Cryptic sequence features within the disordered protein p27Kip1 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 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.

Keywords: disordered regions; motif; p27.

Fig. 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.

Fig. 2.

Design of p27-C permutants to probe effects of charge patterning on conformational features and intracomplex phosphorylation. (A) Sequences of the variants of p27-C including the WT. Hydrophobic residues are shown in black, polar residues in green, positively and negatively charged residues in blue and red, respectively. The κ value of each variant is shown to the Right of each sequence. (B) Each panel shows the NCPR profile along the linear sequence for each p27-C variant. T187 in each NCPR profile is marked using an orange asterisk. The blue and red peaks denote blocks of positive and negative charge, respectively.

Fig. 3.

Dimensions of the p27-C-vXY and p27-vXY variants. (A) R_g values for the p27-C-vXY variants are inversely correlated with κ as indicated by the Pearson r value –0.96. (B) R_g values determined from SAXS measurements for the p27-C variants are plotted against the R_g values estimated from the simulations. The two sets of values are positively correlated (r = 0.97). (C) The influence of charge patterning on dimensions of the p27-C-vXY variants in isolation was reproduced in the context of the full-length p27-vXY variants bound to Cdk2/cyclin A, as shown by strong positive correlation (r = 0.99) between the R_g values determined from SAXS measurements on both sets of constructs. In all plots, the blue dashed line denotes the linear fit to the quantity plotted in the respective ordinate. The error bars for the simulation results are SEs about the mean and for the SAXS they denote SDs based on experiments performed in triplicate.

Fig. 4.

Relative efficiencies of T187 phosphorylation via the in cis mechanisms. A.U., arbitrary units.The error bars denote SDs from three independent experiments. These data were obtained at a concentration of 2 μM for the p27-vXY/Cdk2/cyclin A ternary complex.

Fig. 5.

Correlation between the relative efficiencies of T187 phosphorylation measured via the in cis and in trans mechanisms for different p27-vXY variants. The Pearson r value is 0.89. The blue dashed line denotes the linear fit of the in cis phosphorylation efficiency as a function of the in trans efficiency. Both in cis and in trans phosphorylation data used in this analysis were obtained at 4 μM for the ternary complex. The error bars denote SDs of the values for phosphorylation efficiency measured from three independent experiments.

Fig. 6.

Normalized relative efficiency of T187 phosphorylation plotted against the wNCPR values within auxiliary motifs for p27-vXY and p27-v31^(wNCPR) variants. For p27-vXY variants, the datasets correspond to concentrations of 1, 2, and 4 μM of the 1:1:1 ternary complex of p27-vXY/Cdk2/cyclin A. For each dataset, shown using different colors, we calculated the normalized relative efficiency e by dividing the relative efficiencies by the corresponding concentrations of the ternary complex. Within error, the normalized relative efficiencies are coincident for each p27-vXY variant. The Pearson r value quantifying the positive correlation between e and wNCPR is 0.93. For the linear regression and correlation analysis we left out the data for p27-v78. The values along the ordinate can be written as e = m(wNPCR) + c. Here, m is the slope and c is the intercept. Because wNCPR is a dimensionless quantity the slope quantifies the increase in the normalized efficiency per unit rise in wNCPR and the intercept quantifies the normalized relative efficiency if the wNCPR is zero. The values for m and c are 133.1 A.U.⋅μM^–1 and 80.9 A.U.⋅μM^–1, respectively. This model predicts the normalized relative efficiency of T187 phosphorylation as a function of the wNCPR within the auxiliary motif of p27-vXY. The solid diamonds are data for p27-v31^(–0.30), p27-v31^(–0.10), p27-v31^(0.00), and p27-v31^(+0.25), respectively, and test the generality of the linear model in sequences where the κ value is fixed and wNCPR is varied by changes to the NCPR profile.