Biochemical analysis of the CD45-p56(lck) complex in Jurkat T cells lacking expression of lymphocyte phosphatase-associated phosphoprotein.
In human and mouse lymphocytes the protein tyrosine phosphatase CD45, a key molecule involved in T cell activation, non-covalently associates with the tyrosine kinase p56(lck) and lymphocyte phosphatase-associated phosphoprotein (LPAP), a 32 kDa phosphoprotein of unknown function. In order to gain insight into the function of LPAP we have generated an LPAP-deficient Jurkat variant by means of antisense strategies. Analysis of the CD45-p56(lck) molecular complex in this cell line revealed that loss of LPAP does not alter the expression or the enzymatic activity of CD45 or p56(lck). In addition, the association between CD45 and p56(lck) is not affected in LPAP-deficient T cells. These data suggest that LPAP does not regulate the enzymatic activity of CD45 or p56(lck) and is not required for the association between these two proteins. In order to identify polypeptides that preferentially associate with LPAP we established a Jurkat variant expressing a chimeric receptor which was composed of the extracellular portion of the human HLA-A2.1 molecule and the full-length LPAP protein. Comparative two-dimensional analysis of CD45 and HLA-A2 immunoprecipitates obtained from these cells following metabolic labeling resulted in the identification of a 43 kDa protein that preferentially associates with LPAP under mild detergent conditions.
Bruyns
E
E
Institute of Immunology, Immunomodulation Laboratory, Ruprecht-Karls University of Heidelberg, Germany.
Kirchgessner
H
H
Meuer
S
S
Schraven
B
B
D015180:Electrophoresis, Gel, Two-Dimensional; D015789:HLA-A2 Antigen / Q000276:immunology; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D019169:Jurkat Cells / Q000276:immunology*; D017493:Leukocyte Common Antigens / Q000378:metabolism*; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000378:metabolism*; D046911:Macromolecular Substances; D008565:Membrane Proteins; D008970:Molecular Weight; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010750:Phosphoproteins / Q000172:deficiency / Q000502:physiology*; D019000:Phosphotyrosine / Q000378:metabolism; D011485:Protein Binding; D011993:Recombinant Fusion Proteins; D013601:T-Lymphocytes / Q000276:immunology
D015789:HLA-A2 Antigen; D047908:Intracellular Signaling Peptides and Proteins; D046911:Macromolecular Substances; D008565:Membrane Proteins; C090519:PTPRCAP protein, human; D010750:Phosphoproteins; C513303:Ptprcap protein, mouse; D011993:Recombinant Fusion Proteins; D019000:Phosphotyrosine; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D010749:Phosphoprotein Phosphatases; D017493:Leukocyte Common Antigens