The RACK1 signaling scaffold protein selectively interacts with the cAMP-specific phosphodiesterase PDE4D5 isoform.

Stephen J. Yarwood, Michael R. Steele, Grant Scotland, Miles D. Houslay, and Graeme B. Bolger

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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