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...
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