title: 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.
authors.affiliation: Laboratory of Immunochemistry, Institute of Immunology , Moscow, Russia.
authors.forename: Alexander
authors.initials: A
authors.lastname: Filatov
authors.affiliation: Faculty of Biology, Lomonosov Moscow State University , Moscow, Russia.
authors.forename: Natalia
authors.initials: N
authors.lastname: Kruglova
authors.affiliation: Faculty of Biology, Lomonosov Moscow State University , Moscow, Russia.
authors.forename: Tatiana
authors.initials: T
authors.lastname: Meshkova
authors.affiliation: Laboratory of Immunochemistry, Institute of Immunology , Moscow, Russia.
authors.forename: Dmitriy
authors.initials: D
authors.lastname: Mazurov