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First published online 28 June 2005
doi: 10.1242/jcs.02438

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Proteolytic maturation and activation of autotaxin (NPP2), a secreted metastasis-enhancing lysophospholipase D

Silvia Jansen^1,*, Cristiana Stefan^1,*, John W. M. Creemers², Etienne Waelkens¹, Aleyde Van Eynde¹, Willy Stalmans¹ and Mathieu Bollen^1,

¹ Division of Biochemistry, Flanders Interuniversity Institute for Biotechnology, Faculty of Medicine, K.U.Leuven, B-3000 Leuven, Belgium
² Department for Human Genetics, Flanders Interuniversity Institute for Biotechnology, Faculty of Medicine, K.U.Leuven, B-3000 Leuven, Belgium

View larger version (64K): [in a new window]   Fig. 1. Localization of NPP1 and NPP2 in HEK293 cells. (A) Cells were transiently transfected with HA-NPP1-Myc and NPP2-EGFP (upper three panels) or with NPP2-EGFP alone (lower nine panels). After 24 hours, the cells were fixed with 2% formaldehyde. NPP2-EGFP, HA-NPP1-Myc, the ER-marker BiP, the Golgi-marker Golgin-97 and the trans-Golgi network marker TGN38 were visualized by confocal microscopy using the spontaneous green fluorescence of EGFP, anti-Myc antibodies, anti-BiP antibodies, anti-TGN38 antibodies or anti-Golgin-97 antibodies, as indicated. The right panels represent the merges of the left and middle panels. (B) HEK293 cells were transiently transfected with HA-NPP1-Myc or HA-NPP2-Myc. At the indicated time points, the fusion proteins were detected by immunoblotting of the cell lysates and the culture medium with anti-Myc antibodies.

View larger version (30K): [in a new window]   Fig. 2. Mapping of the polypeptide fragment that determines the extracellular release of NPP2.(A) The domain structure of NPP1 and NPP2. The hydrophobic fragment in the N-terminal domain is represented by a thick black vertical line. (B) Secretion by HEK293 cells of NPP1, NPP2 and the indicated chimaeras of the N-terminal, catalytic and nuclease-like domains of NPP1 and NPP2, as defined in panel A. NPP1-1-2 refers to a fusion of the N-terminal and catalytic domains of NPP1 and the nuclease-like domain of NPP2. The other chimaeras are annotated in the same manner. All proteins were expressed with an N-terminal HA-tag and a C-terminal Myc-tag, and were visualized after 72 hours by immunoblotting with anti-Myc antibodies. (C) Extracellular release of NPP1, NPP2 and the indicated chimaeras of the N-terminal subdomains, all expressed for 72 hours with an N-terminal HA-tag and a C-terminal Myc-tag and visualized by immunoblotting with anti-Myc antibodies. NPP1-212-30-1 refers to NPP1 with its N-terminal hydrophobic subdomain (residues 59-79) swapped for the corresponding subdomain (residues 12-30) of NPP2. The other fusions are annotated in the same manner. (D) Effect of mutation of the N-terminal hydrophobic domain on the secretion of HA-NPP2-Myc. The residues of the hydrophobic domain of NPP2 were replaced four by four by the corresponding residues of NPP1. The secretion of wild-type NPP1, NPP2 and the NPP2 mutants was visualized by immunoblotting with anti-Myc antibodies. All data shown in panels B-D are representative for at least three independent experiments.

View larger version (52K): [in a new window]   Fig. 3. The hydrophobic subdomain determines the trafficking pathways of NPP1 and NPP2. (A) Localization in HEK293 cells of NPP1, NPP2, NPP1-212-30-1 and NPP2-159-79-2, each expressed as fusions with an N-terminal HA-tag and a C-terminal Myc-tag, and detected by confocal fluorescence microscopy with anti-Myc antibodies. (B) An aliquot of the same cells was lysed for immunoblotting with anti-HA and anti-Myc antibodies. All data shown are representative for at least three independent experiments.

View larger version (39K): [in a new window]   Fig. 4. The N-terminus of NPP2 functions as a signal peptide. (A) The N-terminal sequence of NPP2 with the predicted N-terminal (n), hydrophobic (h) and C-terminal (c) fragment of the signal peptide sequence, as predicted by the Signal P program (version 3.1). Also indicated is the predicted cleavage site (arrow). (B) HA-NPP2-Myc, with a Flag epitope inserted after either Gly27 or Phe28, was expressed in HEK 293 cells. 24 hours after transfection the cells were harvested and the lysates were subjected to immunoblotting with anti-Flag M1 and M2 monoclonal antibodies. The M1 antibodies only recognize an N-terminally free Flag-tag, whereas the M2s also recognize an internal Flag-tag. The data are representative for at least three independent experiments.

View larger version (31K): [in a new window]   Fig. 5. NPP2 is processed by furin-like endoproteinases. (A) The N-terminal sequence of NPP2 with the predicted signal peptide cleavage site (white arrow), the consensus site for recognition by furin (underlined) and the predicted furin cleavage site (grey arrow). (B) HA-NPP2-Myc with the Flag-tag inserted after Arg35 was expressed in HEK293 cells. After 24 hours and 72 hours the cell lysates and culture medium were processed for immunoblotting with the anti-Flag M1 and M2 monoclonal antibodies. (C) The same NPP2 construct was also expressed for 72 hours with or without the furin-inhibitor 1-PDX, and the corresponding lysates and media were immunoblotted with the M1 and M2 antibodies. (D) HA-NPP2-Myc with a Flag-tag after Arg35 was expressed in ß-TC3 cells before or after the RNAi-mediated knockdown of furin or PACE4, as indicated. Aliquots of the culture medium were processed for immunoblotting with the M1 and M2 antibodies. The right panels show the effect of the furin or PACE4 shRNAs on the level of overexpressed furin or PACE4, respectively, as detected by immunoblotting. The data are representative for at least three independent experiments.

View larger version (27K): [in a new window]   Fig. 6. Comparison of pro-NPP2 and NPP2. (A) HA-NPP2-Myc was expressed in HEK293 cells with or without the furin-inhibitor 1-PDX, to generate pro-NPP2 and NPP2, respectively. After 24 hours, 48 hours and 72 hours the cell lysates and culture media were processed for immunoblotting with anti-Myc antibodies. (B) Pro-NPP2 and NPP2, obtained after the expression of HA-NPP2-Myc with or without the furin-inhibitor 1-PDX, respectively, were purified on an anti-Myc affinity column. The purified enzymes were assayed for lysophospholipase-D activities. The bar diagrams represent the means ±s.e.m. (n=3) of the enzymatic activities. Pro-NPP2 and NPP2 were assayed at the same concentration, as measured in triplicate by immunoblotting with anti-c-Myc antibodies (inset).

View larger version (32K): [in a new window]   Fig. 7. Cellular and secreted NPP2 differ in their extent of glycosylation. HA-NPP2-Myc was expressed in HEK293 cells with or without 1-PDX, as indicated. The cell lysates and media were processed for immunoblotting with anti-Myc antibodies in the absence or presence of 50 mM 2-mercaptoethanol and before or after a pretreatment for 50 mU of N-glycosidase F, as indicated. The data are representative for at least three independent experiments.

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