Figure 7

Phosphorylation of S463 controls F-actin bundling activity and Arp2/3 interaction of CRN2.

(A) two-step F-actin spin-down assay employing rabbit skeletal muscle G-actin, bovine Arp2/3 complex and purified recombinant CRN2 wild-type (W), phosphomimetic S463D (D) and S463A (A) mutant C-terminal fragments. Coomassie brilliant blue stained SDS-PAGE gels are shown. The left panel (10,000xg first pellet) demonstrates F-actin bundling activity of wild-type and S463A mutant CRN2 in comparison to reduced bundling activity of S463D mutant CRN2. Vice versa the middle panel (100,000xg second pellet) shows increased co-sedimentation of S463D mutant CRN2. Arp2/3 does not influence the results. Right panel, 100,000xg supernatant given as control. As further control, intensities of actin bands were analyzed by densitometry and the measurements demonstrated equal sums for the six triplets n+n'+n''. (B) Arp2/3 immunoblot of a 100,000xg F-actin spin-down experiment. Presence of all CRN2 polypeptides releases Arp2/3 from F-actin into the supernatant. This experiment only allows a qualitative assessment due to difficulties to completely dissolve the pellets in SDS sample buffer and transfer the proteins onto the blot membrane⁸⁸. (C) pull-down assay employing purified recombinant His-tagged CRN2 wild-type as well as S463A and phosphomimetic S463D mutant C-terminal polypeptides coupled to Ni-beads and soluble purified Arp2/3 complex. In comparison to wild-type and S463A mutant CRN2, the S463D mutant shows reduced direct binding to Arp2/3. Arp2/3, p34 immunoblot; CRN2pep, mAb K6-444 immunoblot. Beads, Ni-sepharose beads lacking CRN2; flow-through, Arp2/3 flow-through from these blank beads. (D) co-immunoprecipitations using GFP mAb K3-167-2⁶ coupled to Protein G coated beads and lysates from 293TN cells expressing GFP-tagged full-length wild-type CRN2 as well as S463D and S463A mutants. Immunoblotting was performed with CRN2 mAb K6-444 and p34 pAb (Upstate #07-227). S463D mutant GFP-CRN2 shows reduced interaction with the Arp2/3 complex. Prior to preparation of the lysates cells were treated with latrunculin B to prevent unspecific co-precipitation of proteins tied together via F-actin bridges. (E) bar chart, densitometry analysis of the Arp2/3 signal intensity from three independent experiments, where GFP-CRN2 wild-type and S463D mutant were parallelly immunoprecipitated; one experiment is shown in D. Arp2/3 values are normalized to the respective GFP-CRN2 values.