Binding of phosphatase inhibitor-2 to prolyl isomerase Pin1 modifies specificity for mitotic phosphoproteins.
Inhibitor-2 (I-2) is the most ancient protein that selectively recognizes type-1 protein phosphatase and is phosphorylated by CDK1-cyclinB during mitosis at Thr72 in a conserved PXTP site. Pin1 is a peptide prolyl cis/trans isomerase conserved among eukaryotes that specifically reacts with proteins phosphorylated at Ser/Thr-Pro sites. We tested phospho-T72-I-2 as a substrate for Pin1 and discovered that unphosphorylated I-2 bound Pin1 with micromolar affinity and phosphorylation of the PXTP site or truncation of I-2 reduced binding 10-fold. Ectopic Pin1 coprecipitated endogenous I-2 and ectopic I-2 coprecipitated endogenous Pin1, but only in the absence of detergents, which may account for the interaction not being detected previously. Endogenous I-2 and Pin1 colocalized in HeLa cells and showed nuclear-cytoplasmic redistribution in response to cell density, suggestive of their association in living cells. Recombinant Pin1 binding to different phosphoproteins in mitotic cell extracts was modulated by I-2, and binding to individual mitotic phosphoproteins was increased, decreased or unaffected by I-2, showing that I-2 allosterically modifies Pin1 specificity. This was confirmed by mutation of Ser16 to Ala in the Pin1 WW domain that eliminated I-2 binding and abrogated I-2 effects on Pin1 binding to different phosphoproteins. A S16E mutation to mimic Pin1 phosphorylation restored binding to both I-2 and phospho-T72-I-2, indicating that phosphorylation of both proteins governs their interaction. The results reveal a novel function for I-2, and suggest phosphorylation-dependent regulation of Pin1 specificity during entry and exit of mitosis, in other phases of the cell cycle, and in multiple cell signaling processes.
Center for Cell Signaling, University of Virginia School of Medicine Charlottesville, Virginia 22908, USA.
Mingguang
M
Li
P Todd
PT
Stukenberg
David L
DL
Brautigan
D015153:Blotting, Western; D006367:HeLa Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D008938:Mitosis; D008954:Models, Biological; D009154:Mutation; D000072340:NIMA-Interacting Peptidylprolyl Isomerase; D019696:Peptidylprolyl Isomerase / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012694:Serine / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D000097847:Protein Phosphatase Inhibitory Proteins
D000072340:NIMA-Interacting Peptidylprolyl Isomerase; D019696:Peptidylprolyl Isomerase; D010750:Phosphoproteins; D011506:Proteins; D011994:Recombinant Proteins; D012694:Serine; C024833:protein phosphatase inhibitor-2; D000097847:Protein Phosphatase Inhibitory Proteins; C000606846:PIN1 protein, human