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 beta-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 beta-propeller blades of RACK1 in a manner akin to the interaction of the helical G-gamma signal transducing protein with the beta-propeller protein, G-beta.
Bolger
Graeme B
GB
Veterans Affairs Medical Center, Huntsman Cancer Institute, Division of Oncology, Department of Medicine, University of Utah Health Sciences Center, Salt Lake City, UT 84148, USA. Graeme.Bolger@ccc.uab.edu
McCahill
Angela
A
Yarwood
Stephen J
SJ
Steele
Michael R
MR
Warwicker
Jim
J
Houslay
Miles D
MD
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000502:physiology; D019556:COS Cells; D053420:CRADD Signaling Adaptor Protein; D002522:Chlorocebus aethiops; D000242:Cyclic AMP / Q000235:genetics / Q000378:metabolism*; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D010727:Phosphoric Diester Hydrolases / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D053420:CRADD Signaling Adaptor Protein; C104252:CRADD protein, human; D009363:Neoplasm Proteins; C419368:RACK1 protein, human; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface; D000242:Cyclic AMP; D010727:Phosphoric Diester Hydrolases; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; C517376:PDE4D protein, human; D019204:GTP-Binding Proteins