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PNAS 104 (20): 8328-8333

Copyright © 2007 by the National Academy of Sciences.

Profiling signaling polarity in chemotactic cells

Yingchun Wang{dagger}, Shi-Jian Ding{ddagger}, Wei Wang{dagger}, Jon M. Jacobs{ddagger}, Wei-Jun Qian{ddagger}, Ronald J. Moore{ddagger}, Feng Yang{ddagger}, David G. Camp, II{ddagger}, Richard D. Smith{ddagger}, and Richard L. Klemke{dagger},§

{dagger}Department of Pathology and Moores Cancer Center, University of California at San Diego, La Jolla, CA 92093; and {ddagger}Biological Sciences Division, Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, WA 99354


Figure 1
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Fig. 1. Microporous filter system for purification and mass spectrometry analysis of PD and CB fractions isolated from polarized cells. (A) Three-dimensional reconstruction of a Cos-7 cell expressing GFP and protruding PD through 3.0-µm pores of the filter (dashed red line) to the lower surface. The PD- and CB-localized proteins are differentially harvested, digested with trypsin, and then analyzed by LC-MS/MS (1) or dual-step 18O/16O labeling (2), followed by IMAC to isolate phosphorylated peptides as described in Experimental Procedures. The number of peptides sequenced and proteins identified is shown. (B) (Left) Cos-7 cells on microporous filters were either not treated (NT) or stimulated with a uniform (U) or gradient (G) concentration of LPA (100 ng/ml) for the indicated times and then Western blotted for activated ERK (ERK-P) or total ERK protein. (Right) Cos-7 cells were stimulated (+) or not stimulated (–) with an LPA gradient for 30 min to allow cells to polarize by protruding PD through 3-µm porous filters. CBs on the tops of the filters were then stimulated with LPA for the indicated times before being collected and Western blotted for activated and total ERK. Note that the CB is not responsive to LPA-induced ERK activation in polarized cells.

 

Figure 2
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Fig. 2. Functional comparison of the PD and CB proteomes. (A) The bars represent the quantitative distribution of the number of PD proteins identified by LC-MS/MS relative to corresponding proteins identified in the CB fraction, as determined in Experimental Procedures. (B) The relative abundance can also be indicated from the intensity of bands on Coomassie-blue-stained SDS/PAGE gel for PD and CB proteins (Left) or by quantitative Western blotting of the indicated proteins (Right). RB indicates the PD/CB ratio as measured by Western blot and densitometry. Rm ratio was measured by LC-MS/MS as in A. (C) Comparison of the pathways that are relevant to the CB- and PD-enriched proteins (P < 0.05; the lower P value indicates the pathway is more relevant to the analyzed proteins). The pathways are extracted from 1,088 CB-enriched proteins (PD/CB <0.67) and 1,073 PD-enriched proteins (PD/CB >1.5) using Ingenuity Pathways software.

 

Figure 3
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Fig. 3. Functional comparison of the PD and CB phosphoproteomes. (A) Comparison of the pathways that are relevant to the CB- and PD-enriched phosphoproteins (P < 0.05), as in Fig. 2A. (B) The distribution pattern of kinase phosphorylation motifs and associated kinase classes of identified phosphopeptides. The percentage indicates the ratio of the number of proteins containing a particular motif to the total number of proteins in each fraction.

 

Figure 4
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Fig. 4. Signaling pathways that regulate ERK in response to LPA-induced PD formation. (A) Western blot shows the total and phosphorylation levels of MEK and ERK in PD and CB compartments. (B) Three possible signaling pathways that may activate ERK in response to LPA-induced PD extension. ERK activity can also be negatively regulated by EphA2. (C) Known ERK substrates and positive/negative regulators. The relative abundance of each protein is included in parenthesis. PDU indicates that the protein is unique in PD.

 


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