PTP1B dephosphorylates tyrosine-phosphorylated NSF. A:
top left, recombinant NSF expressed in E. coli BLR(DE3) (0.5
μg) or expressed and tyrosine phosphorylated in E. coli TKB1 (0.5
μg) were resolved on 8% SDS gels, transferred to nitrocellulose, and
immunoblotted (IB) with 4G10 (anti-PY, top) and anti-NSF
(bottom) antibodies. Top right, recombinant phospho-NSF (310
nm) was incubated with 1 μg/ml recombinant wild type PTP1B. At
the indicated time points, aliquots containing 0.7 μg of NSF were mixed
with SDS-PAGE sample buffer, proteins were resolved on 8% SDS gels,
transferred to nitrocellulose, and immunoblotted with 4G10 (anti-PY,
top). The same membranes were probed (without stripping) with anti-NSF
(anti-NSF, bottom) antibodies. Shown is an experiment representative
of five repetitions; quantification (carried out with Image J, freeware from
NIH) of Western blots is depicted as mean ± S.E. from all five
replicates below the immunoblots. B, same as in A, except
that the substrate trapping mutant was used instead of wild type PTP1B. The
experiment was repeated twice. C, two-dimensional electrophoresis of
recombinant, tyrosine-phosphorylated NSF expressed in E. coli TKB1
(20μg, NSF) and solubilized sperm proteins (150μg,
SPERM). The first dimension was carried out on precast IPG strips and
the second dimension on 8% SDS gels. Proteins were transferred to
nitrocellulose, and Western blots were performed with anti-NSF and
anti-phosphotyrosine (anti-PY) antibodies as described. pH is
indicated on top and Mr standards
(×103) are indicated on the left. Shown are images
representative of two independent experiments. D, SLO-permeabilized
human sperm were treated for 15 min at 37 °C in the presence of 3.3
nm anti-PTP1B antibodies or 300 nm PTP1B D181A, followed
by an additional 15 min in the presence of 310 nm recombinant NSF
(black bars). AR was initiated by adding 0.5 mm
CaCl2 and incubating for 15 min at 37 °C. Controls (gray
bars) included AR inhibition by 3.3 nm anti-PTP1B antibodies
or 300 nm PTP1B D181A (anti-PTP1B/PTP1B D181A →
Ca2+); and AR unperturbed by 310 nm NSF (NSF →
Ca2+). The data were normalized as described under
“Experimental Procedures.” Actual percentages of reacted sperm for
control and Ca2+ ranged between 10–38 and 21–52%,
respectively. The data represent the mean ± S.E. of at least three
independent experiments.