Rho-family GTPases regulate many cellular functions. To visualize the activity of Rho-family GTPases in living cells, we developed fluorescence resonance energy transfer (FRET)–based probes for Rac1 and Cdc42 previously (Itoh, R.E., K. Kurokawa, Y. Ohba, H. Yoshizaki, N. Mochizuki, and M. Matsuda. 2002. Mol. Cell. Biol. 22:6582–6591). Here, we added two types of probes for RhoA. One is to monitor the activity balance between guanine nucleotide exchange factors and GTPase-activating proteins, and another is to monitor the level of GTP-RhoA. Using these FRET probes, we imaged the activities of Rho-family GTPases during the cell division of HeLa cells. The activities of RhoA, Rac1, and Cdc42 were high at the plasma membrane in interphase, and decreased rapidly on entry into M phase. From after anaphase, the RhoA activity increased at the plasma membrane including cleavage furrow. Rac1 activity was suppressed at the spindle midzone and increased at the plasma membrane of polar sides after telophase. Cdc42 activity was suppressed at the plasma membrane and was high at the intracellular membrane compartments during cytokinesis. In conclusion, we could use the FRET-based probes to visualize the complex spatio-temporal regulation of Rho-family GTPases during cell division.

The mammalian Chk2 kinase is thought to mediate ATM‐dependent signaling in response to DNA damage. The physiological role of mammalian Chk2 has now been investigated by the generation of Chk2‐deficient mice. Although Chk2−/− mice appeared normal, they were resistant to ionizing radiation (IR) as a result of the preservation of splenic lymphocytes. Thymocytes and neurons of the developing brain were also resistant to IR‐induced apoptosis. The IR‐induced G1/S cell cycle checkpoint, but not the G2/M or S phase checkpoints, was impaired in embryonic fibroblasts derived from Chk2−/− mice. IR‐induced stabilization of p53 in Chk2−/− cells was 50–70% of that in wild‐type cells. Caffeine further reduced p53 accumulation, suggesting the existence of an ATM/ATR‐dependent but Chk2‐independent pathway for p53 stabilization. In spite of p53 protein stabilization and phosphorylation of Ser23, p53‐dependent transcriptional induction of target genes, such as p21 and Noxa, was not observed in Chk2−/− cells. Our results show that Chk2 plays a critical role in p53 function in response to IR by regulating its transcriptional activity as well as its stability.

Genetic etiologies of at least 20% of autosomal dominant cerebellar ataxias (ADCAs) have yet to be clarified. We identified a novel spinocerebellar ataxia (SCA) form in four Japanese pedigrees which is caused by an abnormal CAG expansion in the TATA-binding protein (TBP) gene, a general transcription initiation factor. Consequently, it has been added to the group of polyglutamine diseases. This abnormal expansion of glutamine tracts in TBP bears 47--55 repeats, whereas the normal repeat number ranges from 29 to 42. Immunocytochemical examination of a postmortem brain which carried 48 CAG repeats detected neuronal intranuclear inclusion bodies that stained with anti-ubiquitin antibody, anti-TBP antibody and with the 1C2 antibody that recognizes specifically expanded pathological polyglutamine tracts. We therefore propose that this new disease be called SCA17 (TBP disease).

