Abstract
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.
Highlights
For construction of a retroviral vector expressing hemagglutinin (HA) epitope-tagged M2-PK fused with the nuclear localization signal (NLS) from SV40 T-antigen at its N terminus (HA-NLS-M2-PK), the coding sequence of M2-PK was amplified by PCR and ligated with a sequence encoding SV40 T-antigen NLS cleaved from pLGV-NLS--galactosidase [10] and pEF-HA [11]
PK are high, it is likely that other isoforms (L, R, and M1) of PK can translocate into the nucleus by IL-3
It has been recently shown that growth factors, including IL-3, influence cell growth and survival through effects on glucose metabolism
Summary
Cell Lines—BB13 was derived from an IL-3-dependent hematopoietic cell line, Ba/F3 [8], and ectopically expresses. In the absence of EGF and IL-3, BB13 dies by apoptosis, but cell death is markedly delayed compared with its parental cell line, which does not overexpress Bcl-2. Plasmid Construction—For construction of a retroviral vector expressing GFP fused with the carboxyl (C)-terminal of the full-length M2-PK (M2-PK-GFP), the coding sequence of mouse M2-PK was amplified by PCR from the Ba/F3 cDNA library and ligated with GFP gene from pEGFP-N3 (Clontech) and pMX-neo vector. For construction of a retroviral vector expressing hemagglutinin (HA) epitope-tagged M2-PK fused with the nuclear localization signal (NLS) from SV40 T-antigen at its N terminus (HA-NLS-M2-PK), the coding sequence of M2-PK was amplified by PCR and ligated with a sequence encoding SV40 T-antigen NLS cleaved from pLGV-NLS--galactosidase [10] and pEF-HA [11]. BB13 is a Ba/F3-derived cell line ectopically expressing EGF receptor and antiapoptotic protein Bcl-2. Ba/F3 dies by apoptosis in the absence of IL-3, whereas BB13 proliferates even in the absence of IL-3 if EGF is present in the culture medium [9]
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