Gankyrin is an ankyrin repeat oncoprotein commonly overexpressed in hepatocellular carcinomas. Gankyrin interacts with the S6 proteasomal ATPase and accelerates the degradation of the tumor suppressor Rb. We show here that gankyrin has an antiapoptotic activity in cells exposed to DNA damaging agents. Downregulation of gankyrin induces apoptosis in cells with wild-type p53. In vitro and in vivo experiments revealed that gankyrin binds to Mdm2, facilitating p53-Mdm2 binding, and increases ubiquitylation and degradation of p53. Gankyrin also enhances Mdm2 autoubiquitylation in the absence of p53. Downregulation of gankyrin reduced amounts of Mdm2 and p53 associated with the 26S proteasome. Thus, gankyrin is a cofactor that increases the activities of Mdm2 on p53 and probably targets polyubiquitylated p53 into the 26S proteasome.

Nine residues of Bacillus cereus ATCC7064 oligo-l,6-glucosidase were replaced stepwise with proline residues. Of the nine residues, Lysl21, Glu208 and Glu290 were at second sites of p turns; Asnl09, Glu175 and Thr261 were at N-caps of a helices; Glu216, Glu270 and Glu378 were in coils within loops. The replacements were carried out in the order, Lysl21-.Pro, Glul75-.Pro, Glu29Ct.Pr0, Glu208-Pr0, Glu270+Pro, Glu378-Pr0, Thr261+Pro, Glu216+Pro and Asnl09 -+Pro. The resultant nine active mutant enzymes contained 1-9 more proline residues than B. cereus oligo-l,6-glucosidase. The therrnostability of these mutants was additively enhanced with the increase in the number of proline residues introduced. The increase in the thermostability was most remarkable when proline residues were introduced at second sites of p turns or at N-caps of a

Canonical aversive long-term memory (LTM) formation in Drosophila requires multiple spaced trainings, whereas appetitive LTM can be formed after a single training. Appetitive LTM requires fasting prior to training, which increases motivation for food intake. However, we found that fasting facilitated LTM formation in general; aversive LTM formation also occurred after single-cycle training when mild fasting was applied before training. Both fasting-dependent LTM (fLTM) and spaced training-dependent LTM (spLTM) required protein synthesis and cyclic adenosine monophosphate response element-binding protein (CREB) activity. However, spLTM required CREB activity in two neural populations--mushroom body and DAL neurons--whereas fLTM required CREB activity only in mushroom body neurons. fLTM uses the CREB coactivator CRTC, whereas spLTM uses the coactivator CBP. Thus, flies use distinct LTM machinery depending on their hunger state.

Accumulating evidence suggests that transcriptional regulation is required for maintenance of long-term memories (LTMs). Here we characterize global transcriptional and epigenetic changes that occur during LTM storage in the Drosophila mushroom bodies (MBs), structures important for memory. Although LTM formation requires the CREB transcription factor and its coactivator, CBP, subsequent early maintenance requires CREB and a different coactivator, CRTC. Late maintenance becomes CREB independent and instead requires the transcription factor Bx. Bx expression initially depends on CREB/CRTC activity, but later becomes CREB/CRTC independent. The timing of the CREB/CRTC early maintenance phase correlates with the time window for LTM extinction and we identify different subsets of CREB/CRTC target genes that are required for memory maintenance and extinction. Furthermore, we find that prolonging CREB/CRTC-dependent transcription extends the time window for LTM extinction. Our results demonstrate the dynamic nature of stored memory and its regulation by shifting transcription systems in the MBs.

Cold-inducible RNA-binding protein (Cirp) was the first cold-shock protein identified in mammals. It is structurally quite different from bacterial cold-shock proteins and is induced in response to mild, but not severe, hypothermia. To clarify the physiological function of Cirp in vivo, we produced cirp -knockout mice. They showed neither gross abnormality nor defect in fertility, but the number of undifferentiated spermatogonia was significantly reduced and the recovery of spermatogenesis was delayed after treatment with a cytotoxic agent, busulfan. Cirp accelerated cell-cycle progression from G0 to G1 as well as from G1 to S phase in cultured mouse embryonic fibroblasts. Cirp directly bound to dual-specificity tyrosine-phosphorylation–regulated kinase 1B (Dyrk1b, also called Mirk) and inhibited its binding to p27, resulting in decreased phosphorylation and destabilization of p27. Cirp did not affect binding of Dyrk1b to cyclin D1 but inhibited phosphorylation of cyclin D1 by Dyrk1b, resulting in cyclin D1 stabilization. In the spermatogonial cell line GC-1spg, suppression of Cirp expression increased the protein level of p27, decreased that of cyclin D1, and decreased the growth rate, which depended on Dyrk1b. Consistent changes in the protein levels of p27 and cyclin D1 as well as the percentage of cells in G0 phase were observed in undifferentiated spermatogonia of cirp -knockout mice. In undifferentiated spermatogonia of wild-type mice, Cirp and Dyrk1b colocalized in the nucleus. Thus, our study demonstrates that Cirp functions to fine-tune the proliferation of undifferentiated spermatogonia by interacting with Dyrk1b.