skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Profiling Signaling Polarity in Chemotactic Cells

Abstract

While directional movement requires morphological polarization characterized by formation of a leading pseudopodium at the front and a trailing rear at the back, little is known about how protein networks are spatially integrated to regulate this process. Here, we utilize a unique pseudopodial purification system and quantitative proteomics and phosphoproteomics to map the spatial relationship of 3509 proteins and 228 distinct sites of phosphorylation in polarized cells. Networks of signaling proteins, metabolic pathways, actin regulatory proteins, and kinase-substrate cascades were found to partition to different poles of the cell including components of the Ras/ERK pathway. Also, several novel proteins were found to be differentially phosphorylated at the front or rear of polarized cells and to localize to distinct subcellular structures. Our findings provide insight into the spatial organization of signaling networks that control cell movement and provide a comprehensive profile of proteins and their sites of phosphorylation that control cell polarization.

Authors:
; ; ; ; ; ; ; ; ;
Publication Date:
Research Org.:
Pacific Northwest National Laboratory (PNNL), Richland, WA (US), Environmental Molecular Sciences Laboratory (EMSL)
Sponsoring Org.:
USDOE
OSTI Identifier:
912509
Report Number(s):
PNNL-SA-51866
Journal ID: ISSN 0027-8424; PNASA6; 11391; 400412000; TRN: US200801%%1060
DOE Contract Number:
AC05-76RL01830
Resource Type:
Journal Article
Resource Relation:
Journal Name: Proceedings of the National Academy of Sciences of the United States of America, 104(20):8328-8333; Journal Volume: 104; Journal Issue: 20
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; ACTIN; BIOLOGICAL PATHWAYS; PHOSPHORYLATION; POLARIZATION; PROTEINS; PURIFICATION; Phosphoproteome, IMAC, stable isotope dual labeling, global proteome, cell metastasis, pseudopodia, cell signaling; Environmental Molecular Sciences Laboratory

