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First published online November 7, 2007
doi: 10.1242/10.1242/jcs.008177

Journal of Cell Science 120, 3911-3918 (2007)
Published by The Company of Biologists 2007
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Identification of phosphorylation sites in βPIX and PAK1

Mark W. Mayhew1, Erin D. Jeffery2, Nicholas E. Sherman3, Kristina Nelson3, Joy M. Polefrone2, Stephen J. Pratt1, Jeffrey Shabanowitz2, J. Thomas Parsons4, Jay W. Fox2, Donald F. Hunt2,5 and Alan F. Horwitz1,*

1 Department of Cell Biology, University of Virginia, Charlottesville, VA 22908, USA
2 Department of Chemistry, University of Virginia, Charlottesville, VA 22908, USA
3 Department of W. M. Keck Biomedical Mass Spectrometry Laboratory, University of Virginia, Charlottesville, VA 22908, USA
4 Department of Microbiology, University of Virginia, Charlottesville, VA 22908, USA
5 Department of Pathology, University of Virginia, Charlottesville, VA 22908, USA


Figure 1
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  		Fig. 1. Phosphorylation sites detected in serum-only and EGF-stimulated murine βPIXa. (A) Ser, Thr and Tyr coverage of the FLAG-βPIXa sequence (tag not shown) generated with trypsin. Detected peptides are bold and underlined. Residues not covered are shaded in gray. Observed phosphorylation sites are red. Red brackets above residues indicate that a phosphorylation site could not be unambiguously assigned to the specific amino acid. In total, 91% of the amino acid sequence and 89% of the Ser, Thr and Tyr sites were covered. A total of 16 phosphorylation sites were identified. (B) Mapping of domains and identified phosphorylation sites. MS/MS-identified phosphorylation sites are labeled. Sites labeled with red text were detected only with the treatment of EGF to cells. Sites within red brackets specify ambiguity between two sites.

 

Figure 2
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  		Fig. 2. Phosphorylation sites detected in human PAK1. (A) Ser, Thr and Tyr coverage of the FLAG-PAK1 sequence (tag not shown) generated with trypsin/chymotrypsin. Detected peptides are bold and underlined. Residues not covered are shaded in gray. Observed phosphorylation sites are red. In total, 83% of the amino acid sequence was covered. Thirteen phosphorylation sites were identified. (B) Mapping of domains and identified phosphorylation sites. MS/MS-identified phosphorylation sites are labeled. N, G and P denote the NCK-, GRB2- and βPIX-binding sites, respectively; AI, auto-inhibitory domain. Red brackets indicate the ambiguous site.

 

Figure 3
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  		Fig. 3. Effect of PAK1 T359 and T185 on protrusion. GFP-PAK1 mutants were expressed in CHO cells and protrusion rates determined by kymography (Nayal et al., 2006Go). Rates are in µm/minute. Rates were determined using at least ten cells per mutant.

 




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