A bipartite NLS at the SHP-1 C-terminus mediates cytokine-induced SHP-1 nuclear localization in cell growth control.
SHP-1 protein tyrosine phosphatase is a critical regulator of signaling in hematopoietic cells as illustrated by the lethal hematopoietic disorders in SHP-1-deficient mice. We and others have shown in previous studies that SHP-1 regulates membrane receptor signaling: it binds via its N-terminal region SH2 domains to tyrosine phosphorylated membrane receptors to dephosphorylate key substrates in the receptor complexes. Here we demonstrate that the SHP-1 C-terminal region contains a bipartite NLS that mediates SHP-1 nuclear localization in response to cytokines. This NLS was located within amino acids 576-595 of the PTPase and, when fused by itself to EGFP, targeted the fluorescent protein into the nuclei of transiently transfected NIH3T3 fibroblasts and Bac1.2f5 macrophage cells. When positioned within SHP-1, the activity of the NSL was under tight regulation as indicated by the predominant cytoplasmic distribution of the EGFP/SHP-1 fusion protein in NIH3T3 transfectants and the exclusive cytoplasmic localization of the endogenous SHP-1 in hematopoietic cell line PBLC-1. Activation of the NLS in SHP-1 by IL-4 was demonstrated by increased nuclear localization of the EGFP/SHP-1 fusion protein in NIH3T3 transfectants and of the endogenous SHP-1 protein in PBCL-1 cells at 4, 6 and 8 h post-IL-4 stimulation. SHP-1 nuclear localization in PBCL-1 cells was also induced by IL-7 in a similar manner, suggesting it as a common event in cytokine signaling. In comparison to that of the wild-type phosphatase, an SHP-1 mutant lacking the NLS showed only approximately half of the activity in inhibiting proliferation of NIH3T3 transfectants. These results provide evidence of cytokine-regulated SHP-1 nuclear localization mediated by a bipartite NLS and suggest that SHP-1 regulates nuclear signaling in cell growth control.
Yang
Wentian
W
Department of Cancer Biology, Lerner Research Institute, The Clevelaand Clinic Foundation, Ohio 44195, USA.
Tabrizi
Mina
M
Yi
Taolin
T
D016475:3T3 Cells; D021581:Active Transport, Cell Nucleus / Q000187:drug effects; D000818:Animals; D001483:Base Sequence; D002455:Cell Division / Q000187:drug effects; D016207:Cytokines / Q000494:pharmacology*; D006412:Hematopoietic Stem Cells / Q000378:metabolism; D015847:Interleukin-4 / Q000494:pharmacology; D015851:Interleukin-7 / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D008856:Microscopy, Fluorescence; D019913:Nuclear Localization Signals / Q000187:drug effects / Q000502:physiology*; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D016207:Cytokines; D015851:Interleukin-7; D047908:Intracellular Signaling Peptides and Proteins; D019913:Nuclear Localization Signals; D011993:Recombinant Fusion Proteins; D015847:Interleukin-4; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases; C509248:Ptpn6 protein, mouse