Cryptic sequence features within the disordered protein p27 ^Kip1 regulate cell cycle signaling

Abstract

Peptide motifs embedded within intrinsically disordered regions (IDRs) of proteins are often the sites of posttranslational modifications that control cell-signaling pathways. How do IDR sequences modulate the functionalities of motifs? We answer this question using the polyampholytic C-terminal IDR of the cell cycle inhibitory protein p27Kip1 (p27). Phosphorylation of Thr-187 (T187) within the p27 IDR controls entry into S phase of the cell division cycle. Additionally, the conformational properties of polyampholytic sequences are predicted to be influenced by the linear patterning of oppositely charged residues. Therefore, we designed sequence variants of the p27 IDR to alter charge patterning outside the primary substrate motif containing T187. Computer simulations and biophysical measurements confirm predictions regarding the impact of charge patterning on the global dimensions of IDRs. Through functional studies, we uncover cryptic sequence features within the p27 IDR that influence the efficiency of T187 phosphorylation. Specifically, we find a positive correlation between T187 phosphorylation efficiency and the weighted net charge per residue of an auxiliary motif. We also find that accumulation of positive charges within the auxiliary motif can diminish the efficiency of T187 phosphorylation because this increases the likelihood of long-range intra-IDR interactions that involve both the primary and auxiliary motifs and inhibit their contributions to function. Importantly, our findings suggest that the cryptic sequence features of the WT p27 IDR negatively regulate T187 phosphorylation signaling. Our approaches provide a generalizable strategy for uncovering the influence of sequence contexts on the functionalities of primary motifs in other IDRs.p27 | motif | disordered regions

Selection maintains signaling function of a highly diverged intrinsically disordered region

“…This is consistent with the model for the evolution of phosphorylation sites as a mechanism for modulation of charge (60). The importance of the basal net charge of the Ste50 IDR in signaling function is also consistent with recent studies suggesting that "cryptic" electrostatic properties encoded in amino acid sequences of IDRs are important for their function (61,62). We speculate that the charge of the Ste50 IDR affects interactions with the cell membrane, as has been demonstrated for Ste5 (47), but understanding the precise biophysical and biochemical properties of the Ste50 IDR that translate charge into signaling output is an important area for further study.…”

MLL1 promotes cervical carcinoma cell tumorigenesis and metastasis through interaction with β-catenin

“…We next investigated the detailed mechanism of MLL1 in the regulation of cell cycle. Cell cycle is regulated by numerous proteins, such as CCND1,20 p21,21 p27,22 and CCNE1 23. Among these proteins, CCND1 has been demonstrated to be transcriptional activated by MLL1 transcription and to mediate the effect of MLL1 in promoting cell proliferation.…”

The contribution of intrinsically disordered regions to protein function, cellular complexity, and human disease

“…This can further influence the functional elements (e.g. motifs or posttranslational modification sites) that are embedded within IDRs and can affect critical processes such as the cell cycle [39]. For the same number of charged residues, the charge patterning has also been shown to determine whether the polypeptide segment will be fully extended (e.g.…”