Wakula Paulina, Beullens Monique, Ceulemans Hugo, Stalmans Willy, Bollen Mathieu
Afdeling Biochemie, Faculteit Geneeskunde, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium.
J Biol Chem. 2003 May 23;278(21):18817-23. doi: 10.1074/jbc.M300175200. Epub 2003 Mar 25.
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.
蛋白磷酸酶-1（PP1）的大多数相互作用蛋白都含有一种所谓“RVXF”序列的变体，该序列可与催化亚基的疏水凹槽结合。通过序列比对和定点诱变相结合的方法，我们得以进一步确定这个简并基序的共有序列为[RK]-X(0 - 1)-[VI]-[P]-[FW]，其中X表示任意残基，[P]表示除脯氨酸以外的任意残基。天然存在的RVXF序列对PP1的亲和力各不相同，我们通过交换实验表明，这种结合亲和力是调节因子NIPP1和抑制剂-1抑制效力的重要决定因素。此外，当将RVXF基序（加上前面的丝氨酸）替换为人抑制剂-2的两个不相关的PP1结合序列之一（即KGILK或RKLHY）时，NIPP1-(143 - 224)的抑制作用仍然保留。相反，抑制剂-2的KGILK基序在功能上可以被NIPP1的RVXF基序取代。我们的数据为以下观点提供了额外证据：RVXF和KGILK基序作为PP1的锚定物发挥作用，从而促进决定该酶活性和底物特异性的二级结合位点之间的相互作用。
