Signal transduction in human B cells during aging: alterations in stimulus-induced phosphorylations of tyrosine and serine/threonine substrates and in cytosolic calcium responsiveness.
Protein phosphorylation is considered an early cellular mechanism of signal transduction by surface immunoglobulins (sIg) and other receptors of B cells. Using intact human peripheral blood B cells of young subjects labeled with orthophosphate, increased phosphorylation levels of serine/threonine and tyrosine substrates were demonstrated on indicator phosphoproteins corresponding to the CD20 isoforms and microtubule-associated protein 2 kinase after cross-linking sIg and costimulation with phorbol diesters. By contrast, stimulated B cells from certain elderly subjects displayed substantial alterations in the phosphorylation patterns of serine/threonine or tyrosine indicator phosphoproteins. Also, age-related impairments in sIg stimulated mobilization of cytosolic protein kinase C (PKC) enzymatic activity and in cytosolic calcium [Ca2+]i responses of B cells were observed with the altered phosphorylation reactions. Comparison of the substrate phosphorylation profiles to the proliferative responses of stimulated B cells from individual elderly subjects suggested a model of signal transduction in which differing stimuli have different dependencies on phosphorylation reactions. Diminished proliferative responses after sIg ligation coincided with decreased phosphorylations of either tyrosine or serine/threonine indicator substrates. However, the decreased proliferative responses of B cells from elderly subjects with substantial reductions of tyrosine phosphorylation after sIg ligation were enhanced by the direct stimulation of serine/threonine kinase activity with phorbol diesters or CD40 ligation. Experiments with kinase inhibitors evaluated the relative dependency of different B cell stimuli on tyrosine and serine/threonine phosphorylation reactions. The proliferative responses of normal B cells to sIg ligation were quite sensitive to the tyrosine kinase inhibitor genistein whereas those observed following costimulations with phorbol diesters or CD40 ligation were more resistant. However, treatment of B cells with H7, an inhibitor of PKC activity, led to a more uniform reduction of B-cell responses after different stimuli. Results from RNase protection assays of c-myc expression also suggested that different B-cell stimuli might utilize distinct intracellular signaling pathways. Both the type of stimuli and mode of sIg ligation were important in determining the stimulated levels of c-myc mRNA expression. Thus, the current findings suggest that age-related defects are present in human B cell signaling pathways as reflected by tyrosine and serine/threonine phosphorylation reactions. Also, these age-related defects can coexist with altered mobilization of PKC enzymatic activity and with alterations in [Ca2+]i and proliferative responses.
Whisler
R L
RL
Department of Internal Medicine, College of Medicine, Ohio State University, Columbus 43210.
Liu
B Q
BQ
Newhouse
Y G
YG
Walters
J D
JD
Breckenridge
M B
MB
Grants
I S
IS
D000375:Aging / Q000502:physiology*; D000596:Amino Acids / Q000378:metabolism*; D001402:B-Lymphocytes / Q000502:physiology*; D002118:Calcium / Q000378:metabolism*; D002455:Cell Division / Q000502:physiology; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism*; D006801:Humans; D010766:Phosphorylation; D011493:Protein Kinase C / Q000097:blood / Q000502:physiology; D011505:Protein-Tyrosine Kinases / Q000502:physiology; D012260:Ribonucleases / Q000037:antagonists & inhibitors; D012694:Serine / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D013912:Threonine / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism
D000596:Amino Acids; D013912:Threonine; D014443:Tyrosine; D012694:Serine; D011505:Protein-Tyrosine Kinases; D011493:Protein Kinase C; D012260:Ribonucleases; D002118:Calcium