Lymphocyte phosphatase‐associated phosphoprotein proteoforms analyzed using monoclonal antibodies

Abstract

Phosphatase CD45 regulates the activation of lymphocytes by controlling the level of receptor and signal molecule phosphorylation. However, it remains unknown which molecules mediate the phosphatase activity of CD45. A candidate for such a molecule is a small transmembrane adapter protein called lymphocyte phosphatase-associated phosphoprotein (LPAP). LPAP forms a supramolecular complex that consists of not only CD45 molecule but also CD4 and Lck kinase. The function of LPAP has not been defined clearly. In our study, we determined the pattern of LPAP expression in various cell types and characterized its proteoforms using new monoclonal antibodies generated against the intracellular portion of the protein. We show that LPAP is a pan-lymphocyte marker, and its expression in cells correlates with the expression of CD45. The majority of T, B and NK cells express high levels of LPAP, whereas monocytes, granulocytes, monocyte-derived dendritic cells, platelets and red blood cells are negative for LPAP. Using one- and two-dimensional protein gel electrophoresis, we demonstrate that LPAP has at least four sites of phosphorylation. The resting cells express at least six different LPAP phosphoforms representing mono-, di- and tri-phosphorylated LPAP. T and B cells differ in the distribution of the protein between phosphoforms. The activation of lymphocytes with PMA reduces the diversity of phosphorylated forms. Our experiments on Lck-deficient Jurkat cells show that Lck kinase is not involved in LPAP phosphorylation. Thus, LPAP is a dynamically phosphorylated protein, the function of which can be understood, when all phosphosites and kinases involved in its phosphorylation will be identified.

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

“…LPAP from Jurkat cells and other lymphoblastoid cell lines is phosphorylated [ 27 , 29 ], but whether this holds true for primary cells is unclear. To address this issue, freshly isolated peripheral blood mononuclear cells (PBMCs) were labeled with Cy3/Cy5, lysed, and LPAP was immunoprecipitated and analyzed by SDS-PAGE or difference gel electrophoresis (2D-DIGE).…”

“…The intensity of the first band represents the quantity of the unphosphorylated and pS153 mono-phosphorylated forms. Taking into account our previous results [ 27 ], demonstrating that the level of unphosphorylated LPAP was approximately 15%, we calculated the proportion of the pS153 mono-phosphorylated form to be approximately 40%.…”

“…Labeling of cells with amino reactive fluorescent dyes Cy3 or Cy5 (BioDye, Moscow, Russia) was performed as described previously [ 27 ]. The protocol can be found at http://dx.doi.org/10.17504/protocols.io.h5db826 .…”

“…A new set of anti-LPAP monoclonal antibodies, generated in our laboratory [ 25 ] and tested on the 10 th human leukocyte differentiation antigen (HLDA) [ 26 ], provide an additional instrument for LPAP investigation. Using these antibodies, we identified at least five different LPAP phosphoforms in lymphocytes [ 27 ].…”

Origins and clinical relevance of proteoforms in pediatric malignancies

“…Filatov showed that LPAP has at least four sites of phosphorylation and that resting (non-activated) cells express at least six different LPAP proteoforms depending on phospho-state. Additionally, T and B cells differed in the relative abundance of the various phosphoproteoforms, indicating possible differences in their roles [92].…”