Degeneracy and function of the ubiquitous RVXF motif that mediates binding to protein phosphatase-1.
Most interactors of protein phosphatase-1 (PP1) contain a variant of a so-called "RVXF" sequence that binds to a hydrophobic groove of the catalytic subunit. A combination of sequence alignments and site-directed mutagenesis has enabled us to further define the consensus sequence for this degenerate motif as [RK]-X(0-1)-[VI]-[P]-[FW], where X denotes any residue and [P] any residue except Pro. Naturally occurring RVXF sequences differ in their affinity for PP1, and we show by swapping experiments that this binding affinity is an important determinant of the inhibitory potency of the regulators NIPP1 and inhibitor-1. Also, inhibition by NIPP1-(143-224) was retained when the RVXF motif (plus the preceding Ser) was swapped for either of two unrelated PP1-binding sequences from human inhibitor-2, i.e. KGILK or RKLHY. Conversely, the KGILK motif of inhibitor-2 could be functionally replaced by the RVXF motif of NIPP1. Our data provide additional evidence for the view that the RVXF and KGILK motifs function as anchors for PP1 and thereby promote the interaction of secondary binding sites that determine the activity and substrate specificity of the enzyme.
Wakula
Paulina
P
Afdeling Biochemie, Faculteit Geneeskunde, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium.
Beullens
Monique
M
Ceulemans
Hugo
H
Stalmans
Willy
W
Bollen
Mathieu
M
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D019556:COS Cells; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016384:Consensus Sequence; D004722:Endoribonucleases; D005982:Glutathione Transferase / Q000235:genetics; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D054645:Protein Phosphatase 1; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins; D011817:Rabbits; D011993:Recombinant Fusion Proteins; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D000097847:Protein Phosphatase Inhibitory Proteins
D002352:Carrier Proteins; D004722:Endoribonucleases; D005982:Glutathione Transferase; D047908:Intracellular Signaling Peptides and Proteins; D010446:Peptide Fragments; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D011506:Proteins; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; C021042:protein phosphatase inhibitor-1; C024833:protein phosphatase inhibitor-2; D000097847:Protein Phosphatase Inhibitory Proteins; C097081:PPP1R8 protein, human