Increased ubiquitination and reduced expression of LCK in T lymphocytes from patients with systemic lupus erythematosus.
OBJECTIVE
To explore regulation of proximal signaling and composition of lipid rafts in T lymphocytes from patients with systemic lupus erythematosus (SLE).


METHODS
The expression, phosphorylation, and degradation of lipid raft-associated signaling molecules in T lymphocytes from 50 patients with SLE compared with 28 healthy controls and 22 rheumatoid arthritis patients were investigated. Lipid raft and nonraft fractions from T cells were isolated by ultracentrifugation. Proteins in the lipid raft and nonraft fractions were analyzed by Western blotting and probed for phosphotyrosine activity and for LCK, LAT, and CD3 epsilon. Immunoprecipitation experiments were performed to assess protein ubiquitination in T cell lysates. T cell phenotype and levels of intracellular LCK were determined by flow cytometry.


RESULTS
LCK, an essential signaling molecule for T cell activation, was significantly reduced in both lipid raft and nonraft fractions of T lymphocytes from patients with active SLE compared with controls, and the reduction was independent of treatment. To identify the likely causes of reduced LCK, we explored the possibility that chronic activation of T lymphocytes underlies LCK degradation. The results revealed an increase in protein ubiquitination, and specifically LCK ubiquitination, in T cells from SLE patients. However, our findings suggest that the increase in ubiquitination is independent of T cell activation.


CONCLUSIONS
LCK is reduced in T cell lipid rafts from patients with SLE. This reduction appears to be independent of activation and may be associated with abnormal ubiquitin-mediated regulation mechanisms.
Jury
Elizabeth C
EC
Royal Free and University College Medical School, University College, London, UK. e.jury@ucl.ac.uk
Kabouridis
Panagiotis S
PS
Abba
Amina
A
Mageed
Rizgar A
RA
Isenberg
David A
DA
D048868:Adaptor Proteins, Signal Transducing; D000328:Adult; D000368:Aged; D015153:Blotting, Western; D017252:CD3 Complex; D002352:Carrier Proteins / Q000378:metabolism; D002458:Cell Fractionation; D005434:Flow Cytometry; D006801:Humans; D050356:Lipid Metabolism; D008180:Lupus Erythematosus, Systemic / Q000201:enzymology*; D008213:Lymphocyte Activation; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000378:metabolism*; D008565:Membrane Proteins; D008875:Middle Aged; D010750:Phosphoproteins / Q000378:metabolism; D019000:Phosphotyrosine / Q000378:metabolism; D011233:Precipitin Tests; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000201:enzymology*; D014452:Ubiquitins / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D017252:CD3 Complex; C115335:CD3E protein, human; D002352:Carrier Proteins; C110810:LAT protein, human; D008565:Membrane Proteins; D010750:Phosphoproteins; D011948:Receptors, Antigen, T-Cell; D014452:Ubiquitins; D019000:Phosphotyrosine; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck)