Phosphotyrosine of macrophage by low-density lipoproteins from hemodialysis patients.
BACKGROUND
Because of the possible importance of tyrosine phosphorylation in the signal transduction process, we investigated whether an interaction of low-density lipoprotein (LDL) from hemodialysis patients (HD-LDL) and human macrophages induces tyrosine-phosphorylated proteins in the macrophages.


METHODS
Human monocyte-derived macrophages were incubated with HD-LDL (100 micrograms/ml) or native LDL (100 micrograms/ml) for 15 minutes at 37 degrees C. Whole cells were lyzed with Tris-HCl buffer containing vanadate and Triton X-100. After centrifugation, lyzed proteins were divided into Triton-soluble and -insoluble fractions. Both fractions (soluble and insoluble) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were electroblotted onto a polyvinylidene difluoride (PVDF) membrane. Immunoblotting was performed using an antibody against phosphotyrosine or c-Src.


RESULTS
Several proteins in the range 40 to 100 kDa were found to be phosphorylated constitutively in the macrophages and not affected by the addition of HD-LDL. HD-LDL did not induce any tyrosine-phosphorylated proteins either in the soluble or insoluble fractions. Macrophages pretreated with tyrosine kinase inhibitor genestein drastically inhibited tyrosine phosphorylation of these proteins. The nonreceptor tyrosine kinase, c-Src p60, was also strongly tyrosine phosphorylated in the macrophages, and this was not enhanced by the stimulation of HD-LDL.


CONCLUSIONS
These data suggest that tyrosine autophosphorylated proteins may play a role in the early step of signal transduction in the macrophages.
Sonobe
M
M
Third Department of Internal Medicine, Wakayama Medical College, Japan. sonobem@wakayama-med.ac.jp
Kimura
K
K
Kishino
M
M
Tone
Y
Y
Yamada
Y
Y
Mune
M
M
Yukawa
S
S
Nishide
I
I
D004791:Enzyme Inhibitors / Q000494:pharmacology; D019833:Genistein / Q000494:pharmacology; D006801:Humans; D015151:Immunoblotting; D008077:Lipoproteins, LDL / Q000494:pharmacology*; D008264:Macrophages / Q000187:drug effects* / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D019000:Phosphotyrosine / Q000378:metabolism*; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism; D006435:Renal Dialysis; D014443:Tyrosine / Q000378:metabolism
D004791:Enzyme Inhibitors; D008077:Lipoproteins, LDL; D019000:Phosphotyrosine; D014443:Tyrosine; D019833:Genistein; D016392:Proto-Oncogene Proteins pp60(c-src)