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Identification of the Molecular Partners of Lymphocyte Phosphatase-Associated Phosphoprotein (LPAP) That Are Involved in Human Lymphocyte Activation

  • MOLECULAR MECHANISMS OF ADAPTIVE IMMUNITY
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Abstract

Lymphocyte phosphatase-associated phosphoprotein (LPAP) is a small transmembrane protein that is found in lymphocytes and is tightly associated with the phosphatase CD45. The function of LPAP is still unknown. Studies of the LPAP interactome may reveal new details of how C45 and lymphocyte signaling in general are regulated. LPAP binding partners were sought using coimmunoprecipitation coupled with mass spectrometry, stabilization of protein complexes with chemical crosslinkers, and Blue Native electrophoresis. In addition to CD45, several proteins were identified as LPAP partners, including CD71, CD98, cytoskeletal proteins, the amino acid transporter SLC1A4, and the cell signaling component HS1. It was confirmed that more than 70% of LPAP molecules were bound with CD45 in a 1 : 1 complex. The effect of CD45 on LPAP was studied in CEM and Jurkat cells with a CD45 knockout. The LPAP levels in the cells were 10% of the level in wild-type cells. In the absence of CD45, LPAP phosphorylation at Ser-153 and Ser-163 was not affected, whereas phosphorylation at Ser-99 and Ser-172 decreased significantly. Based on the results, CD45 was assumed to play a role in regulating LPAP expression and phosphorylation status.

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Funding

This work was supported by the Russian Foundation for Basic Research (project no. 18-34-00705 for proteomics experiments; project no. 17-04-00526 for construction of knockout and stably transfected cells).

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Authors and Affiliations

  1. Institute of Immunology National Research Center, Federal Medical-Biological Agency, 115522, Moscow, Russia

    N. A. Kruglova & A. V. Filatov

  2. Immunology Department, Biological Faculty, Moscow State University, 119234, Moscow, Russia

    N. A. Kruglova & A. V. Filatov

  3. Orekhovich Institute of Biomedical Chemistry, Russian Academy of Medical Sciences, 119121, Moscow, Russia

    A. T. Kopylov

Authors
  1. N. A. Kruglova
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  2. A. T. Kopylov
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  3. A. V. Filatov
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Correspondence to N. A. Kruglova.

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The authors declare that they have no conflict of interest. This article does not contain any studies involving animals or human participants performed by any of the authors.

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Translated by T. Tkacheva

Abbreviations: CIP, calf intestinal alkaline phosphatase; co-IP/MS, coimmunoprecipitation coupled with mass spectrometry; DTME, dithiobis(maleimidoethane); DTT, dithiothreitol; DSP, dithiobis(succinimidyl propionate); FIPA, fluorescence immunoprecipitation assay; IP, immunoprecipitation; LC–MS, liquid chromatography–mass spectrometry; LPAP, lymphocyte phosphatase-associated phosphoprotein; PAGE, polyacrylamide gel electrophoresis; PMA, phorbol 12-myristate 13-acetate; R6G rhodamine 6G, succinimidyl ester; RIPA, radioimmunoprecipitation assay; SDS, sodium dodecyl sulfate; WB, Western blotting.

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Kruglova, N.A., Kopylov, A.T. & Filatov, A.V. Identification of the Molecular Partners of Lymphocyte Phosphatase-Associated Phosphoprotein (LPAP) That Are Involved in Human Lymphocyte Activation. Mol Biol 53, 739–747 (2019). https://doi.org/10.1134/S002689331905011X

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  • DOI: https://doi.org/10.1134/S002689331905011X

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