The Loss of PIN1 Deregulates Cyclin E and Sensitizes Mouse Embryo Fibroblasts to Genomic Instability

Abstract

During the G 0 /G 1 -S phase transition, the timely synthesis and degradation of key regulatory proteins is required for normal cell cycle progression. Two of these proteins, c-Myc and cyclin E, are recognized by the Cdc4 E3 ligase of the Skp1/Cul1/Rbx1 (SCF) complex. SCF Cdc4 binds to a similar phosphodegron sequence in c-Myc and cyclin E proteins resulting in ubiquitylation and degradation of both proteins via the 26 S proteosome. Since the prolyl isomerase Pin1 binds the c-Myc phosphodegron and participates in regulation of c-Myc turnover, we hypothesized that Pin1 would bind to and regulate cyclin E turnover in a similar manner. Here we show that Pin1 regulates the turnover of cyclin E in mouse embryo fibroblasts. Pin1 binds to the cyclin E-Cdk2 complex in a manner that depends on Ser 384 of cyclin E, which is phosphorylated by Cdk2. The absence of Pin1 results in an increased steady-state level of cyclin E and stalling of the cells in the G 1 /S phase of the cell cycle. The cellular changes that result from the loss of Pin1 predispose Pin1 null mouse embryo fibroblasts to undergo more rapid genomic instability when immortalized by conditional inactivation of p53 and sensitizes these cells to more aggressive Ras-dependent transformation and tumorigenesis.Mitogenic stimuli initiate a sequence of events that result in the entry of quiescent cells into S phase (1). Critical for this important transition is the ordered synthesis and degradation of transcription factors such as c-Jun and c-Myc (Myc) and cyclins such as cyclin D and cyclin E (1). Deregulation of the turnover of these proteins, such that they remain active at inappropriate times during cell cycle progression, is frequently found in human cancer. Thus, an understanding of the molecular mechanisms that regulate protein turnover is crucial to provide insight into the oncogenic process. Three proteins important for the progression of cells into S phase, c-Jun, c-Myc, and cyclin E, are ubiquitylated by a common member of the Skp1/ Cul1/Rbx1 (SCF) 3 group of ubiquitin enzymes in which the F-box component, which serves as the ubiquitin E3 ligase, is Cdc4 (SEL-10, Fbw7, Ago) (2). SCF Cdc4 binds to a component of each protein that has been termed the "phosphodegron" (3) to promote the ubiquitylation and degradation of these proteins via the 26 S proteosome. c-Jun and Myc have an additional binding protein in common, the peptidyl prolyl cis/trans-isomerase Pin1, which binds and isomerizes prolyl bonds in the context of phospho-Ser/ Thr-Pro motifs (4, 5).Previously, we described the mechanism by which Pin1 promotes Myc degradation (5). The Cdc4 phosphodegron of Myc is present in a domain termed Myc box 1 (MB1), containing the sequence LPpTPPLpSP (where pT represents phosphothreonine and pS represents phosphoserine), in which the two phosphorylation events occur sequentially and are catalyzed by ERK (Ser 62 ) and GSK3␤ (Thr 58 ), respectively (6, 7). Pin1 binds to the doubly phosphorylated motif in a manner requiring phospho-Thr 58 and promotes a conformationa...

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

“…4, A and B); as previously reported, in our Pin1-KO MEFs, the cyclin D1 levels are markedly decreased (21), and cyclin E is significantly increased (41) (Fig. 4A).…”

“…As an example of this, the overexpression of Pin1 in mammalian cells leads to a G 2 arrest, whereas its inhibition causes mitotic arrest (16). Moreover, Pin1 regulates the turnover of c-Myc and cyclin E (49, 41), both of which play critical roles in the G 1 /S phase transition, and the cyclin E protein has been shown to be further destabilized by Pin1 in MEFs (41). On the other hand, we have also reported that Pin1 can regulate cyclin D1 through both transcriptional and translational mechanisms (17)(18)21).…”

Active Mek2 as a regulatory scaffold that promotes Pin1 binding to BPGAP1 to suppress BPGAP1-induced acute Erk activation and cell migration

“…Such non-canonical binding by Pin1 has also been observed with BNIP-H (or caytaxin) (Buschdorf et al, 2008), A3G cytidine deaminase (Watashi et al, 2008) and the Pro-X-Thr-Pro recognition motif of phosphatase inhibitor-2, but independent of phosphorylation (Li et al, 2008). In addition, Pin1 can bind to the pThr-Gly motif of cyclin E (Yeh et al, 2005). These results strongly support the versatility of Pin1 in regulating a wide spectrum of cellular targets and processes.…”

The Prolyl Isomerase Pin1 Targets Stem-Loop Binding Protein (SLBP) To Dissociate the SLBP-Histone mRNA Complex Linking Histone mRNA Decay with SLBP Ubiquitination

“…Pin1 catalyzes cis-trans prolyl isomerization in its substrates, facilitating conformational changes that regulate protein function. Proteins identified as Pin1 targets include mitotic phosphoproteins (4,17,60), transcription factors (14,34,49), and cell cycle regulators (46,63). Pin1 is specific for phosphorylated Ser/ Thr-Pro sequences and acts along with kinases and phosphatases to exert its biochemical effects.…”