On Forgiveness

On Forgiveness is a young woman\u27s thoughts on forgiveness - of the self and of others - based on several key moments in her life. Told through a series of memories, the essay wrestles with the power of forgiveness and how it can be misunderstood in difficult moments

Delineation of RAID1, the RACK1 interaction domain located within the unique N-terminal region of the cAMP-specific phosphodiesterase, PDE4D5

Background The cyclic AMP specific phosphodiesterase, PDE4D5 interacts with the β-propeller protein RACK1 to form a signaling scaffold complex in cells. Two-hybrid analysis of truncation and mutant constructs of the unique N-terminal region of the cAMP-specific phosphodiesterase, PDE4D5 were used to define a domain conferring interaction with the signaling scaffold protein, RACK1. Results Truncation and mutagenesis approaches showed that the RACK1-interacting domain on PDE4D5 comprised a cluster of residues provided by Asn-22/Pro-23/Trp-24/Asn-26 together with a series of hydrophobic amino acids, namely Leu-29, Val-30, Leu-33, Leu-37 and Leu-38 in a 'Leu-Xaa-Xaa-Xaa-Leu' repeat. This was done by 2-hybrid analyses and then confirmed in biochemical pull down analyses using GST-RACK1 and mutant PDE4D5 forms expressed in COS cells. Mutation of Arg-34, to alanine, in PDE4D5 attenuated its interaction with RACK1 both in 2-hybrid screens and in pull down analyses. A 38-mer peptide, whose sequence reflected residues 12 through 49 of PDE4D5, bound to RACK1 with similar affinity to native PDE4D5 itself (Ka circa 6 nM). Conclusions The RACK1 Interaction Domain on PDE4D5, that we here call RAID1, is proposed to form an amphipathic helical structure that we suggest may interact with the C-terminal β-propeller blades of RACK1 in a manner akin to the interaction of the helical G-γ signal transducing protein with the β-propeller protein, G-β