Sj. Tumminia et P. Russell, ALPHA-B-CRYSTALLIN ACCUMULATION IN HUMAN ASTROGLIOMA CELL-LINE U373MGIS STRESS-DEPENDENT AND PHOSPHORYLATION-INDEPENDENT, Journal of Biochemistry, 116(5), 1994, pp. 973-979
alpha B-crystallin, a major lens protein, is present in non-lenticular
tissues. Although alpha B-crystallin possesses chaperone-like activit
y, its physiological significance outside the lens is unclear. Recent
studies suggest that stress-induced mechanisms of alpha B-crystallin a
ccumulation are dependent upon the type of insult. To further investig
ate this possibility, the response of alpha B-crystallin to different
stress conditions was examined in the human astroglioma U373MG cell li
ne which constitutively expresses alpha B-crystallin. Exposure of this
cell line to 25 mu M cobalt chloride and heat stress resulted in incr
eased accumulation of alpha B-crystallin. Exposure to cobalt chloride
resulted in a 6-fold (water-soluble) and 5-fold (water-insoluble) incr
ease in alpha B-crystallin, 4 h after the initial exposure to cobalt.
The water-soluble fraction of U373 cells subjected to heat shock at 42
degrees C for 30 min and harvested after 1 h exhibited a 4.5-fold inc
rease in the level of alpha B-crystallin over the control levels. Appr
oximately 2 h after the removal of the cells from the heat stress, the
level of alpha B-crystallin in the urea-soluble fraction increased 3-
fold. Although alpha B-crystallin accumulation initially increased in
response to both the cobalt and heat shock challenges, analysis of the
pattern of protein accumulation indicates that there are differences
in the mechanism of alpha B-crystallin induction which are dependent o
n the type of stress. The concentration of alpha B-crystallin in both
the water-soluble and water-insoluble fractions of heat-stressed cells
showed that alpha B-crystallin concentrations returned to basal level
s within 4 h after the initial shock, while levels of alpha B-crystall
in remained high in the water-soluble fraction of cobalt-stressed cell
s 72 h after the initial stress. Since the role of phosphorylation in
chaperone function is controversial, U373 cells were examined for indu
ced forms of phosphorylated alpha B-crystallin. Isoelectric focusing g
el electrophoresis followed by Western analysis indicated that the pre
dominant form of alpha B-crystallin was unphosphorylated although smal
l amounts of the phosphorylated form were found in all conditions. The
phosphorylation state of alpha B-crystallin did not change after expo
sure to stress conditions indicating that the protective role of alpha
B-crystallin is phosphorylation-independent under these conditions.