Pin1 Catalyzes Conformational Changes of Thr-187 in p27Kip1 and Mediates Its Stability through a Polyubiquitination Process

The cis-trans peptidylprolyl isomerase Pin1 plays a critical role in regulating a subset of phosphoproteins by catalyzing conformational changes on the phosphorylated Ser/Thr-Pro motifs. The phosphorylation-directed ubiquitination is one of the major mechanisms to regulate the abundance of p27^Kip1. In this study, we demonstrate that Pin1 catalyzes the cis-trans conformational changes of p27^Kip1 and further mediates its stability through the polyubiquitination mechanism. Our results show that the phosphorylated Thr-187-Pro motif in p27^Kip1 is a key Pin1-binding site. In addition, NMR analyses show that this phosphorylated Thr-187-Pro site undergoes conformational change catalyzed by Pin1. Moreover, in Pin1 knock-out mouse embryonic fibroblasts, p27^Kip1 has a shorter lifetime and displays a higher degree of polyubiquitination than in Pin1 wild-type mouse embryonic fibroblasts, suggesting that Pin1 plays a critical role in regulating p27^Kip1 degradation. Additionally, Pin1 dramatically reduces the interaction between p27^Kip1 and Cks1, possibly via isomerizing the cis-trans conformation of p27^Kip1. Our study thus reveals a novel regulatory mechanism for p27^Kip1 stability and sheds new light on the biological function of Pin1 as a general regulator of protein stability.Cellular differentiation and cell cycle inhibition are tightly controlled via sensitive molecular mechanisms. p27^Kip1, a member of the Cip/Kip family, is an essential cell cycle inhibitor that functions largely during the G₀/G₁ phase where it promotes the assembly of the cyclin D1-CDK4 complex and inhibits the kinase activity of the cyclin E-CDK2 complex in the G₁-S phase (1–4). Several review articles have elegantly summarized and discussed the detailed cellular functions of p27^Kip1 (1–6). p27^Kip1 is also a phosphoprotein with multiple Ser/Thr phosphorylation sites, including Ser-10, Ser-178, and Thr-187, followed by a proline residue. Hence, these motifs are potential substrate sites for proline-directed kinases (5, 6). Compared with Ser-178, which has not yet been well studied, the phosphorylation of Ser-10 and Thr-187 has been well characterized to be important for the regulation of p27^Kip1 function. For instance, Ser-10 has been found to be the major phosphorylation site of p27^Kip1 (7) and to play an important role in regulating cell migration (8–10), although the regulation of Ser-10 phosphorylation is still not completely defined (11, 12).In contrast to Ser-10 and Thr-178, Thr-187 is the best characterized phosphorylation site on p27^Kip1 and is known to regulate the complex formation of p27^Kip1-cyclin E-CDK2 (1–2). In addition, it is also widely accepted that Thr-187 plays a crucial role in determining the abundance of mature p27^Kip1 proteins. The phosphorylation of Thr-187 directs p27^Kip1 to an SCF^Skp2 ubiquitin ligase complex (consisting of Skp2-Skp1-Cks1-Cul1-Roc1), which in turn promotes the polyubiquitination and degradation of p27^Kip1 (13, 14). The crystal structure of the Skp1-Skp2-Cks1-p27^Kip1 phosphopeptide complex shows that p27^Kip1 binds both Cks1 and Skp2 and that the C terminus of Skp2 and Cks1 forms the substrate recognition core of the SCF complex (15). Furthermore, the structure of this complex has revealed that the phosphorylation of Thr-187 in p27^Kip1 is recognized by the phosphate-binding site of Cks1, indicating that Cks1 is not only a facilitator but also an indispensable component in p27^Kip1 degradation machinery (15).Pin1 is a unique peptidyl-prolyl isomerase (PPIase)² that recognizes only the phosphorylated Ser/Thr motif preceding a proline residue (16). In addition, Pin1 is very prominent in isomerizing the cis-trans conformation of prolyl-peptidyl bonds in its substrates, resulting in either the modification of their function (e.g. c-Jun (17), β-catenin (18), Bax (19), and Notch1 (20)) or modulation of their stability (e.g. cyclin D1 (21), p53 (22, 23), and NF-κB (24)). Loss of Pin1 in mice results in several phenotypes similar to those of cyclin D1-null mice (21) and neuronal degenerative phenotypes (25–28), suggesting the conformational changes mediated by Pin1 may be crucial for the normal functioning of cells. Additionally, Pin1 also plays important roles in cancer and other cellular events, which have been extensively discussed in several recent review articles (29–33).In this study, we show that Pin1 binds to p27^Kip1, mainly through the phosphorylated Thr-187-Pro motif, and causes subsequent prolyl isomerization of this cell cycle protein. Moreover, we also find that Pin1 can protect p27^Kip1 from degradation. Importantly, we demonstrate that by catalyzing conformational changes in p27^Kip1, Pin1 hinders its association with Cks1, resulting in a reduction of polyubiquitination of p27^Kip1 and protecting its degradation by SCF^Skp2 complexes. Our results suggest that the cis-trans isomerization catalyzed by Pin1 represents a novel regulatory mechanism during post-phosphorylation of proteins and polyubiquitination-directed degradation pathways.