Abstract
The Cytoplasmic Polyadenylation Element Binding proteins are RNA binding proteins involved in the translational regulation of mRNA. During cell cycle progression, CPEB1 is labeled for degradation by phosphorylation-dependent ubiquitination by the SCFβ−TrCP ligase. The peptidyl-prolyl isomerase Pin1 plays a key role in CPEB1 degradation. Conditioned by the cell cycle stage, CPEB1 and Pin1 interactions occur in a phosphorylation-independent or -dependent manner. CPEB1 contains six potential phosphorylatable Pin1 binding sites. Using a set of biophysical techniques, we discovered that the pS210 site is unique, since it displays binding activity not only to the WW domain but also to the prolyl-isomerase domain of Pin1. The NMR structure of the Pin1 WW-CPEB1 pS210 (PDB ID: 2n1o) reveals that the pSerPro motif is bound in trans configuration through contacts with amino acids located in the first turn of the WW domain and the conserved tryptophan in the β3-strand. NMR relaxation analyses of Pin1 suggest that inter-domain flexibility is conferred by the modulation of the interaction with peptides containing the pS210 site, which is essential for degradation.
Highlights
The Cytoplasmic Polyadenylation Element Binding (CPEB) proteins activate RNA translation, promoting the elongation of the poly(A) tail of messenger RNAs
Since target recognition of Pin[1] is achieved by the interaction of the WW domain with the protein partners, we prepared an 15N-labeled WW domain sample and monitored the chemical shift perturbations induced in the WW domain by the presence of the ligands (1:1 WW: ligand ratio) using HSQC experiments (Fig. 1b,c)
The affected residues of the WW domain are located at the beginning of the first β -strand (Glu[12]; we use one-letter code for CPEB1 amino acids and three-letter code for Pin[1] residues) and second loop (His[27], Ile28), which are in spatial proximity to each other, and residues at the end of the third β -strand (Ser[32], Gln[33], Trp[34] and Glu35)
Summary
The Cytoplasmic Polyadenylation Element Binding (CPEB) proteins activate RNA translation, promoting the elongation of the poly(A) tail of messenger RNAs. Partial destruction of CPEB1 is necessary for the transition from Metaphase I (MI) to Metaphase II (MII) and for the functional substitution of CPEB1 by CPEB46 This mechanism requires the phosphorylation of CPEB1 to create the binding site for the SCFβ-TrCP ubiquitin ligase and guarantees the polyadenylation of various CPE-containing mRNAs, a process regulated by the levels of different CPEBs in vivo in a time-specific manner. Configuration of the pSer-Pro motifs being crucial for SCFβ-TrCP ubiquitin ligase recognition[11,12,13] In this context, it is not surprising that phosphorylated CPEB1 has recently been identified as a target for the peptidyl-prolyl isomerase Pin[1] and that the isomerase protein regulates CPEB1 destruction[14]. Together with the available information in the literature, contribute to a better understanding of the role of Pin[1] in ubiquitination and CPEB1 protein degradation
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