v-Crk was identified originally as an oncogene product of the CT10 retrovirus and became the first example of an adaptor protein which consists mostly of SH2 and SH3 domains (24). The cellular homolog of v-Crk has been isolated from chickens, humans, and mice (22,33,36). Alternative splicing of the human CRK gene yields two forms of translation products, designated the 28-kDa CRK-I and 40-and 42-kDa CRK-II proteins (22). Microinjection of CRK induces neuronal differentiation of PC12 cells, and overexpression of v-Crk accelerates the neuronal differentiation of the PC12 cells induced by nerve growth factor and epidermal growth factor (EGF), which trigger the cognate tyrosine kinase receptors (13,44). This CRK-dependent differentiation requires Ras, activation of which is enhanced by overexpression of CRK (20, 44). Moreover, two guanine nucleotide exchange proteins for the Ras family protein, mSos and C3G, have been shown to bind to the SH3 domain of CRK (20). These results have assigned CRK a position between receptor-type tyrosine kinases and the Ras family proteins in the signal transduction pathway.CRK may also be involved in signalling from focal adhesions, which not only anchor cells to the extracellular matrix but also play a pivotal role in cell differentiation, migration, and proliferation (4, 16, 37). Binding of integrin to the extracellular matrix induces activation of focal adhesion tyrosine kinase bound to the cytoplasmic domain of the integrin ␤ subunit. Activated and autophosphorylated focal adhesion tyrosine kinase, in turn, phosphorylates paxillin, a protein bound to focal adhesion tyrosine kinase and vinculin (40). Tyrosine phosphorylation of p130 cas is also induced by integrin engagement (31). Because paxillin and p130 cas are two of the three major phosphotyrosine-containing proteins in Crk-transformed cells and bind to the SH2 domain of Crk (21, 26), Crk seems to have an important role in signalling from focal adhesions.The adaptor proteins, including Crk, Grb2/Ash, and Nck, perceive signals from a number of tyrosine kinases by the interaction of SH2 with phosphotyrosine-containing peptides (34). The signals are next transmitted to the proteins bound to the SH3 domains through proline-rich sequences (5, 34). By using far Western blotting, we and others have previously shown that the SH3 domain of CRK binds to 135-to 145-, 160-, and 180-kDa proteins (7,44). The 135-to 145-kDa proteinsis designated C3G and has been shown to be a guanine nucleotide exchange protein for Rap1 (45). The identities of the 160-and 180-kDa proteins have not been reported. Other proteins known to bind to the SH3 domain of Crk include Sos (7,20), Abl (8), and Eps15 (43).A function of an adaptor protein is to recruit cytoplasmic enzymes bound to its SH3 domain to the cell membrane (34). Thus, the membrane targeting of the SH3-binding proteins mimics the activation of SH3-binding proteins. A typical example is Sos, a guanine nucleotide exchange protein for Ras, which binds to the SH3 domains of Grb2. Membrane targeting of...

CRK protein, together with GRB2/ASH and Nck proteins, belongs to the adaptor-type Src homology (SH)2-containing molecules, which transduce signals from tyrosine kinases. Here another guanine nucleotide-releasing protein (GNRP), C3G, has been identified as a CRK SH3-binding protein. The nucleotide sequence of a 4.1-kb C3G cDNA contains a 3.2-kb open reading frame encoding a 121-kDa protein, and antibodies against C3G have been shown to detect a protein of 130-140 kDa. The carboxyl terminus of C3G has a peptide sequence homologous to GNRPs for Ras, and the expression of this carboxyl terminus region suppresses the loss of CDC25 function in the yeast Saccharomyces cerevisiae. The C3G protein expressed in Escherichia coli binds to CRK and GRB2/ASH proteins. Mutational analysis of C3G assigns the SH3 binding region to a 50-amino acid region containing a proline-rich sequence. The mRNAs of both the C3G and CRK proteins are expressed ubiquitously in human adult and fetal tissues. The results of these studies suggest that the complex of CRK and C3G, or GRB2/ASH and C3G, may transduce the signals from tyrosine kinases to Ras in a number of different tissues.Growth factors elicit various responses through the activation of receptor-type and non-receptor-type tyrosine kinases (1). A group of cytoplasmic enzymes containing common amino acid sequences, designated Src homology (SH)2 and SH3 domains, play a pivotal role in transducing signals from the tyrosine kinases (2, 3). The SH2 domain responds to the signals from tyrosine kinases by binding to the tyrosinephosphorylated proteins, including the tyrosine kinases themselves. Some of the signals are also transmitted to proteins bound to the SH3 domains, but much less information is available on SH3-mediated signaling.The v-Crk protein was originally identified as an oncoprotein of a chicken retrovirus, CT10 (4). The protooncogene product v-Crk represents a newly emerging class of proteins consisting mostly of the SH2 and SH3 domains (5, 6). These proteins, now known as adaptorproteins, include Nck, GRB2/ ASH, Sem-5, and Drk (2, 7, 8). Sem-5 of Caenorhabditis elegans and Drk of Drosophila melanogaster appear to be homologues of mammalian GRB2/ASH. All of the adaptor proteins may be involved in the growth of fibroblasts. Overexpression or microinjection ofCRK, Nck, and GRB2 induces transformation of rat 3Y1 fibroblasts or DNA replication in mouse 3T3 fibroblasts (6,9,10). One common feature of the adaptor proteins may be signal transmission to Ras. The sem-5 gene of C. elegans has been mapped genetically downstream of let-23 tyrosine kinase and upstream of let-60 Ras-like protein (11). Similarly, drk of Drosophila is mapped between sevenless (sev) receptor tyrosine kinase and son of sevenless (sos), which encodes a guanine nucleotide-releasing protein (GNRP) for Rasl. Recently, the Sos protein ofDrosophila was shown to bind to the Drk protein, which contains SH2 and SH3 domains (7,8). Anti-Ras antibody also inhibited neuronal differentiation of PC12 cells induced by th...

