Cryptic sequence features within the disordered protein p27Kip1 regulate cell cycle signaling

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 p27^Kip1 (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.Intrinsically disordered proteins and regions (IDPs/IDRs) serve as scaffolds for short linear motifs, which are highly conserved functional sequence modules that are typically 3- to 10-residues long (1, 2). Motifs often mediate protein–protein interactions and they encompass sites of specific posttranslational modifications that control a range of regulatory functions in cells (3). An example of cellular regulation that is achieved through posttranslational modifications of specific residues in motifs is the control of cell cycle arrest through Tyr and Thr phosphorylation in linear motifs within the cell cycle inhibitor protein p27^Kip1.Progression through the mammalian cell cycle is controlled by the interplay between cyclin-dependent kinases (Cdks), cyclins, and the Cip/Kip family of Cdk inhibitors that includes p27^Kip1—referred to hereafter as p27 (4). In isolation, p27, which is disordered (5), encompasses two functional domains. The N-terminal domain (p27_1–95) folds upon binding to Cdk2/cyclin A, resulting in complete kinase inhibition. Conversely, whereas p27_1–95 folds upon binding to Cdk2/cyclin A, the C-terminal domain (p27_96–198) remains disordered and participates in a phosphorylation/ubiquitination-signaling cascade (6) (Fig. 1). Proliferative signals cause the activation of nonreceptor tyrosine kinases (NRTKs) that phosphorylate p27 within the Cdk2-bound N-terminal domain at Tyr-88 (Y88) (7). Phosphorylation of Y88 leads to partial restoration of the kinase activity of Cdk2 that is achieved without releasing p27 from the complex with Cdk2/cyclin A; this triggers intracomplex phosphorylation of Thr-187 (T187) near the C terminus of p27, creating a phosphodegron site (8). Subsequent recruitment of the E3 ubiquitin ligase, SCF^Skp2 (9, 10), catalyzes polyubiquitination of three lysine-containing motifs within p27_96–198 (11). Polyubiquitinated p27 is then selectively degraded by the 26S proteasome, releasing fully active Cdk2/cyclin A to drive progression of the cell cycle into S phase (7).Open in a separate windowFig. 1.The p27 IDR integrates proliferative signals from NRTKs to release active Cdk2/cyclin A via T187 phosphorylation. Proliferative signals activate NRTKs that phosphorylate p27 at Y88, partially restoring Cdk2 activity (step 1). This allows for intracomplex phosphorylation of p27 at T187 by Cdk2 and creates a phosphodegron (step 2). Recruitment of the SCF^Skp2 E3 ubiquitin ligase leads to ubiquitination of lysine residues within p27-C (step 3) followed by selective degradation of p27 by the proteasome (step 4) and release of the fully active Cdk2/cyclin A. The primary sequence of the p27 IDR is shown with positively and negatively charged residues depicted in blue and red, respectively. The primary substrate motif is highlighted in yellow.T187 is part of the primary substrate motif T₁₈₇-P₁₈₈-K₁₈₉-K₁₉₀. Phosphorylation of T187 is the key signaling step that leads to p27 degradation and full activation of Cdk2/cyclin A, and this signaling reaction is enabled by disorder within p27_96–198 (6). Here, we investigate the impact of changes to sequence-encoded disorder on the conformational properties of p27_96–198 and the efficiency of T187 phosphorylation. This investigation was motivated by recent studies showing that the conformational properties and the degree of disorder of IDPs/IDRs are influenced by sequence attributes such as the net charge per residue (NCPR) and the fraction of charged residues (12–18). Importantly, a majority of IDPs/IDRs, including p27_96–198, are polyampholytes that are enriched in both positively and negatively charged residues (19–21). In polyampholytes, the sequence patterning of oppositely charged residues is predicted to modulate the degree of chain expansion/compaction and the amplitudes of conformational fluctuations (20). Alterations to the linear patterning of oppositely charged residues leads to changes in the sequence-specific NCPR profiles and these changes can be quantified using a single parameter κ (20). In sequences with low κ values, the oppositely charged residues are well mixed. In such sequences, intrachain electrostatic repulsions and attractions are mutually screened and the result is a heterogeneous ensemble of expanded, random coil-like conformations (20). In contrast, in sequences with high κ values, the opposite charges are segregated within the sequence and electrostatic attractions dominate. The result is an ensemble of compact conformations.We used sequence design to uncover the role of charge patterning on the conformations of p27_96–198 and the impact, if any, on the efficiency of T187 phosphorylation. Specifically, we generated distinct variants of p27_96–198 by manipulating the charge patterning of its sequence between residues 100 and 180 while keeping the amino acid composition fixed. The sequence of the primary motif and its immediate sequence context, namely, positions 181–198, were also held fixed in the designed variants. In a motif-centric model, the efficiency of T187 phosphorylation should depend only on the presence or absence of the primary substrate motif and be insensitive to sequence changes within the unconstrained region of p27. However, contrary to a purely motif-centric model, we have uncovered cryptic sequence features within p27_96–198 that regulate T187 phosphorylation. Our findings suggest that the p27_96–198 IDR is not just a passive tether between the primary motifs encompassing Y88 and T187. Instead, the sequence features within p27_96–198 actively regulate the coupling of Y88 and T187 phosphorylation.