p21GTP complexes were present in growing cells than in quiescent cells. When quiescent cells were stimulated with fetal bovine serum to promote DNA synthesis, p2l-GTP increased --fold. Among a number of purified growth factors, platelet-derived growth factor enhanced the formation of p2lFGTP, whereas the combination of bombesin and insulin, which also induces DNA synthesis, did not. These results strongly suggest that p21 is a transducer of the growth signal from the platelet-derived growth factor receptor in Swiss 3T3 cells and that the signal is transmitted through a p2FGTP complex.The ras protooncogene product (p21) is a GTP-binding protein involved in the transduction of signals controlling cellular proliferation and differentiation (1). The activity of a GTPbinding protein is regulated by the bound guanine nucleotide, GDP or GTP. The protein-GDP complex is an inactive conformation and nucleotide exchange from GDP to GTP activates the protein. The active protein-GTP complex specifically interacts with an effector molecule, which transduces the signal downstream. Hydrolysis of the bound GTP to GDP and Pi converts the protein-guanine nucleotide complex to an inactive form. Thus, the activity of a GTPbinding protein is regulated at two steps-i.e., the GDP/GTP

The rcs gene product @21) specifically binds GDP or GTP. In analogy with the reaction mechanism of other GTP-binding proteins, only the GTP-bound conformation is believed to be the biologically active one. Previously, we reported that not only oncogenic p2 1 (Val-12) but also proto-oncogenic p2 I (Gly-12) could induce morphological differentiation in rat pheochromocytoma PC12 cells when microinjected in the complexed form with GTPyS [(1987) Mol. Cell. Biol. 7, 455345561.In the present report we transformed PC12 cells with the oncogenic ru.t gene placed under the metallothionein I promoter.It was found that the transformed cells, when induced with Cd z+ differentiated in the absence of , NGF. Then we analyzed the guanine nucleotide bound to p21 in the intact PC12 cells. It was found that conditionally induced p2 1 (Val-12) was mostly present in the GTP-bound form, whereas the endogenous p2 1 (Gly-12) was in the GDPbound form. These results indicate again that p21 .GTP induces the morphological differentiation of PC12 cells. rus protein; Nucleotide exchange; (PC12 cell)

Nanos is expressed in the primordial germ cells (PGCs) and also the germ cells of a variety of organisms as diverse as Drosophila, medaka fish, Xenopus and mouse. In Nanos3-deficient mice, PGCs fail to incorporate into the gonad and the size of the testis and ovary is thereby dramatically reduced. To elucidate the role of Nanos in an amphibian species, we cloned Nanos3 cDNA from the testis of the R. rugosa frog. RT-PCR analysis showed strong expression of Nanos3 mRNA in the testis of adult R. rugosa frogs, but expression was not sexually dimorphic during gonadal differentiation. In Nanos3-knockdown tadpoles produced by the CRISPR/Cas9 system, the number of germ cells decreased dramatically in the gonads of both male and female tadpoles before sex determination and thereafter. This was confirmed by three dimensional imaging of wild-type and Nanos3 knockdown gonads using serial sections immunostained for Vasa, a marker specific to germ cells. Taken together, these results suggest that Nanos3 protein function is conserved between R. rugosa and mouse.