Adult T-cell leukemia-lymphoma (ATLL) is a group of T-cell malignancies caused by infection with human T-lymphotropic virus type I (HTLV-I). Although the pathogenesis of ATLL remains incompletely understood, the viral regulatory protein Tax is centrally involved in cellular transformation. Here we describe the generation of HTLV-I Tax transgenic mice using the Lck proximal promoter to restrict transgene expression to developing thymocytes. After prolonged latency periods, transgenic mice developed diffuse large-cell lymphomas and leukemia with clinical, pathological and immunological features characteristic of acute ATLL. Transgenic mice were functionally immunocompromised and they developed opportunistic infections. Fulminant disease also developed rapidly in SCID mice after engraftment of lymphomatous cells from transgenic mice. Flow cytometry showed that the cells were CD4(-) and CD8(-), but CD44(+), CD25(+) and cytoplasmic CD3(+). This phenotype is indicative of a thymus-derived pre-T-cell phenotype, and disease development was associated with the constitutive activation of NF-kappaB. Our model accurately reproduces human disease and will provide a tool for analysis of the molecular events in transformation and for the development of new therapeutics.

C3G is a guanine nucleotide exchange factor (GEF) for Rap1, and is activated via Crk adaptor protein. To understand the physiological role of C3G, we generated C3G knockout mice. C3G ±/± homozygous mice died before embryonic day 7.5. The lethality was rescued by the expression of the human C3G transgene, which could be excised upon the expression of Cre recombinase. From the embryo of this mouse, we prepared ®broblast cell lines, MEF-hC3G. Expression of Cre abolished the expression of C3G in MEF-hC3G and inhibited cell adhesion-induced activation of Rap1. The Cre-expressing MEF-hC3G showed impaired cell adhesion, delayed cell spreading and accelerated cell migration. The accelerated cell migration was suppressed by the expression of active Rap1, Rap2 and R-Ras. Expression of Epac and CalDAG-GEFI, GEFs for Rap1, also suppressed the accelerated migration of the C3G-de®cient cells. This observation indicated that Rap1 activation was suf®cient to complement the C3G de®ciency. In conclusion, C3G-dependent activation of Rap1 is required for adhesion and spreading of embryonic ®broblasts and for the early embryogenesis of the mouse.

Many receptors for neuropeptides and hormones are coupled with the heterotrimeric G(i) protein, which activates the p42/44 mitogen-activated protein kinase (ERK/MAPK) cascade through both the alpha- and betagamma-subunits of G(i). The betagamma-subunit activates the ERK/MAPK cascade through tyrosine kinase. Constitutively active G(alpha)i2 (gip2) isolated from adrenal and ovarian tumours transforms Rat-1 fibroblasts and also activates the ERK/MAPK cascade by an unknown mechanism. The ERK/MAPK pathway is activated by Ras, and is inhibited when the low-molecular-mass GTP-binding protein Rap1 antagonizes Ras function. Here we show that a novel isoform of Rapl GTPase-activating protein, called rap1GAPII, binds specifically to the alpha-subunits of the G(i) family of heterotrimeric G-proteins. Stimulation of the G(i)-coupled m2-muscarinic receptor translocates rap1GAPII from the cytosol to the membrane and decreases the amount of GTP-bound Rap1. This decrease in GTP-bound Rap1 activates ERK/MAPK. Thus, the alpha-subunit of G(i) activates the Ras-ERK/MAPK mitogenic pathway by membrane recruitment of rap1GAPII and reduction of GTP-bound Rap1.