Fig 1: CCM3 binding to striatin is affected by the same deletions that reduce Mst3 binding. (A) Three days after transfection with HA-tagged wild-type and mutant striatins, HEK293 cells stably expressing FLAG-CCM3 were lysed and FLAG-CCM3 immune complexes were isolated, separated by SDS-PAGE, and immunoblotted. Lysate levels of the transfected HA-tagged striatins are shown for comparison to the amount of HA-striatins that coimmunoprecipitated with FLAG-CCM3. (B) Relative binding of wild-type and mutant striatins to FLAG-CCM3 was measured as described in the legend to Figure 2B, except that in this experiment the values were normalized to expression of the HA-constructs in the cell lysates. The error bars represent the standard deviation of three independent experiments. **, p ≤ 0.01 relative to wild-type.
Fig 2: Model of human wild-type striatin complex, based on results from previous studies and the current study. Schematic of wild-type human striatin, drawn to scale, showing the locations of its previously published protein-interaction motifs/domains and residues or domains determined in this study to be important for binding of the striatin-associated proteins. Previously published domains: Caveolin (Cav)-binding motif (aa53-63); C-C, coiled-coil region (aa70-116); Calmodulin (CaM)-binding domain (aa149-166); WD-repeat domain (aa419-780). The Cav-binding motif and CaM-binding domain are indicated by arrows with corresponding proteins below. Estrogen receptor alpha (not shown) has been found to bind to striatin amino acids 1-203 [23]. Upper labels (from this study): Names are as indicated. Residues important for the binding of these proteins are indicated by arrows or by brackets. Mst4 binding was not affected as dramatically as Mst3 by Δ(227-309) so it may have an overlapping, but not identical, binding site. Note: The amino acid stretch in striatin important for Mst3/CCM3 association contains a small region of amino acid homology with PP2A B' B-type subunits that we previously identified [8]. Whether these residues play a role in binding Mst3/CCM3 remains to be tested.
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