Figure 5
Figure 5. Identification of PECAM-1 serine phosphorylation sites and characterization of phosphoserine-specific antibodies. (A) Identification of S702 and S707 as major sites of PECAM-1 serine phosphorylation. Levels of incorporation of radiolabeled phosphate (32P) into PECAM-1 immunoprecipitated from untreated (UN) or OA-treated HEK cells expressing WT or mutant forms of human PECAM-1. Mutant forms of PECAM-1 included Y663,686F(12S), in which tyrosine residues at positions 663 and 686 were substituted with phenylalanine; Y663,686F(12S→A), which contained the tyrosine-to-phenylalanine substitutions, as well as substitution of alanine for all 12 serine residues; and Y663,686F(11S→A), which contained the tyrosine-to-phenylalanine substitutions at positions 663 and 686, as well as serine-to-alanine substitutions at all but 1 position. At 5 of these positions (620, 670, 687, 702, and 707), serine was found in all of the species in which PECAM-1 had thus far been sequenced, and at 4 of these positions (611, 626, 669, and 673), serine was less well conserved (D.K.N., unpublished observations). Note that the WT and Y663,686F(12S), but not Y663,686F(12S→A), forms of PECAM-1 were phosphorylated in both untreated and OA-treated HEK 293T cells. Reintroduction of single serine residues revealed preferential serine phosphorylation of PECAM-1 at positions 702 > 707 ≫ 673 (★). (B) Characterization of PECAM-1 pS702- and pS707-specific polyclonal antibodies. Western blot analysis of PECAM-1 immunoprecipitates from HEK 293T transfectants described in panel A revealed that affinity-purified rabbit polyclonal anti-pS702 (top) and anti-pS707 (middle) IgGs both recognized WT and Y663,686F(12S) but not Y663,686F(12S→A) forms of PECAM-1. Reintroduction of a single serine residue at position 702 restored recognition of PECAM-1 by the pS702-specific but not the pS707-specific IgG. Similarly, reintroduction of a single serine residue at position 707 restored recognition of PECAM-1 by the pS707-specific but not the pS702-specific IgG. The presence of PECAM-1 in all PECAM-1 immunoprecipitates was confirmed by binding of a phosphorylation-independent PECAM-1-specific antibody (bottom). (C) Characterization of the PECAM-1 pY686-specific polyclonal antibody. Western blot of PECAM-1 immunoprecipitates from HEK 293T cells transfected with WT, Y663F, Y686F, or Y663,686F mutant forms of PECAM-1 revealed that rabbit polyclonal anti-pY686 (top) recognized WT and Y663F but not Y686F or Y663,686F forms of PECAM-1. Tyrosine phosphorylation of WT, Y663F, and Y686F but not Y663,686F forms of PECAM-1 was confirmed by Western blot analysis with pY20 (middle). The presence of PECAM-1 in all PECAM-1 immunoprecipitates was confirmed by binding of a phosphorylation-independent PECAM-1–specific antibody (bottom).

Identification of PECAM-1 serine phosphorylation sites and characterization of phosphoserine-specific antibodies. (A) Identification of S702 and S707 as major sites of PECAM-1 serine phosphorylation. Levels of incorporation of radiolabeled phosphate (32P) into PECAM-1 immunoprecipitated from untreated (UN) or OA-treated HEK cells expressing WT or mutant forms of human PECAM-1. Mutant forms of PECAM-1 included Y663,686F(12S), in which tyrosine residues at positions 663 and 686 were substituted with phenylalanine; Y663,686F(12S→A), which contained the tyrosine-to-phenylalanine substitutions, as well as substitution of alanine for all 12 serine residues; and Y663,686F(11S→A), which contained the tyrosine-to-phenylalanine substitutions at positions 663 and 686, as well as serine-to-alanine substitutions at all but 1 position. At 5 of these positions (620, 670, 687, 702, and 707), serine was found in all of the species in which PECAM-1 had thus far been sequenced, and at 4 of these positions (611, 626, 669, and 673), serine was less well conserved (D.K.N., unpublished observations). Note that the WT and Y663,686F(12S), but not Y663,686F(12S→A), forms of PECAM-1 were phosphorylated in both untreated and OA-treated HEK 293T cells. Reintroduction of single serine residues revealed preferential serine phosphorylation of PECAM-1 at positions 702 > 707 ≫ 673 (★). (B) Characterization of PECAM-1 pS702- and pS707-specific polyclonal antibodies. Western blot analysis of PECAM-1 immunoprecipitates from HEK 293T transfectants described in panel A revealed that affinity-purified rabbit polyclonal anti-pS702 (top) and anti-pS707 (middle) IgGs both recognized WT and Y663,686F(12S) but not Y663,686F(12S→A) forms of PECAM-1. Reintroduction of a single serine residue at position 702 restored recognition of PECAM-1 by the pS702-specific but not the pS707-specific IgG. Similarly, reintroduction of a single serine residue at position 707 restored recognition of PECAM-1 by the pS707-specific but not the pS702-specific IgG. The presence of PECAM-1 in all PECAM-1 immunoprecipitates was confirmed by binding of a phosphorylation-independent PECAM-1-specific antibody (bottom). (C) Characterization of the PECAM-1 pY686-specific polyclonal antibody. Western blot of PECAM-1 immunoprecipitates from HEK 293T cells transfected with WT, Y663F, Y686F, or Y663,686F mutant forms of PECAM-1 revealed that rabbit polyclonal anti-pY686 (top) recognized WT and Y663F but not Y686F or Y663,686F forms of PECAM-1. Tyrosine phosphorylation of WT, Y663F, and Y686F but not Y663,686F forms of PECAM-1 was confirmed by Western blot analysis with pY20 (middle). The presence of PECAM-1 in all PECAM-1 immunoprecipitates was confirmed by binding of a phosphorylation-independent PECAM-1–specific antibody (bottom).

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