Constitutive and activation-dependent phosphorylation of lymphocyte phosphatase-associated phosphoprotein (LPAP)

PLoS One. 2017 Aug 21;12(8):e0182468. doi: 10.1371/journal.pone.0182468. eCollection 2017.

Abstract

Lymphocyte phosphatase-associated phosphoprotein (LPAP) is a small transmembrane protein expressed exclusively in lymphocytes. LPAP is a component of a supramolecular complex composed of the phosphatase CD45, the co-receptor CD4, and the kinase Lck. In contrast to its immunologically important partners, the function of LPAP is unknown. We hypothesized that the biological role of LPAP may be determined by analyzing LPAP phosphorylation. In the present study, we identified LPAP phosphorylation sites by site-directed mutagenesis, phospho-specific antibodies, and protein immunoprecipitation coupled to mass spectrometry analysis. Our results confirmed previous reports that Ser-99, Ser-153, and Ser-163 are phosphorylated, as well as provided evidence for the phosphorylation of Ser-172. Using various SDS-PAGE techniques, we detected and quantified a set of LPAP phosphoforms that were assigned to a combination of particular phosphorylation events. The phosphorylation of LPAP appears to be a tightly regulated process. Our results support the model: following phorbol 12-myristate 13-acetate (PMA) or TCR/CD3 activation of T cells, LPAP is rapidly dephosphorylated at Ser-99 and Ser-172 and slowly phosphorylated at Ser-163. Ser-153 exhibited a high basal level of phosphorylation in both resting and activated cells. Therefore, we suggest that LPAP may function as a co-regulator of T-cell signaling.

MeSH terms

  • Cell Line
  • Electrophoresis, Polyacrylamide Gel
  • Flow Cytometry
  • Humans
  • Intracellular Signaling Peptides and Proteins / genetics
  • Intracellular Signaling Peptides and Proteins / metabolism*
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism*
  • Mutagenesis, Site-Directed
  • Phosphorylation
  • Tandem Mass Spectrometry

Substances

  • Intracellular Signaling Peptides and Proteins
  • Membrane Proteins
  • PTPRCAP protein, human

Grant support

This work was supported by the Russian Science Foundation (project 14-15-00702) (http://www.rscf.ru/prjcard?rid=14-15-00702) and a grant from the Russian Foundation for Basic Research No. 17-04-00526. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.