Citation Formats

Wang, Yingchun, Ding, Shi-Jian, Wang, Wei, Jacobs, Jon M., Qian, Weijun, Moore, Ronald J., Yang, Feng, Camp, David G., Smith, Richard D., and Klemke, Richard L. Profiling Signaling Polarity in Chemotactic Cells. United States: N. p., 2007. Web. doi:10.1073/pnas.0701103104.
Wang, Yingchun, Ding, Shi-Jian, Wang, Wei, Jacobs, Jon M., Qian, Weijun, Moore, Ronald J., Yang, Feng, Camp, David G., Smith, Richard D., & Klemke, Richard L. Profiling Signaling Polarity in Chemotactic Cells. United States. doi:10.1073/pnas.0701103104.
Wang, Yingchun, Ding, Shi-Jian, Wang, Wei, Jacobs, Jon M., Qian, Weijun, Moore, Ronald J., Yang, Feng, Camp, David G., Smith, Richard D., and Klemke, Richard L. Tue . "Profiling Signaling Polarity in Chemotactic Cells". United States. doi:10.1073/pnas.0701103104.
@article{osti_912509,
title = {Profiling Signaling Polarity in Chemotactic Cells},
author = {Wang, Yingchun and Ding, Shi-Jian and Wang, Wei and Jacobs, Jon M. and Qian, Weijun and Moore, Ronald J. and Yang, Feng and Camp, David G. and Smith, Richard D. and Klemke, Richard L.},
abstractNote = {While directional movement requires morphological polarization characterized by formation of a leading pseudopodium at the front and a trailing rear at the back, little is known about how protein networks are spatially integrated to regulate this process. Here, we utilize a unique pseudopodial purification system and quantitative proteomics and phosphoproteomics to map the spatial relationship of 3509 proteins and 228 distinct sites of phosphorylation in polarized cells. Networks of signaling proteins, metabolic pathways, actin regulatory proteins, and kinase-substrate cascades were found to partition to different poles of the cell including components of the Ras/ERK pathway. Also, several novel proteins were found to be differentially phosphorylated at the front or rear of polarized cells and to localize to distinct subcellular structures. Our findings provide insight into the spatial organization of signaling networks that control cell movement and provide a comprehensive profile of proteins and their sites of phosphorylation that control cell polarization.},
doi = {10.1073/pnas.0701103104},
journal = {Proceedings of the National Academy of Sciences of the United States of America, 104(20):8328-8333},
number = 20,
volume = 104,
place = {United States},
year = {Tue May 15 00:00:00 EDT 2007},
month = {Tue May 15 00:00:00 EDT 2007}
}
  • No abstract prepared.
  • Distinct genes encode {alpha} and {beta} platelet-derived growth factor (PDGF) receptors that differ in their abilities to be triggered by three dimeric forms of the PDGF molecule. The authors show that PDGF-receptor mitogenic function can be reconstituted in a naive hematopoietic cell line by introduction of expression vectors for either {alpha} or {beta} PDGF receptor cDNAs. Thus, each receptor is independently capable of coupling with mitogenic signal-transduction pathways inherently present in these cells. Activation of either receptor also resulted in chemotaxis, alterations in inositol lipid metabolism, and mobilization of intracellular Ca{sup 2+}. The magnitude of these functional responses correlated wellmore » with the binding properties of the different PDGF isoforms to each receptor. Thus, availability of specific PDGF isoforms and relative expression of each PDGF-receptor gene product are major determinants of the spectrum of known PDGF responses.« less
  • After exposure to low density lipoprotein (LDL) that had been minimally modified by oxidation (MM-LDL), human endothelial cells (EC) and smooth muscle cells (SMC) cultured separately or together produced 2- to 3-fold more monocyte chemotactic activity than did control cells or cells exposed to freshly isolated LDL. This increase in monocyte chemotactic activity was paralleled by increases in mRNA levels for a monocyte chemotactic protein 1 (MCP-1) that is constitutively produced by the human glioma U-105MG cell line. Antibody that had been prepared against cultured baboon smooth muscle cell chemotactic factor (anti-SMCF) did not inhibit monocyte migration induced by themore » potent bacterial chemotactic factor f-Met-Leu-Phe. However, anti-SMCF completely inhibited the monocyte chemotactic activity found in the media of U-105MG cells, EC, and SMC before and after exposure to MM-LDL. Moreover, monocyte migration into the subendothelial space of a coculture of EC and SMC that had been exposed to MM-LDL was completely inhibited by anti-SMCF. Anti-SMCF specifically immunoprecipitated 10-kDa and 12.5-kDa proteins from EC. Incorporation of (35S)methionine into the immunoprecipitated proteins paralleled the monocyte chemotactic activity found in the medium of MM-LDL stimulated EC and the levels of MCP-1 mRNA found in the EC. We conclude that SMCF is in fact MCP-1 and MCP-1 is induced by MM-LDL.« less
  • N-Formylmethionylleucylphenylalanine (fMLP) induces chemotaxis in leukocytes, the response being mediated by peptide binding to a receptor on the plasma membrane. In tumor cells, this peptide has been reported to induce cellular swelling and chemotaxis in vitro and to enhance the localization of circulating tumor cells in vivo. In the Boyden chamber, the authors evaluated the migratory responses of Walker carcinosarcoma 256 cells to varying concentrations of fMLP. Sigmoidal dose-response curves were obtained with the dose of chemotactic factor that elicits a half-maximal chemotactic response of 5.0 +/- 2.5 X 10(-8) M. Checkerboard analysis indicated that these responses were dependent uponmore » a concentration gradient of fMLP with increases in migration of circa 2 to 2.5 times that of random movement. To examine the binding of fMLP, the tumor cells were incubated with 5 X 10(-9) M fML-(/sup 3/H)P in Hanks balanced salt solution. Specific binding (0.5 to 1% of total radioligand, to whole cells inhibited by 5 X 10(-6) M fMLP) approached equilibrium after 4 to 6 h at 4 degrees C and after 6 to 10 h at 22 degrees C. Autoradiographic studies demonstrated heterogeneous binding of the peptide by tumor cells and also showed its intracellular localization. In homogenates of Walker cells prepared in 0.1 M Tris HCl, pH 7.4, with 10 mM MgCl2 and bovine serum albumin (1 mg/ml), specific binding of approximately 0.5% of total fML-(/sup 3/H)P reached equilibrium after 60 min at 4 degrees C. In whole cells and homogenates, binding was reversible by addition of unlabeled fMLP.« less
  • The authors' have shown previously that formyl peptide chemotactic receptors (FPCR) of human phagocytic cells contain at least two asparagine-linked oligosaccharide chains located at the distal end of the receptor. The requirement of these N-linked oligosaccharide chains for expression and function of FPCR was investigated in HL-60 cells induced to differentiate by N/sup 6/,O/sup 2/-dibutyryladenosine 3',5'-monophosphate (Bt/sub 2/cAMP) in the presence or absence of 5 ..mu..g/ml tunicamycin. Tunicamycin did not prevent the changes in morphology associated with Bt/sub 2/cAMP-induced differentiation of HL-60 cells. Autoradiographic analysis after SDS-PAGE of FPCR affinity labeled with N-formyl-Nle-Leu-Phe-Nle-(/sup 125/I) iodo-Tyr-Lys (formyl /sup 125/I-hexapeptide) and ethylenemore » glycol bis(succinimidyl succinate) demonstrated that greater than 95% of FPCR expressed by tunicamycin-treated cells completely lacked N-linked oligosaccharide (M/sub r/ 32,000), and no fully glycosylated FPCR (M/sub r/ 62,000 to 85,000) was detectable. Scatchard analysis of formyl /sup 125/I-hexapeptide binding indicated the presence of two classes of binding sites for both control and tunicamycin-treated cells. Both control and tunicamuycin-treated cells augmented superoxide anion release, exhibited a migratory response, and showed a transient rise in intracellular free Ca/sup 2 +/ upon stimulation with N-formyl-Nle-Leu-Phe. However, the responses of the tunicamycin-treated cells were less than that of the control cells.« less