Regulation of cell proliferation by interleukin-3-induced nuclear translocation of pyruvate kinase.

Extracellular signaling molecules bound to cell surface receptors can regulate nuclear function with consequences for cell proliferation, differentiation, and function. To regulate nuclear function, signals must be transduced across the nuclear envelope to propagate the signal from the cytoplasm to the nucleus. Therefore, many signaling responses induce the nuclear translocation of transcription factors, kinases, and others. By using inducible translocation trap, a reporter gene-based system to detect inducible nuclear translocation, we found that the M2 isoform of pyruvate kinase, a key enzyme in glycolysis, translocates into the nucleus by interleukin-3, but not by epidermal growth factor, stimulation. The C domain of the M2 isoform of pyruvate kinase was sufficient for interleukin-3-induced nuclear translocation. Interleukin-3-induced nuclear translocation of the M2 isoform of pyruvate kinase was dependent on the activation of Jak2. Overexpression of the M2 isoform of pyruvate kinase protein fused with a nuclear localization signal enhanced cell proliferation in the absence of interleukin-3, suggesting that the nuclear pyruvate kinase plays an important role in cell proliferation.

HIV Nef-mediated CD4 down-regulation is adaptor protein complex 2 dependent.

Nef is a crucial viral protein for HIV to replicate at high titers and in the development of AIDS. One Nef function is down-regulating CD4 from the cell surface, which correlates with Nef-enhanced viral pathogenicity. Nef down-regulates CD4 by linking CD4 to clathrin-coated pits. However, the mechanistic connection between the C-terminal dileucine motif of Nef and the component(s) of the clathrin-coated pits has not been pinpointed. In this report we used two AP-2 complex-specific inhibitors: a dominant negative mutant of Eps15 (Eps15DIII) that binds to the alpha subunit of AP-2 complex and a small interference RNA that is specific for the mu2 subunit of AP-2 complex. We show that both HIV Nef- and SIV Nef-mediated CD4 down-regulations were profoundly blocked by the synergistic effect of Eps15DIII and RNA interference of AP-2 expression. The results demonstrate that HIV/SIV Nef-mediated CD4 down-regulation is AP-2 dependent. We also show that the PMA-induced CD4 down-regulation was blocked by these two inhibitors. Therefore, PMA-induced CD4 down-regulation is also AP-2 dependent. The results demonstrate that, like the tyrosine sorting motif-dependent endocytosis (for which the transferrin receptor and the epidermal growth factor receptor are the two prototypes), dileucine sorting motif-dependent endocytosis of Nef and CD4 are also AP-2 dependent.

Inducible translocation trap: a system for detecting inducible nuclear translocation.

Here we report a general system, inducible translocation trap (ITT), for identification of proteins that translocate into the nucleus following signal transduction from cell surface receptors. ITT consists of a retroviral cDNA expression library of fusion proteins consisting of a LexA DNA binding domain, the transactivation domain of a transcriptional activator, and proteins encoded by cDNA inserts. The retroviral library is then transduced into cell lines containing a reporter gene with LexA binding sites in its promoter. Cells expressing the reporter gene by extracellular stimuli are then selected by flow-cytometric sorting. By using ITT, we identified cDNA encoding Stat1 in a screen of proteins which translocate into the nucleus by IFNgamma, indicating that this system can be used for isolation of nuclear translocating proteins induced by extracellular stimuli. ITT may be a useful tool for dissecting dynamic translocation in various biological systems.