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.
Highlights
Lymphocyte phosphatase-associated phosphoprotein (LPAP), encoded by the gene protein tyrosine phosphatase, receptor type C associated protein (PTPRCAP), is a 206 amino acid small transmembrane protein with an apparent molecular weight of 32 kDa [1]
Treatment of LPAP with calf intestinal phosphatase (CIP) resulted in disappearance of the lower band and an increase in the intensity of the upper band, indicating that the lower band corresponded to the phosphorylated protein
In order to determine the phosphorylated sites of LPAP in activated peripheral blood mononuclear cells (PBMCs), we examined the effects of phorbol 12-myristate 13-acetate (PMA) treatment on the LPAP 2D-SDS-PAGE pattern
Summary
Lymphocyte phosphatase-associated phosphoprotein (LPAP), encoded by the gene protein tyrosine phosphatase, receptor type C associated protein (PTPRCAP), is a 206 amino acid (aa) small transmembrane protein with an apparent molecular weight of 32 kDa [1]. LPAP is located predominantly on the plasma membrane as part of a supramolecular complex and is tightly bound to the phosphatase CD45 via its transmembrane domain [2]. Two segments of LPAP are identified as putative binding domains, including a WW domain [3] and an acidic domain required for interaction with the kinase Lck [4,5]. Another component of the supramolecular complex is the co-receptor CD4 [6].
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