Page 14909: Line 14 of the
Abstract should read "with high affinity (Ka
approximately 7 nM) . . . . ."
Page 14914, line 3 and in the legend
to Fig. 5: "EC50 of 7.4 ± 1.1 pM" should
be "EC50 of 7.4 ± 1.1 nM." Also, the x axis in Fig. 5B should be revised so that the
values are increased 1000-fold (i.e. the range becomes 10-E9
to 10-E5 instead of 1-E12 to 10-E8). The revised Fig. 5 and its legend
are shown below.
These corrections do not affect the
conclusions of the paper.
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Fig. 5.
Interaction of recombinant forms of
PDE4D5 and RACK1, as purified from E. coli.
A, fusions between GST and RACK1, and also between MBP
and PDE4D3 or PDE4D5, were expressed and purified from E. coli (see "Experimental Procedures"). GST alone
(i.e. not as a fusion) was expressed and purified in an
identical manner. Cell lysates (lanes b) and purified
proteins (lanes e) obtained after elution from the
appropriate affinity column were run on SDS-PAGE and stained with
Coomassie Blue. The species were purified to apparent homogeneity as
analyzed by SDS-PAGE. The positions of the arrows mark the
relative molecular weight of the purified proteins as follows:
GST, 27.3 ± 1.1 kDa; GST-RACK1, 59.8 ± 1.4 kDa;
MBP-4D3, 128 ± 3.8 kDa; MBP-4D5, 135.3 ± 2.6 kDa. These data are typical of experiments done at least three times.
B, the interaction of E. coli-purified
recombinant PDE4D5 and RACK1 was tested in an ELISA as described under
"Experimental Procedures." The MBP-PDE4D5 fusion bound to GST-RACK1
in a dose-dependent manner, with an EC50 of 7.4 ± 1.1 nM (mean ± S.D.; n = 3 separate experiments).
As a control, parallel experiments were performed for MBP-PDE4D3.
C, dose-response curves were calculated for the inhibition
of PDE4D5 by rolipram at a concentration of substrate (cAMP) of 1.0 µM. Assays were performed on MBP-PDE4D5 ("4D5-MBP")
alone, and also on MBP-PDE4D5 complexed with GST-RACK1. Assays were
performed using an excess of GST-RACK1 so that all of the PDE4D5 would
be complexed with RACK1 (see "Experimental Procedures"). In
pull-down experiments, all of the PDE4D5 could be shown to complex with
GST-RACK1 under these conditions (data not shown). As a control, assays
were performed with GST alone, added at comparable levels. The
IC50 values for rolipram inhibition were 0.13 ± 0.1, 0.16 ± 0.05, and 0.52 ± 0.07 µM for MBP-PDE4D5 alone,
MBP-PDE4D5 mixed with GST, and MBP-PDE4D5 mixed with GST-RACK1,
respectively (mean ± S.D., n = 3). These values are
significantly different (MBP-PDE4D5 alone compared with MBP-PDE4D5
complexed with GST-RACK1; p < 0.005, t test).
Protein assays were performed, and molar concentrations were determined
on the basis of the calculated molecular weights.
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