In addition to the SH3 binding region, multiple regions within the N-terminal noncatalytic portion of the cAMP-specific phosphodiesterase, PDE4A5, contribute to its intracellular targeting.
The long cyclic AMP (cAMP)-specific phosphodiesterase isoform, PDE4A5 (PDE4A subfamily isoform variant 5), when transiently expressed in COS-7 cells, was shown in subcellular fractionation studies to be associated with both membrane and cytosol fractions, with immunofluorescence analyses identifying PDE4A5 as associated both with ruffles at the cell margin and also at a distinct perinuclear localisation. Deletion of the first nine amino acids of PDE4A5 (1) ablated its ability to interact with the SH3 domain of the tyrosyl kinase, LYN; (2) reduced, but did not ablate, membrane association; and (3) disrupted the focus of PDE4A5 localisation within ruffles at the cell margin. This deleted region contained a Class I SH3 binding motif of similar sequence to those identified by screening a phage display library with the LYN-SH3 domain. Truncation to remove the PDE4A5 isoform-specific N-terminal region caused a further reduction in membrane association and ablated localisation at the cell margin. Progressive truncation to delete the PDE4A long isoform common region and then the long isoform-specific UCR1 did not cause any further change in membrane association or intracellular distribution. However, deletion up to the super-short form splice junction generated an entirely soluble 'core' PDE4A species. We propose that multiple sites in the N-terminal noncatalytic portion of PDE4A5 have the potential to associate with intracellular structures and thus define its intracellular localisation. At least two such sites lie within the PDE4A5 isoform-specific N-terminal region and these appear to be primarily responsible for targeting PDE4A5 to, and organising it within, the cell margin; one is an SH3 binding motif able to interact with LYN kinase and the other lies within the C-terminal portion of the PDE4A5 unique region. A third membrane association region is located within the N-terminal portion of UCR2 and appears to be primarily responsible for targeting to the perinuclear region. Progressive N-terminal truncation, to delete defined regions of PDE4A5, identified activity changes occurring upon deletion of the SH3 binding site region and then upon deletion of the membrane association site region located within UCR2. This suggests that certain of these anchor sites may not only determine intracellular targeting but may also transduce regulatory effects on PDE4A5 activity.
Beard
Matthew B
MB
Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Davidson Building, Institute of Biomedical and Life Sciences, University of Glasgow, G12 8QQ, Scotland, Glasgow, UK.
Huston
Elaine
E
Campbell
Lachlan
L
Gall
Irene
I
McPhee
Ian
I
Yarwood
Stephen
S
Scotland
Grant
G
Houslay
Miles D
MD
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D020134:Catalytic Domain; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D003600:Cytosol / Q000201:enzymology; D007425:Intracellular Membranes / Q000201:enzymology; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D019151:Peptide Library; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D017384:Sequence Deletion; D018909:src Homology Domains; D019061:src-Family Kinases / Q000737:chemistry / Q000378:metabolism
D019151:Peptide Library; C060902:lyn protein-tyrosine kinase; D019061:src-Family Kinases; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4