Serine phosphorylation of the GPIbα 580-590 sequence in resting platelets and requirement for 14-3-3ζ binding. (A) The rabbit antiserum pSGPIb1 was obtained by immunization with the P1P2 phosphopeptide (LVAGRRPpSALpS) and its reactivity was analyzed by ELISA. Dilutions of the serum were incubated with immobilized GPIbα peptides and revealed with GAR-HRP. pSGPIb1 recognized P1P2 and the monophosphorylated P1 (LVAGRRPpSALS) and P2 (LVAGRRPSALpS) peptides but not the nonphosphorylated ØP (LVAGRRPSALS) peptide. Results are the mean (± SEM) of 3 separate experiments, and NI represents nonimmune serum from the same animal. (B) Triton X-100 lysates of resting platelets were separated by 4% to 15% SDS-PAGE and immunoblotted with pSGPIb1, pSer609, or an antibody against the 3 GPIb-IX subunits. GPIbα was heavily labeled by pSGPIb1, suggesting Ser phosphorylation of the 580-590 domain. In accordance with previous reports, labeling with pSer609 showed that GPIbα was also phosphorylated at Ser609. (C) Triton X-100 lysates of resting platelets were treated or not with 0.3 U/mL PAP for 30 minutes at 37°C. Proteins were separated by 4% to 15% SDS-PAGE, transferred to PVDF membranes, and probed with pSer609 and pSGPIb1. Reactivity to the antibodies was lost in samples treated with PAP, confirming phosphorylation at both sites. (D) The proportion of phosphorylated GPIbα was estimated by immunodepleting the cell lysates with an excess of the phosphospecific antibody pSGPIb1 (middle panel) or pSer609 (right panel) and performing a second immunoprecipitation with an anti-GPIbβ antibody. Products of the first and second immunoprecipitations were revealed with a polyclonal anti-GPIb-IX antibody. In both cases, a similar small proportion of GPIb-IX was revealed following the second immunoprecipitation, indicating that most GPIbα subunits were phosphorylated at both sites. The left panel corresponds to a control where the first depletion was performed in the presence of a nonimmune serum, and results are from 1 experiment representative of 4 independent assays. (E) In GST-14-3-3ζ pull-down experiments, proteins were precipitated from the same lysates as in panel C and probed with an antibody against the GPIb-IX complex. GST-14-3-3ζ precipitated GPIbα in the absence of PAP treatment but not after its dephosphorylation by PAP. A negative control using GST alone is shown in the left lane. Results in panels B-E are from 1 experiment representative of 3.
